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					                  3nd Annual Institute for Molecular Virology Symposium
                              “Host Interactions and Structure”
                                        May 14th, 2006
                              Coffman Memorial Union Theater
                                          8am-6pm

                                    Symposium Program


7:30-7:55         Registration
                  Bagels, pastries and coffee

7:55-8:00         Welcome and Opening Remarks (Louis Mansky)

8:00-9:00         Morning Keynote Lecture: Jack Johnson
                  Intro by Dan Cohen
                  “Biophysical Studies of Viruses and their Maturation: Insights into
                  Elegantly Programmed Nanomachines”

Morning Session

Convenors: Mark Stenglein and Rebecca LaRue

9:00-9:20         Jinghua Tang, Paul J. Jardine, Norman Olson, Shelley Grimes,
                  Dwight L. Anderson and Timothy S. Baker
                  “Bacteriophage ø29 at subnanometer resolution: visualization of the
                  DNA double helix poised for release”

9:20-9:40         Yan Chen, Bin Wu, Karin Musier-Forsyth, Joachim D. Mueller
                  “Determining the stoichiometry of GAG protein in Viral Like Particle
                  using fluorescence fluctuation spectroscopy”

9:40-10:00        My Vo, Ioulia Rouzina, Karin Musier Forsyth
                  “Mg effects of Chaperone Activity of HIV NC: Importance of NC-
                  Induced Nucleic Acid Aggregation”

10:00-10:30       Morning break and poster viewing
                  Coffee, cookies

10:30-10:50       Jun Han, Yue Wang and Kay S. Faaberg
                  “Identification of Nonessential Regions of the Nsp2 Replicase Protein
                  of PRRSV Strain VR-2332 for Replication in Cell Culture”

10:50-11:10       Lenka Sedlackova and Stephen A. Rice
                  “HSV-1 ICP27 is required for the virion incorporation of two viral
                  transcriptional activators, ICP0 and ICP4”

11:10-11:30       Alistair McGregor, Yeon Choi, Jodi Anderson, Mike Leviton, Mike
                  McVoy, Mark Schleiss
                  “A chimeric GPCMV encoding HCMV UL97 renders GPCMV
                  susceptible to ganciclovir and maribavir while retaining ability to
                  disseminate in the animal host”


                                                                                          1
11:30-11:50      Ryuta Sakuma, Josh A. Noser, Seiga Ohmine and Yasuhiro Ikeda
                 “Rhesus monkey TRIM5alpha restricts HIV-1 production through
                 rapid degradation of viral Gag polyproteins”

                                 11:50-1:00 Lunch

Afternoon Session

Convenors: Rodrigo Giacaman and Casey Dorr

1:00-1:20        Guylaine Haché and Reuben S. Harris
                 “VIF-deficient HIV-1 can resist APOBEC3G”

1:20-1:40        Anjalee Vacharaksa, Anil C. Asrani, Mark C. Herzberg and Karen F.
                 Ross
                 “HIV-1 Infection in Keratinocytes Reduces Resistance to Candida
                 Invasion”

1:40-2:00        Irene J. Dorweiler, Azah Tabah, Casey Dorr and Louis M. Mansky
                 “Interplay of cell type and drug resistance on HIV-1 genetic variation”

2:00-2:20        Afternoon break and poster viewing
                 Coffee, snacks

2:20-2:40        Masato Yammamoto
                 “Conditionally Replicative Adenovirus (CRAd) for Gastrointestinal
                 Cancers”

2:40-3:00        Jorge Reyes del Valle, Patricia Devaux, Nicholas J. Wegner,
                 Gregory Hodge, Michael B. McChesney, Roberto Cattaneo
                 “A vectored measles vaccine induces anti-hepatitis B surface antigen
                 responses while protecting macaques against measles challenge”

3:00-4:00        Afternoon Keynote Lecture: Peter Palese
                 Intro by April Schumacher
                 “Influenza virus pandemics: past and future"

4:00             Closing Remarks (Louis Mansky)

4:00-6:00        Reception & Poster Session




                                                                                           2
IMV Advisory Committee:

Wade Bresnahan
Kay Faaberg
Paul Jardine
Reuben Harris
James Lokensgard
Louis Mansky
Michael Murtaugh
Irene Dorweiler
Guylaine Hache
Patrycja Lech

Academic financial support for the 2007 IMV Symposium was provided by:

Academic Health Center
School of Dentistry
School of Veterinary Medicine.

Corporate financial support was provided by:

Zeiss Technologies
Guava Technologies




                                                                         3
                    3rd Annual Institute for Molecular Virology Symposium
                                “Host Interactions and Structure”
                                          May 14th, 2007
                                         Talk Abstracts



Morning Keynote Talk: Biophysical Analysis of Virus Particles and their Maturation:
Insights into Elegantly Programmed Nano Machines

John E. Johnson, Department of Molecular Biology, The Scripps Research Institute

Abstract: Complex virus particles progress through a series of maturation intermediates
between initial assembly and formation of the infectious virion. The process is required to
accommodate the weak, equilibrium interactions between subunits that minimize errors in
assembly and the need for a robust particle that will tolerate the hostile environment outside of
the cell. The trajectory for maturation is launched after a signal is transmitted that the
organizational phase of subunit assembly (called a procapsid) is complete. What follows is a
cascade of highly choreographed subunit rotations and translations that totally remodels the
particle, with dramatic increases in buried surface area between subunits, a change in particle
size of 20% or more and the activation of auto-catalytic chemistry that “arms” the virus for
infection. In many cases the dynamics of such stunning machines is encoded in only one or two
gene products assembled in multiples of 60 copies on icosahedral surface lattices. The seminar
will address this process for a ds DNA bacteriophage, HK97, where an expression/assembly
system was developed that allows the entire progression to be studied in vitro. (1-7)To
compliment this contrived in vitro system we did the cryoEM structure of an authentic, mature
dsDNA bacteriophage, P22, without applying icosahedral symmetry, allowing the direct
observation of packaged dsDNA, the packaging motor and the accessory proteins required for
infectivity (8).

1 Gertsman et. al. 2006 The structure of a virus assembly intermediate: bacteriophage HK97
prohead II. J. Mol. Biol. Submitted
2. Wikoff et. al. 2000. Topologically linked protein rings in the bacteriophage HK97 capsid.
Science 289:2129-2133.
3. Wikoff et. al. 2006. Time-resolved molecular dynamics of bacteriophage HK97 capsid
maturation interpreted by electron cryo-microscopy and X-ray crystallography. J Struct Biol
153:300-6.
4. Gan et. al. 2006. Capsid conformational sampling in HK97 maturation visualized by X-ray
crystallography and cryo-EM. Structure accepted for publication
5. Lee et. al. 2005. Cooperative reorganization of a 420 subunit virus capsid. J Mol Biol
352:723-35.
6. Gan et. al. 2004. Control of crosslinking by quaternary structure changes during
bacteriophage HK97 maturation. Mol Cell 14:559-69.
7. Lee et. al. 2006. A quorum of covalent cross-links in HK97 bacteriophage capsids spring-
loads a conformational switch. J. Mol. Biol. Submitted
8. Lander et. al. 2006. The structure of an infectious P22 virion shows the signal for headful
DNA packaging. Science 312:1791-5.




                                                                                                    4
Bacteriophage ø29 at subnanometer resolution: visualization of the DNA double helix
poised for release
Author(s): Jinghua Tang, Paul J. Jardine, Norman Olson, Shelley Grimes, Dwight L. Anderson
and Timothy S. Baker

Abstract: Asymmetric cryoEM-3D reconstructions of fiberless mature and empty virions of the
Bacillus subtilis bacteriophage ø29 yielded subnanometer (~8-9Å) resolution in the region of the
head-tail junction. Fitting of the dodecameric head-tail connector (gp10) crystal structure into the
cryoEM reconstruction density showed good alignment of the multiple alpha-helices of the
central region of the connector with corresponding cylindrical features in the reconstruction,
validating the resolution of the map. However, the wide end of the connector that is embedded
in the head was distorted, and the narrow end of the connector, which contacts both the lower
collar (gp11) of the tail and the appendages (gp12*), was substantially rearranged in both maps
when compared to the connector crystal structure. The conformation of the twelve appendages
that radiate from the head-tail region appeared to be dictated by their interaction with the head
shell protein (gp8) and not the head fibers (gp8.5) as reported previously. Appendages
predominantly adopted a "down" conformation, with the large lobe of the appendage aligned
parallel to the tail axis, whereas the remaining appendages projected out from the tail axis in an
"up" position, approximately perpendicular to the tail axis. The "down" appendages showed
contact at the connecting arm with the gp8 capsid, and appendages in the "up" position showed
no such contact. Continuous density in the fiberless virion, ascribed to the right end of the
dsDNA genome and its convalently-linked, terminal protein gp3, ran along the central channel
that traversed the connector, collar, tail tube and a portion of the tail knob. The gp3 was
assigned to the column of more diffuse density, beginning near the top of the tail knob and
extending upward toward the head, ending in the tail tube short of the lower collar. Density at
this point became more defined and consistent in size and shape with a segment of helical
DNA, traversing the lower collar and connector and merging with the shells of DNA in the head.
The lower end of the connector and the collar together formed a large central cavity along the
channel axis. Here the DNA deviated from an otherwise straight path to form a toroid of ~60Å
diameter. This extreme DNA conformation maybe generated, in part, from compression of the
linear DNA in the channel due to the high pressure within the head. Extra tension stored in the
right end of the packaged genome may facilitate initial penetration of the DNA-gp3 complex into
the host during infection.




                                                                                                       5
Determining the stoichiometry of GAG protein in Viral Like Particle using fluorescence
fluctuation spectroscopy
Author(s): Yan Chen, Bin Wu, Karin Musier-Forsyth, Joachim D. Mueller

Abstract: Retroviral GAG protein, a 55 kDa protein, is sufficient to drive viral like particle (VLP)
assembly. Determining the stoichiometry of GAG in virions has served as an internal standard
for assessing the copy number of many other proteins present in virions, and has been carried
out using either biochemical or electron microscopy techniques. Here, we demonstrate that we
are able to accurately determine the stoichiometry of GAG by using fluorescence fluctuation
spectroscopy. The fluorescence fluctuations within the small excitation volume generated by
two-photon excitation provide, via fluorescence correlation spectroscopy (FCS) and photon
counting histogram (PCH) analysis, a unique way to study biological phenomena on the single
molecule level. We use fluorescence tagged GAG protein, and apply both PCH and
autocorrelation analysis to study VLPs obtained from COS cells. We show that tagging GAG
with GFP does not change the stoichiometry of GAG in VLP by systematically diluting GFP
tagged GAG with untagged GAG. The stoichiometry of GAG determined from PCH is
approximately 750 copies in a VLP particle. Surprisingly, the stoichiometry of Gag in VLP highly
depends on the DNA concentration that used in transfection. The stoichiometry of Gag could
increase from 750 copies to 2000 copies depending on the transfection condition. We will
discuss the implication of our finding. In conclusion, fluorescence fluctuation spectroscopy
presents a novel way to study the stoichiometry of proteins in a viral particle.




                                                                                                       6
Mg effects of Chaperone Activity of HIV NC: Importance of NC-Induced Nucleic Acid
Aggregation.
Author(s): My Vo, Ioulia Rouzina, Karin Musier Forsyth

Abstract: In this work we study effects of Na+, Mg2+ and HIV-1 nucleocapsid protein (NC) on
annealing kinetics of complementary DNA and RNA hairpins of either 30 (mini-TAR) or 59
(TAR) nucleotides (nt) in length from HIV genome. We use gel-shift assay to follow annealing
kinetics in the whole range of nucleic acid (NA) saturation with NC, including high NC leading to
NA aggregation, which we also directly characterize in a parallel sedimentation assay. We
show, that while in the absence of NC the ability of Na+ and Mg2+ cations to facilitate hairpin
annealing is rather limited, its facilitation by NC reached 103-105-fold, dependent on type of NA
molecule. Titration with NC or NC/ Mg2+ competition studies show that the annealing kinetics
depends only on the level of NA saturation with NC. NC competes with Mg2+ or Na+ for
sequence none-specific NA binding as a simple trivalent cation. Upon its saturation NC induces
attraction between NA molecules of ~0.2 kcal/mol/nt, in agreement with the hypothesis of
electrostatic mechanism of NC-induced NA aggregation. The hairpins anneal via the two-step
process, involving formation of annealing intermediate in pre-equilibrium to conversion to
extended duplex. The major effect of NC is on the bimolecular association step of the annealing
intermediate that is facilitated 100- to 1000- fold, while effect of NC on monomolecular rates is
only within ~3-fold. Shorter hairpins anneal faster at low NC, due to less entropy loss upon their
complex nucleation. DNA mutation studies show that the longer hairpins undergo the annealing
pathway switch with the increasing NC binding. The annealing proceeds via the extended loop-
loop intermediate at low NC and via the stem ends at high NC. This switch happens due to the
strong NC-induced stabilization of annealing intermediate, which involves much shorter NA
region at high NC.




                                                                                                     7
Identification of Nonessential Regions of the Nsp2 Replicase Protein of PRRSV Strain
VR-2332 for Replication in Cell Culture
Author(s): Jun Han, Yue Wang and Kay S. Faaberg

Abstract: The nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome
virus (PRRSV) is a multi-domain protein and has been shown to undergo remarkable genetic
variation, primarily in its middle region, while exhibiting high conservation in the N-terminal
putative protease domain and the C-terminal predicted transmembrane region. A reverse
genetic system of PRRSV North American prototype VR-2332 was developed to explore the
importance of different regions of nsp2 for viral replication. A series of mutants with in-frame
deletions in the nsp2 coding region were engineered and infectious viruses were subsequently
recovered from transfected cells and further characterized. The results demonstrated that the
cysteine protease domain (PL2), the PL2 downstream flanking sequence (aa181-323) and the
putative transmembrane domain were critical for replication. By contrast, the segment of nsp2
preceding the PL2 domain (aa13-35) was dispensable for viral replication, and the nsp2 middle
hypervariable region (aa324-813) tolerated 100aa or 200aa deletions but could not be removed
as a whole; the largest deletion was about 400aa (nsp2”324-726). Characterization of the
mutants demonstrated that those with small deletions possessed growth kinetics and RNA
expression profiles similar to the parental virus, while the nsp2”324-726 mutant displayed
decreased cytolytic activity on MARC-145 cells and did not develop visible plaques.




                                                                                                   8
HSV-1 ICP27 is required for the virion incorporation of two viral transcriptional
activators, ICP0 and ICP4.
Author(s): Lenka Sedlackova and Stephen A. Rice

Abstract: Herpes simplex virus type 1 (HSV-1) immediate-early (IE) proteins ICP0 and ICP4 are
key inducers of viral gene transcription. These two proteins are characteristically observed in
the nuclei of HSV-1 infected cells early in infection, but become progressively more cytoplasmic
as the infection ensues. This relocalization was previously shown to be dependent on another
IE protein, ICP27, which is itself an important viral gene activator. ICP27 null mutants fail to
replicate in Vero cells due to a tight block to late gene expression. Here we identify an HSV-1
ICP27 deletion mutant, d3-4 (expressing an ICP27 polypeptide having a small N-terminal
deletion), which replicates to near wild-type (WT) levels in Vero cells but which is completely
unable to relocalize ICP0 and ICP4 to the cytoplasm. This finding demonstrates that ICP27's
effect on ICP4/0 localization is distinct from its role in viral gene induction. Since low levels of
ICP4 and ICP0 are incorporated into the tegument layer of mature virions, and final
tegumentation occurs in the cytoplasm, we hypothesized that ICP27 could be required for the
virion incorporation of ICP4 and ICP0. To test this, the composition of WT and d3-4 virions was
compared. Although both types of virion had similar amounts of several structural proteins
(including capsid protein VP5, tegument protein VP16, and envelope glycoprotein D), d3-4
virions were completely devoid of ICP0 and had greatly decreased levels of ICP4. Thus, our
results demonstrate that ICP27, itself not a virion component, is a required co-factor for the
efficient virion incorporation of ICP0 and ICP4.




                                                                                                       9
A chimeric GPCMV encoding HCMV UL97 renders GPCMV susceptible to ganciclovir and
maribavir while retaining ability to disseminate in the animal host
Author(s): Alistair McGregor, Yeon Choi, Jodi Anderson, Mike Leviton, Mike McVoy, Mark
Schleiss

Abstract: Species-specific constraints preclude the study of human cytomegalovirus (HCMV) in
animal models and necessitate the use of animal species CMVs to model human disease.
However, animal CMV vary in their susceptibility to antivirals that are efficacious for HCMV. For
example, guinea pig cytomegalovirus (GPCMV) is uniquely valuable for modeling congenital
CMV infection but is not sensitive to ganciclovir (GCV), the most common HCMV antiviral in
clinical use, or maribavir (MBV), a new generation antiviral currently in phase II trials. The
activity of both drugs is linked to the viral kinase, pUL97: MBV is thought to act by inhibiting its
kinase activity, whereas GCV must be phosphorylated by pUL97 to be active. We hypothesized
that the GPCMV homolog encoded by GP97 might be (1) insensitive to inhibition by MBV, and
(2) unable to phosphorylate GCV. If so, susceptibility to both drugs could be improved by
engineering a chimeric GPCMV in which GP97 is replaced with UL97. The GPCMV genome,
cloned as a BAC, was modified to introduce UL97 from HCMV Towne strain. Recombinant BAC
colonies in E.coli were selected by co-insertion of a kanamycin-resistance marker and verified
by restriction profile analysis. The in-frame insertion of UL97 into the GP97 locus was verified
and transfection of BAC DNA onto guinea pig lung fibroblasts regenerated a viable
GPCMV/UL97 chimeric virus. Growth kinetics demonstrated near wild type levels of growth for
the chimera, whereas a GP97 null mutant grew poorly, indicating that UL97 can functionally
complement the growth deficiency that results from the loss of GP97. Expression of the UL97
protein was verified by by western immunoblotting using HCMV pUL97-specific antisera. The
chimeric virus showed increased susceptibility to both GCV and MBV. Intraparitoneal challenge
of cyclophosphamide-immunocompromised strain 2 guinea pigs resulted in dissemination to
principle target organs and mortality, whereas animals treated with GCV (25 mg/kg/day) had
reduced mortality and reduced viral dissemination in comparison with the placebo group.




                                                                                                   10
Rhesus monkey TRIM5alpha restricts HIV-1 production through rapid degradation of viral
Gag polyproteins
Author(s): Ryuta Sakuma, Josh A. Noser, Seiga Ohmine and Yasuhiro Ikeda

Abstract: Rhesus monkey TRIM5alpha (TRIM5arh) restricts HIV-1 infection at a post-entry, pre-
integration stage in the viral life cycle through recognition of the incoming capsid and promoting
its premature disassembly. TRIM5a comprises a RBCC (RING, B-box and coiled-coil motifs)
domain and a B30.2(SPRY) domain. Sequences in the B30.2(SPRY) domain dictate the
potency and specificity of the restriction. Since TRIM5arh targets incoming mature HIV-1 capsid,
but not precursor Gag, it was assumed that TRIM5arh did not affect HIV-1 production. Here, we
present surprising findings that TRIM5arh blocks HIV-1 production. Disruption of endogenous
TRIM5arh expression with the TRIM5a-specific siRNA or shRNA constructs increased HIV-1
virion production from infectious molecular clones up to 20-fold in rhesus monkey cells. In
contrast, the TRIM5a siRNA treatment did not affect production of SIVMAC or SIVAGM.
Expression of the siRNA-resistant TRIM5arh mutant abolished the effects of TRIM5a siRNA
treatment on HIV-1 production, indicating that TRIM5arh specifically restricts HIV-1 production
in rhesus monkey cells. Over-expression of TRIM5arh in 293T cells reduced HIV-1 production
from infectious clones up to 200-fold, while its human orthologue (TRIM5ahu) only marginally
reduced the production. Immunoblotting analysis of producer cell lysates showed the reduction
of HIV-1 p24 and precursor Gag in the presence of TRIM5arh. A pulse-chase stability assay
revealed that HIV-1 Gag polyprotein was rapidly turned over in the presence of TRIM5arh.
When the TRIM5arh-mediated Gag degradation was suboptimal, intact and truncated forms of
TRIM5arh were readily detected in the HIV-1 virus-like particles, suggesting that TRIM5arh
interacts with HIV-1 Gag polyprotein before or during Gag assembly. Intriguingly, a panel of the
chimeric TRIM5arh-TRIM5ahu proteins revealed that the specificity determinant for restriction
lies in the RBCC domain, but not in the B30.2(SPRY) domain. Taken together, we conclude that
TRIM5arh can block HIV-1 production through a mechanism distinct from the well-known
TRIM5a-mediated post-entry restriction of incoming HIV-1 capsid. With a greater understanding
of how TRIM5arh blocks HIV-1 production, it may be possible to develop a novel molecular
strategy to inhibit HIV-1 replication.




                                                                                                 11
Vif-Deficient HIV-1 can resist APOBEC3G
Author(s): Guylaine Haché and Reuben S. Harris

Abstract: APOBEC3G is an anti-retroviral cellular enzyme that can inhibit the replication of Vif-
deficient HIV-1 by hypermutating nascent cDNA. In a normal infection, Vif functions to
effectively neutralize APOBEC3G by marking it for degradation. Nevertheless, the remarkable
potency of APOBEC3G has encouraged the development of therapeutic strategies that would
prevent its neutralization. For instance, compounds that inhibit Vif would theoretically render the
virus sensitive to APOBEC3G. If such drugs were to be discovered, an important question is
whether such a _Vif-deficient_ virus might ultimately be able to evolve to resist APOBEC3G (of
course, in addition to the compound itself)? To begin to answer this question, APOBEC3G was
expressed stably and constitutively in a T cell line and HIV growth experiments were performed.
The parental cell line was fully permissive for HIV(delta-Vif) growth, whereas the APOBEC3G-
expressing derivatives were completely non-permissive. Vif-proficient virus was able to replicate
efficiently on both the parental and clonal lines with replication peaking approximately 7-10 days
post-infection. In most continuous culture experiments, HIV(delta-Vif) failed to replicate on the
APOBEC3G-expressing clones. However, after more than 28 days of continuous culture, a
sizable fraction of the APOBEC3G-expressing cultures showed signs of HIV(delta-Vif)
replication suggesting the emergence of APOBEC3G-resistant virus. This possibility was
confirmed using cell-free supernatants to infect new APOBEC3G-expressing cells. Robust
replication of the resulting virus was observed within 15 days. The molecular nature of some of
the mutations that confer resistance to APOBEC3G and how these studies impact our current
understanding of the APOBEC3G-dependent restriction mechanism will be discussed.




                                                                                                  12
HIV-1 Reduced the Resistance to Candida Invasion in TERT-2
Author(s): Vacharaksa A., Asrani AC., Herzberg MC., and Ross KF.

Abstract: Background: Candidiasis is a life-threatening consequence of the emergence of AIDS
in HIV-positive patients. The appearance of oral candidiasis may reflect inadequate host innate
immune response. Calprotectin (S100A8/A9; MRP8/14) expressed in gingival keratinocytes is
an innate immune factor with anti-fungal activity that may contribute to resistance of invasion
and colonization by C. albicans. We hypothesized that HIV-1 down-regulates calprotectin in oral
keratinocytes to reduce resistance to Candida invasion and colonization. Methods: The
immortalized oral keratinocyte OKF6/TERT-2 cell line (TERT-2) was grown in monolayer and
incubated with HIVIIIb or HIVBaL at a MOI (TCID50/cell) of 0.01 for 3, 6, 12, 24, and 48 h.
Expression of S100A8- and A9-specific mRNAs was quantified by SYBR Green Q-PCR. To test
the resistance of TERT-2 to Candida, TERT-2 cells were grown overnight in either 96-well or
24-transwell plates. Cells were incubated with HIV-1 for 48 h before adding C. albicans (strain
10261, 5957, or 6284; MOI 10:1). After 24 h, cell damage by C. albicans was estimated as LDH
release into culture supernatants (96-well plates). To detect C. albicans traversing the TERT
monolayer after 24 h, media from the lower chamber of the transwell system was plated and
Candida were enumerated. Results: S100A8- and A9-specific mRNAs were constitutively
expressed at all time points and did not appear to be regulated by HIV-1. TERT-2 cells
incubated with HIVIIIb, however, showed 11, 12, and 14 % more cytotoxicity in response to
Candida strains 5957, 10261, 6284, respectively, and 11, 16 and 9 % more, respectively, if
incubated with HIVBaL. After incubation with HIV-1, TERT-2 monolayers allowed more C.
albicans to traverse, ranging from 20 to more than 1000 cells depending on the strain.
Conclusion: HIVIIIb and HIVBaL did not appear to regulate constitutive expression of
calprotectin-specific mRNA by TERT-2 oral keratinocytes. None-the-less, TERT-2 cells
incubated with HIV-1 for 48 h showed reduced ability to resist cell damage mediated by C.
albicans. HIV-1, therefore, is able to modulate innate immune resistance of oral keratinocytes to
C. albicans.




                                                                                                13
Interplay of cell type and drug resistance on HIV-1 genetic variation
Author(s): Irene J. Dorweiler, Azah Tabah, Casey Dorr and Louis M. Mansky

Abstract: An undesirable outcome to highly active antiretroviral therapy (HAART) is the
selection and outgrowth of drug resistant virus. Therapy failure has spawned intense interest in
the identification of new antivirals as well as a better understanding of the selection and
evolution of HIV-1 drug resistance. We have extensively investigated HIV mutagenesis in the
context of drug therapy using sophisticated vector systems that monitor mutations in single
round replication assays. We previously discovered that drug-resistance conferring mutations in
reverse transcriptase could significantly alter (increase or decrease) the virus mutation rate.
Furthermore, we have found that nucleoside reverse transcriptase inhibitors as well as non-
nucleoside reverse transcriptase inhibitors can alter the HIV-1 mutation rate by mechanisms
including dNTP pool depletion or imbalances. Our recent attention has been directed at the
analysis of cell type on HIV mutagenesis in the context of drug therapy. To do this, we have
developed a new HIV vector system for more rapid and efficient tracking of mutations during
virus replication. Our current findings suggest that alteration of nucleotide metabolism and
expression of host cell proteins can impact HIV mutagenesis and can be exaggerated in the
context of drug treatment and/or drug resistant virus. Our findings are clinically relevant and
suggest approaches for improving the effectiveness and endurance of HAART.




                                                                                               14
Conditionally Replicative Adenovirus (CRAd) for Gastrointestinal Cancers
Author(s): Masato Yamamoto

Abstract: The clinical outcome of advanced gastrointestinal (GI) cancers (especially pancreatic
and esophageal cancers) is dismal despite the advance of current therapeutic strategies.
Cancer gene therapy is a category of new therapeutics, among which conditionally replicative
adenovirus (CRAd) is one promising strategy to overcome current obstacles of cancer gene
therapy. Various CRAds have been developed for GI cancer treatment by taking advantage of
the replication biology of adenovirus. Some CRAds have already been tested in clinical trials but
have fallen short of initial expectations. Current concerns for clinical applicability are therapeutic
potency, replication selectivity, and interval endpoints in clinical trials. Also, improvement of
animal experiment models is needed for deeper understanding of CRAd biology. Despite these
obstacles, CRAds continue to be an exciting area of investigation with great potential for clinical
utility. Further virological and oncological research will eventually lead to full realization of the
therapeutic potential of CRAds in the field of GI cancers.




                                                                                                     15
A vectored measles vaccine induces anti-hepatitis B surface antigen responses while
protecting macaques against measles challenge
Author(s): Jorge Reyes del Valle, Patricia Devaux, Nicholas J. Wegner, Gregory Hodge,
Michael B. McChesney, Roberto Cattaneo

Abstract: We hypothesize that the measles virus (MV) vaccine, which has an outstanding
efficacy and safety record, is an ideal platform for the development of pediatric vaccines eliciting
immunity against other pathogens. We have produced an infectious cDNA from a vial of vaccine
(Moraten/Schwartz strain) and generated from it an infectious virus with vaccine identical coding
capacity. On the basis of this cDNA we then designed and generated recombinant MV that
express HBsAg at three different levels: about 200, 100 and 50 ng/ml in media of infected cells.
HBsAg was secreted as empty particles of approximately 1.12-1.14 mg/ml density. All viruses
replicated at similar levels. To asses their efficacy in inducing a humoral immune response
against HBsAg after single inoculation, we inoculated genetically modified mice susceptible to
MV infection. These Ifnarko-CD46Ge mice express CD46 with human like tissue specificity and
have a defective interferon alpha-beta receptor. Groups of ten mice inoculated with
MVvac2(HBsAg)P, the vector expressing HBsAg at the highest levels, showed a robust
response with anti-HBsAg levels of 590 mIU/ml in average. While, the mice immunized with the
virus expressing two times less HBsAg had a response 15 times lower. Mice immunized with
the virus expressing four times less HBsAg had a marginal response. Subsequently, we
immunized groups of four Rhesus monkeys (Macaca mulatta), MV natural host, with MVvac2,
and the two higher HBsAg expressing viruses. The anti-MV immune response was equivalent
between the groups. Starting at week 4 post inoculation (PI), we detected an anti-HBsAg
response in 2 monkeys inoculated with MVvac2(HBsAg)P, this response increased steadily
reached 180 and 340 mIU/ml 12 weeks PI. Six to ten weeks PI all the animals were challenged
with wild type MV and none of them developed disease. Thus, one dose of a vectored measles
vaccine induced anti HBsAg responses while protecting macaques against measles challenge.




                                                                                                   16
Afternoon Keynote Talk: Influenza Virus Pandemics: Past and Future
Peter Palese, Department of Microbiology, Mount Sinai School of Medicine

Abstract: The influenza pandemic of 1918/1919 was a unique event in recorded history, costing
on the order of 50 million lives within the time span of only several months. The virus is
obviously extinct, but the advent of reverse genetics techniques for negative strand RNA viruses
(a family of viruses which includes the influenza viruses) makes it possible to reconstruct
infectious influenza viruses. Based on the nucleotide sequence that was obtained from RNA
fragments present in lung samples of victims of the 1918 influenza virus, we succeeded in
rebuilding the extinct pandemic virus entirely from commercially available oligonucleotides. This
virus turned out to be highly virulent in the mouse model, more than any other human influenza
virus strain tested. It also was shown to be highly pathogenic for chicken embryos and grew in
human tissue culture cells to high titers and replicated in cells in the absence of trypsin (also
indicative of high virulence). On the other hand, the 1918 virus was shown to be sensitive to the
FDA-approved antivirals (amantadine as well as the neuraminidase inhibitors). We were also
able to show that vaccines worked perfectly well in protecting mice against a challenge with a
virus containing the 1918 hemagglutinin and neuraminidase genes. We also postulate that the
human population, having experienced infections with currently circulating H1N1 viruses, is
partially immune to a 1918 or 1918-like virus. The availability of the 1918 virus will help us to
better understand the molecular basis of virulence and the mechanisms by which pandemic
influenza viruses are transmitted from human to human and from one species to another. In
addition, these studies will expand our knowledge of the biological and molecular properties of
pandemic influenza viruses in general and this in turn will provide us with better prevention and
treatment strategies for the future.




                                                                                                17
               3rd Annual Institute for Molecular Virology Symposium
                           “Host Interactions and Structure”
                                     May 14th, 2007
                                   Poster Abstracts

1   Title: A Gradual Expansion Model for APOBEC3 Gene Evolution is Suggested by
    the Genomic Sequences of Pig, Sheep and Cattle
    Author(s): Rebecca S. LaRue, Stefán R. Jónsson, Tim Smith, Valgerdur Andrésdottir,
    and Reuben S. Harris

    Abstract: The genomic sequences of mice and humans revealed a dramatic expansion
    in the number of APOBEC3 genes _ from one in mice to seven in humans.
    Chimpanzees also appear to have seven APOBEC3 genes. An obvious question is
    therefore whether this expansion occurred gradually during the evolution of mammals
    or whether it occurred recently (possibly even primate-specific). To begin to address
    this question, we used degenerate PCR, RACE and database searching to assess the
    number of APOBEC3 genes in representative artiodactyls cattle, pigs and sheep.
    Several APOBEC3 sequences were recovered but these approaches proved far from
    definitive. We therefore used APOBEC3-specific hybridization to identify pig and sheep
    BACs harboring this locus. The full DNA sequence spanning this genomic region was
    determined by sequencing BAC fragments. Interestingly, sheep proved to have two
    APOBEC3 genes, whereas pigs had only one. During the course of this work, the cattle
    genome project released a draft sequence, which also indicated the presence of two
    APOBEC3 genes. These data therefore suggest that the common ancestor of primates
    and artiodactyls had two APOBEC3 genes and that during mammalian evolution the
    domesticated pigs have since lost one. Together with the fact that APOBEC3 genes
    from both artiodactyls and primates show evidence of positive selection these data
    further suggest the presence of an ongoing selective pressure that has facilitated the
    gradual expansion of APOBEC3 gene number during the evolution of mammals.

2   Title: Genetic Dissection of Chemoresistance Mechanisms and Development of
    Reverse Strategy in Leukemia Using Cell Culture and Mouse Transplantation
    Models
    Author(s): Bin Yin, Miechaleen D. Diers, Susan K. Rathe, Shannon M. Delaney, M.
    Eileen Dolan, Scott W. Lowe and David A. Largaespada

    Abstract: Chemoresistance remains a major barrier to successful clinical treatment of
    acute myeloid leukemia (AML). With the existing regimen including cytarabine and
    doxorubicin, there is still a large percentage of patients who do not reach complete
    remission or die from relapse. However, the underlying molecular mechanisms of
    chemoresistance in AML are largely unknown. Previously, we reported that loss of
    tumor suppressor genes, Nf1 and Trp53, can confer upon myeloid leukemia cells
    partial resistance to cytarabine (Ara-C), a mainstay of AML chemotherapy. Microarray
    gene expression profiling revealed a few genes/pathways altered in resistant leukemia
    cells, including a group of lysosome function-related genes, and an important regulator
    of apoptosis, Bcl-xL. We now report that Nutlin-3, a p53-stabilizing compound, can
    increase sensitivity of Ara-C in both Ara-C-sensitive and -resistant leukemia cell lines.
    Chloroquine which can collapse the pH gradient of lysosomes and induce their swelling
    was also found to sensitize myeloid cell lines to Ara-C. Furthermore, we have
    examined the possibility of modeling AML chemoresistance in mouse, with the intention
    to identify and characterize AML relapse-related genetic abnormalities. To study the
    pharmacokinetics of Ara-C, mice were intraperitoneally injected with Ara-C at 250
    mg/kg. Sera were taken 10 min, 30 min, 1 hr, 2 hr, 4 hr and 8 hr after Ara-C injection,
    and subjected to HPLC analysis. Ara-C peaked at 10 minutes and was greatly reduced
    by 30 minutes, and went undetectable after 1 hour. To study the pharmacodynamics of
    Ara-C, leukemia cells were treated with Ara-C at 0, 2.5uM, 5uM and 10uM for 24 hours,     18
    but no AraCTP was detected at any of these concentrations, suggesting that Ara-C
    may be activated and catalyzed within 24 hours or values are below the level of
    detection. In addition, cell doses within the range of 1 x 102 ~ 1 x 106 were tested to
    Ara-C, leukemia cells were treated with Ara-C at 0, 2.5uM, 5uM and 10uM for 24 hours,
    but no AraCTP was detected at any of these concentrations, suggesting that Ara-C
    may be activated and catalyzed within 24 hours or values are below the level of
    detection. In addition, cell doses within the range of 1 x 102 ~ 1 x 106 were tested to
    determine the appropriate amount of donor leukemia cells transplanted.
    Transplantation of 1 x 104 ~ 1 x 105 of leukemia cells caused moribund within 3~4
    weeks. This model is under further improvement including testing of tumor response to
    compounds such as MEK inhibitors, Bcl-xL inhibitors and Nutlin-3, and for investigation
    of whether treatment response in leukemia is affected by change in expression level of
    Bcl-xL and Trp53.

3   Title: Presence of haptoglobin alpha1S subunit in serum is associated with
    acute PRRSV infection.
    Author(s): J.S. Gnanandarajah*, C. Dvorak, C. Johnson, M.P. Murtaugh. Dept. of
    Veterinary and Biomedical Sciences, University of Minnesota, Saint Paul, MN.

    Abstract: In porcine reproductive and respiratory syndrome (PRRS), the current
    diagnosis is based on screening for antibodies by ELISA and confirming the presence
    of the organism by PCR. However, detection of antibodies is not possible before seven
    days after infection and PCR is not feasible as an on-site field test. An early warning
    biomarker of infection would improve diagnosis and facilitate better preventive
    strategies. Here, we hypothesized that PRRSV infection produces a serum protein
    profile characteristic of early infection. The objective was to obtain a serum profile of
    low molecular weight proteins in PRRSV-infected and non-infected porcine sera by
    mass spectrometry. The sera from pigs infected with various isolates of PRRSV (n=25)
    and controls (n=34) were processed to remove high molecular weight proteins using
    organic solvent precipitation. The extracted low molecular weight proteins were
    desalted in a C4 resin column, mixed with sinapic acid and analyzed on MALDI-TOF.
    Comparative analysis of serum protein profiles revealed that a protein with an m/z
    value of 9244±2 appeared in PRRS sera within one day of infection, with sensitivity of
    0.92 and specificity of 0.94 at seven days of infection. When sera from pigs infected
    with non-PRRSV pathogens (n=24) were included in the analysis the specificity
    dropped to 0.83. The 9244±2 was identified through SDS-PAGE and LC/MS/MS as the
    alpha1S subunit of porcine haptoglobin. This was further confirmed by immunoblotting
    the organic solvent-extracted serum proteins with anti-porcine haptoglobin antibody.
    The results suggest that the alpha1S subunit of haptoglobin, a well known acute phase
    protein, is a potential protein biomarker for acute PRRS. The presence of free alpha1S
    subunit in serum of PRRSV infected pigs may provide new insights into biochemical
    processing of haptoglobin and its role in PRRSV pathogenesis.


4   Title: Porphyromonas gingivalis co-infection increases release of infectious HIV
    by oral keratinocytes
    Author(s): Rodrigo A. Giacaman, Anil C. Asrani, Kristin H. Gebhard, Elizabeth A.
    Dietrich, Anjalee Vacharaksa, Karen F. Ross and Mark C. Herzberg

    Abstract: Oral keratinocytes become infected by HIV-1 (Vacharaska, et al., submitted),
    but systemic infection occurs infrequently by the oral route. Occurrence may be
    increased by concomitant bacterial infection of the oral tissues, since co-infection and
    inflammation of some cell types increases HIV-1 replication. Porphyromonas gingivalis
    (Pg), a putative periodontal pathogen, was, therefore, hypothesized to modulate the
    outcome of HIV infection in oral epithelial cells. Monolayers of TERT-2 cells, an
    immortalized oral epithelial cell line, were exposed to Pg for 3 h, washed, exposed for 6
    h to either X4- or R5-type HIV-1, washed and maintained in keratinocyte growth media
    for up to 54 hours. Based upon real-time PCR detection of HIVgag RNA, TERT-2 cells
    that were pre-incubated with Pg showed increased harbored X4- and R5-type HIV-1, as      19
    compared to virus-only controls. HIVgag RNA detection, however, gradually decreased
    after 7 h, until signals were barely detectable from 18 to 54 h. Conversely, culture
    supernatants from the TERT-2 infection showed increasing levels of HIV p24 protein by
    that were pre-incubated with Pg showed increased harbored X4- and R5-type HIV-1, as
    compared to virus-only controls. HIVgag RNA detection, however, gradually decreased
    after 7 h, until signals were barely detectable from 18 to 54 h. Conversely, culture
    supernatants from the TERT-2 infection showed increasing levels of HIV p24 protein by
    ELISA until 12 h, remaining constant through 54 h, independent of the presence of Pg.
    TERT-2 culture supernatants were also inoculated onto permissive TZM-bl HIV-
    infection reporter cells (CXCR4+ CCR5+ CD4+). In TERT-2 cells, co-infection by Pg
    and R5-type HIV-1 induced release of more infectious virions than Pg and X4-HIV-1 or
    HIV-1 alone. In these conditions, TERT-2 cells expressed multiply spliced RNA below
    the limits of detection, suggesting that HIV does not replicate. Pg co-infection,
    therefore, appears to induce oral keratinocytes to increase harbored HIV in an R5-
    selective manner, which may be mediated by Pg up-regulation of the HIV co-receptor
    CCR5 (Giacaman, et al., submitted). Pg may also trigger cellular mechanisms to harbor
    HIV without endosome-lysosome degradation, since Pg increases release of infectious
    HIV, without increasing transcription of viral RNA. Thus, Pg infection in periodontal
    disease could increase the risk of oral infection by HIV-1. Supported by NIH/NIDCR
    R01DE015503.

5   Title: Comprehensive analyses of diverse lentiviral and retroviral vectors for
    post-entry restriction by TRIM5alpha and implications for gene therapy
    Author(s): Dyana T. Saenz and Eric M. Poeschla

    Abstract: We analyzed HIV-1, FIV, EIAV, N-MLV and NB-(i.e., Moloney)-MLV vectors
    for post-entry restriction by human (Ref1) and monkey (Lv1) activities and by the
    respective species-specific TRIM5alpha proteins that mediate them. RT-normalized
    preparations were compared. FIV, EIAV and HIV-1 vector infectivity varied widely but
    concordantly in numerous non-primate mammalian cell lines. In contrast, in diverse
    human and monkey cells, relative restriction of FIV and EIAV compared to HIV-1 varied
    from none to substantial, with the greatest nonprimate vector infectivity deficits
    observed in human T cell lines. Using permissive (restriction factor-lacking) non-
    primate cells engineered to express human or rhesus TRIM5alpha, we compared
    restricted (HIV-1, FIV, EIAV, and N-MLV) and unrestricted (NB-MLV) retroviruses
    across wide ranges of viral inputs. EIAV, FIV and N-MLV vectors were restricted by
    rhesus and human TRIM5alpha, while HIV was only significantly restricted by the
    rhesus allele and NB-MLV was unrestricted. For FIV, human TRIM5alpha was less
    restricting than rhesus TRIM5alpha, while EIAV was approximately equivalently
    restricted by both alleles. N-MLV was much more proportionately restricted than FIV or
    EIAV by human TRIM5alpha. Rhesus and human TRIM5alpha proteins restricted in a
    saturable manner, since endogenous and introduced TRIM5alpha restrictions could be
    titrated by co-transduction with FIV, HIV-1 or EIAV virus-like particles. We achieved
    stable RNAi knockdown of endogenous rhesus TRIM5alpha in FRhK4 cells, which
    released the Lv1 restriction, resulting in marked increases in all lentiviral vector
    infectivities while having no effect on negative control NB-MLV. Interestingly, in canine
    cells but not feline cells, human TRIM5alpha supported minimal restriction of FIV
    compared to significant restriction of EIAV, a phenomenon which is under ongoing
    mechanistic investigation. We conclude that human and rhesus TRIM5alpha proteins
    restrict both FIV and EIAV vectors, with the degree of restriction in different human cell
    lines varying from none to substantial. Human T cells are more susceptible to
    transduction by HIV vector than either non-primate vector. Stable knockdown of rhesus
    TRIM5alpha releases LV1 restrictions. Differential restrictions in canine cells raise the
    possibility that these proteins may not function autonomously or that a canine factor
    may interfere. Further development of lentiviral vector gene therapy will benefit from
    informed appraisal of the consequences of Ref1/human TRIM5alpha restriction, which
    may be advantageous or disadvantageous depending on the particular application.

                                                                                             20
6   Title: tBacillus subilis Bacteriophage Phi29 Gp13 in Tail Assembly
    Author(s): Daniel Cohen, Stephen Erickson and Dwight Anderson
    Abstract: The Bacillus subtilis bacteriophage ø29 provides a genetic and structural
    model for dsDNA virus assembly and the assembly of large complexes of protein and
    nucleic acids. Assembly of ø29 proteins is sequential and orderly to form a complex
    protein shell surrounding a densely packed genome that is injected through the tail into
    the host during infection. Briefly, the major capsid protein, gene product 8 (gp8),
    interacts with gp7, gp8.5 and gp10 to form the prohead into which the genome is
    packaged by a noncoding RNA and gp16 upon ATP hydrolysis. The tail (gp9, gp11,
    gp12*) is assembled onto the DNA-filled head to make infectious particles. Another
    essential ø29 protein, gp13, was found to interact with gp9 in the cytoplasm (Garcia et
    al, Virology 125: 18-30, 1983). However, gp13 was not considered a structural ø29
    component by available methods nor was its mechanism of action described. We
    hypothesize that gp13 is an essential structural component of the phage tail and that it
    modulates the infection process in addition to its previously ascribed assembly role. We
    have employed western blot analysis, antibody decoration transmission electron
    microscopy (TEM) and immuno-gold-TEM to test the hypothesis that gp13 is a
    component of infectious ø29 particles. Crystal structure alignment, zymography and
    thin layer chromatography (TLC) were used to investigate gp13 function. We report
    gp13 staining of purified wild-type phage particles, DNA-emptied particles and purified
    phage tails, but not the prohead pre-packaging/pre-tail intermediate or gp13 deficient
    particles by western blotting using a gp13-specific rabbit antiserum. Additionally, ø29
    particles form tail-bound rosettes with gp13-specific antibodies that are visualized by
    negative-stain transmission electron microscopy. Structural prediction and crystal
    alignments of gp13 show lysozyme and metalloendopeptidase homology, however
    zymography and TLC do not identify the gp13 substrate. These findings are consistent
    with the hypothesis that gp13 is a novel ø29 structural component that localizes to the
    distal ø29 tail and may serve to modulate phage-mediated peptidoglycan degradation.

7   Title: Inhibition of toll-like receptor 2 signaling in primary murine microglia
    Author(s): Rajagopal N. Aravalli, Genya Gekker, Shuxian Hu, and James R.
    Lokensgard

    Abstract: Microglia respond to herpes simplex virus (HSV)-1 by producing
    proinflammatory cytokines and chemokines. Following this inflammatory burst, the
    microglial cells undergo apoptotic cell death. We have recently demonstrated that both
    virus-induced immune mediator production and apoptosis were mediated through Toll-
    like receptor (TLR) 2 signaling. Based upon these findings, we hypothesized that
    inhibition of TLR2 signaling may serve as a means to alleviate excessive
    neuroinflammation. In the present study, we have cloned the ORFs of four vaccinia
    virus proteins which could potentially disrupt TLR signaling, and overexpressed them in
    both primary murine microglial cells and in a cell line stably expressing murine TLR2.
    Using an NF-ºB-driven luciferase reporter gene assay, we show that, upon stimulation
    with HSV and Listeria monocytogenes, all four viral proteins inhibited TLR2 signaling,
    with different specificities in murine microglia and the TLR2 expressing cell line. This
    report is the first to demonstrate endogenous inhibition of TLR signaling in primary
    microglial cells.

8   Title: Localized Populations of CD8- MHC Class I tetramer+ SIV-Specific T Cells
    in Lymphoid Follicles and Vaginal Epithelium
    Author(s): Jung Joo Hong, Teresa L. Mattila, Cara B. White, Aaron Hage, Matthew R.
    Reynolds , David I. Watkins, Christopher J. Miller, Elizabeth Connick, Ashley T. Haase,
    and Pamela J. Skinner

    Abstract: Background: We investigated the in situ localization of SIV specific T cells in
    tissues from SIV infected macaques during the natural course of infection in order for 21
    this information that can to serve as the basis for comparison for future vaccine studies.
    During the course of our studies, we identified localized subpopulations of SIV specific
    T cells that showed apparently down-modulated surface expression of CD8 molecules
    tissues from SIV infected macaques during the natural course of infection in order for
    this information that can to serve as the basis for comparison for future vaccine studies.
    During the course of our studies, we identified localized subpopulations of SIV specific
    T cells that showed apparently down-modulated surface expression of CD8 molecules
    in B cell follicles and in vaginal / cervical epithelium. Methods: Fresh tissues from
    SIVmac239 infected macaques were stained with MamuA01 tetramers loaded with SIV
    gag, tat, and or an irrelevant peptide. Sections were counterstained with CD8, CD20, or
    gamma-delta TCR antibodies. Results: The majority SIV gag and tat tetramer stained T
    cells were localized throughout CD8+ zones of all tissues examined and were CD8+. In
    addition, subpopulations of gag and tat tetramer stained cells that did not stain positive
    with CD8 antibodies were detected in CD8 negative regions of lymph tissues and
    largely CD8 negative regions of the vaginal and cervical epithelium. Lymph tissues
    stained with tetramers and CD20 (B cell marker) showed tetramer+ cells in B cell
    follicles, suggesting that the observed tetramer+CD8- populations of cells in lymph
    tissues were localized to B cell follicles. Triple labeling with tetramers, CD8 antibodies
    and in situ hybridization staining of SIV which stains follicles confirmed these findings
    indicating that indeed tetramer+CD8- cells are localized to B cell follicles in lymphoid
    tissues. Gamma delta TCR-antibody staining in lymph nodes showed no co-labeling
    with tetramer stained cells, indicating that the tetramer+CD8- population of cells were
    not gamma delta T cells. Negative control staining with tetramers loaded with an
    irrelevant peptide did not show staining of tetramer+CD8- cells as seen with virus
    specific tetramers . These results indicate that tetramer+CD8- cells are not NK cells
    non-specifically binding to MHC tetramers. Subpopulations of MHC-tetramer+ CD8-
    cells were detected during both the acute as well as chronic stages of infections and in
    both vaginally as well as rectally inoculated macaques, indicating that time post
    infection and route of inoculation did not markedly effect this population of cells.
    Conclusions: This is the first description of a subpopulation of MHC-tetramer+CD8-
    cells that localize to B cell follicles and the genital epithelium. These studies have led
    us to hypothesize that antigen specific CD8+ T cells down modulate CD8 upon entering
    B cell follicles or upon entering the epithelial layer of genital tissues in macaques,
    perhaps preventing unwanted cellular lysis and modulating immune activation in these
    specific tissue locations. Future studies are warranted to further characterize this
    unique subpopulation of cells.

9   Title: Virion-specific gene expression response of oral keratinocytes to HIV-1
    Author(s): K. F. Ross, A. Khammanivong, A.C. Asrani, R. Giacaman-Sarah, A.
    Vacharaska, K. Gebhard, E. Dietrich, M. C. Herzberg

    Abstract: The oral mucosal epithelium is directly exposed to Human Immunodeficiency
    Virus (HIV) during at-risk sexual activity and through breast-feeding. Recent studies
    have shown that oral epithelial cells are able to harbor and effectively transmit the virus
    to peripheral blood mononuclear cells (PBMCs) in an in vitro co-culture system. Within
    three hours of exposure, HIV-1 integrates into the oral keratinocyte genome as
    confirmed by nested-RT-PCR. We have noted that these cells regulate the expression
    of innate immune molecules in the presence of HIV-1. Little is known, however, about
    the transcriptional response of oral keratinocytes to the virus. We hypothesized that
    oral keratinocytes respond to HIV, exclusive of analytes derived from propagating cells,
    with a virion-specific gene expression signature. Methods: HIV-1IIIb was propagated in
    activated PBMCs, harvested and titered by standard methods. To wash the virus free
    of PBMC analytes, HIV was filtered and concentrated by Centricon Plus-20 (MWCO
    100,000; Millipore). The presence of PBMC analytes associated with HIV was
    confirmed by SDS-PAGE. Concentrated and unconcentrated HIV-1IIIb was adjusted to
    an MOI of 0.01 and incubated 6 and 24 h with the immortalized oral keratinocyte cell
    line TERT-2. No virus controls included media from activated PBMCs before and after
    concentration. From infected and uninfected keratinocytes, mRNA was harvested and
    cDNA synthesized to generate biotinylated-cRNA. The labeled cRNA was then                  22
    hybridized to Affymetrix Human Genome U133 Plus 2.0 Arrays. Differential expression
    was confirmed by t-test comparisons with p-values < 0.05 and fold-change e 2.0 as our
    cut-off criteria, with all expression levels normalized to the housekeeping gene, _-actin.
     cDNA synthesized to generate biotinylated-cRNA. The labeled cRNA was then
     hybridized to Affymetrix Human Genome U133 Plus 2.0 Arrays. Differential expression
     was confirmed by t-test comparisons with p-values < 0.05 and fold-change e 2.0 as our
     cut-off criteria, with all expression levels normalized to the housekeeping gene, _-actin.
     The stringent cut-off criteria suggested that important genes may be missed, but the
     false discovery rate would be very low. Data mining and analyses were performed
     using Genedata Expressionist and Agilent GeneSpring GX. Two algorithms were
     developed and compared to isolate the virion-specific transcriptional response. Results:
     In response to HIV-1IIIb we found 83 unique known genes to be significantly regulated
     in 6 h (28 down-regulated and 55 up-regulated) and 66 unique known genes were
     significantly regulated at 24 h (18 down and 48 up). Five genes (CXCL9, CXCL10,
     CXCL11, GRTP1 AND UBD) were regulated at both 6h and 24h. Among the responses
     of functional groups, genes encoding innate immune molecules (e.g., hBD-2, S100A8
     and S100A9) and cytokines were significantly down-regulated in response to HIV-1IIIb.
     Conclusions: HIV-1IIIb infects oral keratinocytes to significantly regulate about 144
     genes. In general, innate immune function appears compromised suggesting that HIV-
     infected oral keratinocytes will be directly and autonomously more susceptible to
     secondary opportunistic infections. Supported by NIH/NIDCR R01DE015503.

10   Title: Effects of Cytomegalovirus Infection and Interferon Gamma on Neural
     Stem Cells
     Author(s): Zibing Jiang, Maxim C-J Cheeran, Shuxian Hu, Genya Gekker, Thomas
     Bakken and James R Lokensgard

     Abstract: Cytomegalovirus (CMV) is the leading infectious cause of congenital brain
     abnormalities in the United States. Neural stem cells (NSC), found in both fetal and
     adult mammalian brains, are known for their ability to generate new neuronal cells
     during development and post-injury. To better understand the consequences of
     cytomegalovirus infection on brain development and repair, studies were done on
     cultured murine neural stem cells derived from embryonic 14.5 day fetal brain tissue.
     NSC cultures were characterized by immunostaining for nestin expression as well as
     their potential to differentiate into astrocytes, neurons and oligodendrocytes. To
     investigate susceptibility of NSC to murine CMV (MCMV), both in vivo and in vitro
     infection models were used. In addition, the effects of inflammatory mediators on NSC
     proliferation and induction of MHC class I (MHC-I) expression were investigated.
     Following intra-cerebroventricular infection with MCMV it was found that nestin-positive
     cells, in the subventricular zone, were infected with the virus. To further determine
     susceptibility of murine NSCs to MCMV infection, one step growth curves and viral
     gene expression profiles were performed on NSC monolayer cultures. MCMV
     replicated productively in NSC, mounting 3-4 Log increases in titers over time. Viral gB
     expression peaked at 3 days post infection (d p.i.). It is known from previous studies
     that MCMV infection induces inflammatory mediators, such as interferon gamma (IFN-
     g) and tumor necrosis factor-alpha (TNF-a) within the brain. The present study found
     that IFN-_ and TNF-a enhanced MHC I expression on NSCs. On the other hand,
     MCMV infection was shown to inhibit both constitutive and IFN-_-induced MHC-I
     expression on NSCs. To determine if cytokines altered NSC proliferation, IFN-g, TNF-
     a, interleukin (IL)-1b, and IL-10 treated 4-day old cultures were evaluated using MTT
     and LDH assays. IFN-g, but not TNF-a, IL-1b, or IL-10, suppressed NSC proliferation in
     vitro. Subsequent analysis of LDH release from IFN-g treated cultures showed no
     change in enzyme levels when compared to untreated cells, suggesting that IFN-g may
     inhibit cell division. It can be concluded from these studies that NSCs are susceptible to
     MCMV infection and that inflammatory mediators, like IFN-g, may alter stem cell
     function during viral encephalitis.


                                                                                              23
11   Title: Dysregulated IFN-gamma responses during lethal cytomegalovirus brain
     infection of IL-10-deficient mice
     Author(s): M.C-J. Cheeran, S. Hu, J.M. Palmquist, G. Gekker, and J.R. Lokensgard

     Abstract: MCMV brain infection induces chemokine production which precedes the
     infiltration of CD3+ lymphocytes. We hypothesized that an absence of anti-
     inflammatory cytokines would result in sustained proinflammatory neuroimmune
     responses. Direct icv injection into IL-10 KO mice produced an unexpected result: while
     wild-type animals cleared MCMV, the infection was 90% lethal in IL-10 KOs. Identical
     infection of IL-4 KOs did not produce lethal disease. Infected brain tissue from both
     wild-type and IL-10 KO animals was assessed for cytokine and chemokine levels, as
     well as viral gene expression. The data show vastly elevated levels of IFN-gamma,
     CXCL9, and CXCL10, as well as IL-6 in brain homogenates obtained from IL-10 KO
     animals. However, MCMV viral load, gB mRNA levels, and the titers of infectious virus
     were similar in both IL-10 KO and wild-type animals. These data demonstrate that
     MCMV brain infection of IL-10-deficient mice causes lethal disease, which occurs in the
     presence of a dysregulated IFN-mediated neuroimmune response.

12   Title: APOBEC3G separation of function mutants define activities necessary for
     HIV restriction
     Author(s): Mark D. Stenglein, April Schumacher, Guylaine Haché, Phil Gross, and
     Reuben S. Harris

     Abstract: The APOBEC3s are a family of polynucleotide cytosine deaminases. Several
     of these proteins, including APOBEC3G, have been shown to block the replication of
     HIV-1 (_vif). It is thought that this block occurs during reverse transcription, when
     APOBEC3G mutates cytosines in the nascent viral DNA to uracil. Published reports
     have attributed several activities to APOBEC3G. These include: cytosine deaminase
     enzymatic activity; nucleic acid binding; dimerization; and a cytoplasmic sub-cellular
     localization. Using fluorescence microscopy and APOBEC3G-GFP fusion proteins, we
     identified a 65 amino acid region of the protein that determines its cytoplasmic
     localization. We then constructed a panel of missense mutants in this region, and
     characterized the activities of these mutants. Using this approach, we identified several
     classes of mutants. This has allowed us to draw several conclusions concerning the
     interrelationship of APOBEC3G_s activities: " Neither a strict cytoplasmic localization
     nor dimerization is required for APOBEC3G to block HIV replication " Dimerization
     appears to help retain APOBEC3G in the cytoplasm. Furthermore, some of these
     mutants are unable to inhibit HIV replication. This suggests that these mutants are
     deficient in an unspecified activity required for HIV restriction. We are in the process of
     better characterizing these mutants

13   Title: Virus specific CD8 T cells are required for a successful SIV vaccine
     Author(s): A. Hage, T. Mattila, J. Hong, C. White, C. Miller, A. Haase, P. Skinner

     Abstract: As of 2006, over 25 million people have died from HIV. Today, with nearly 40
     million people living with HIV it is more important than ever to discover a vaccine for
     HIV. As the majority of new HIV cases involve women being infected through vaginal
     intercourse it is imperative that a model be used to emulate this mode of transmission.
     Rhesus macaque monkeys that have been vaginally inoculated with SIV mac239, a
     cloned natural infection, provide an excellent model for the natural HIV infection. In
     macaques vaginally infected with SIV mac239, virus spreads from the portal of entry to
     distal lymphoid tissue within a week after infection. However, a virus specific CD8+
     response does not materialize until around 18 to 21 days post infection. Thus CD8 T
     cells arise too late to prevent the virus from spreading and seeding itself within lymph
     tissues. A quicker virus specific CD8+ response induced by a vaccine may allow for
     greater control of SIV viremia. The most successful SIV vaccines to date are             24
     attenuated SIV viruses including the non-pathogenic SHIV virus. The goal of our
     studies is to identify the immune correlates of a successful SIV vaccine. We
     hypothesize that a successful SIV vaccine requires the early and strong induction of
     greater control of SIV viremia. The most successful SIV vaccines to date are
     attenuated SIV viruses including the non-pathogenic SHIV virus. The goal of our
     studies is to identify the immune correlates of a successful SIV vaccine. We
     hypothesize that a successful SIV vaccine requires the early and strong induction of
     SIV specific CD8 T cells. For these studies, rhesus macaques were intravenously
     vaccinated with non-pathogenic SHIV 89.6, and then challenged intravaginally with SIV
     mac239 and sacrificed at 3, 7, 10, 14, or 90 days post challenge. Control animals were
     not vaccinated and intravaginally infected with SIV mac239. To stain virus specific CD8
     T cells in situ, fresh lymph and genital tissues were sectioned and stained with MHC-
     class 1 tetramers and CD8 antibodies. Early results show that in SHIV 89.6 vaccinated
     and SIVmac239 challenged macaques SIV gag and tat specific CD8 T cells become
     detectable as early as 3 days post challenge in LNs and spleen, and by 7 days post
     challenge in the genital tissues. The virus specific CD8 T cell response was most
     robust at 14 days post challenge, and was largely gone by 3 months post challenge.
     These results support our hypothesis and indicate that successful vaccination is
     dependent upon a robust early virus specific CD8+ T cell response.

14   Title: A Screen For Mll-AF9 Cooperating Mutations in Leukemogenesis Using
     MLV-Based Mutagenesis
     Author(s): Rachel J. Bergerson, Lara S. Collier, Raha Allaei, Kevin Silverstein, Anne-
     Francoise Lamblin, Linda Wolff, John H. Kersey, David J. Adams, and David A.
     Largaespada

     Abstract: Mice with a Mll-AF9 knock-in develop mostly develop acute myeloid leukemia
     (AML), the phenotype observed in human patients with this translocation. The
     MOL4070LTR (M4070) retrovirus is a chimera between Moloney and 4070A Murine
     Leukemia Viruses and has been shown to be capable of inducing lymphoid or myeloid
     disease in mice. We hypothesized that infection with this retrovirus could cooperate
     with MLL-AF9 to accelerate the onset of AML by causing cooperating somatic
     mutations via insertional mutagenesis. We bred Mll-AF9 heterozygous C57BL/6J males
     to wild type 129/SvJ females, and injected the 280 offspring at three days of age with
     either M4070 virus (212) or a control supernatant (68). Infected Mll-AF9 mice succumb
     to disease with a significantly reduced latency period compared to controls (p less than
     .0001), indicating that M4070 infection causes significant leukemia acceleration in
     these mice. The gross pathology and surface immunophenotype analysis indicates the
     majority of infected Mll-AF9 animals present myeloid leukemia while most infected wild
     type animals present lymphoid leukemia. Retroviral insertion sites were cloned from our
     accelerated leukemia tissues using a linker mediated cloning protocol to identify the
     genes most frequently mutated in these animals. From more than 20,000 sequence
     reads, 4,731 non-redundant proviral insertion events and 141 common insertion sites
     (CIS) were identified. Several CIS are enriched in infected Mll-AF9 AMLs. We plan to
     verify nearby genes for cooperation with MLL-AF9 in AML development using mouse
     modeling and by examining the expression of these genes in human AML with MLL
     gene translocations.

15   Title: In situ analysis of HIV- and SIV-specific CD8+ T cells
     Author(s): Teresa Mattila, Qingsheng Li, Aaron Hage, Joy Folkvord, Elizabeth
     Connick, Christopher Miller, Ashley Haase, Pamela Skinner

     Abstract: Because the human immunodeficiency virus (HIV) has caused over 25
     million deaths worldwide and its incidence continues to grow, it is imperative that a
     vaccine be developed that will reduce its spread. For unknown reasons, CD8+ T cell
     response to HIV infection is insufficient, and virus is never cleared. Therefore, it is
     important to identify the deficits in this vital component of the adaptive immune system
     which prevent it from controlling HIV infection. The objective of our study is to gain
     understanding of why virus-specific CD8+ T cells cannot clear virus, and to identify     25
     correlates of a successful vaccine. Our central hypothesis is that a successful HIV
     vaccine requires a strong, early induction of virus-specific CD8+ T cells that occurs
     prior to dissemination and seeding of virus in lymphoid B cell follicles (BCF). HIV
     understanding of why virus-specific CD8+ T cells cannot clear virus, and to identify
     correlates of a successful vaccine. Our central hypothesis is that a successful HIV
     vaccine requires a strong, early induction of virus-specific CD8+ T cells that occurs
     prior to dissemination and seeding of virus in lymphoid B cell follicles (BCF). HIV
     replicates primarily inside BCF after the acute stages of infection, resulting in a
     reservoir of virus that seeds infection throughout the remainder of the patient_s life.
     One reason for this may be that BCF are relatively immune privileged sites into which
     virus-specific CD8+ T cells seldom enter. We visualized the spatial relationships
     between virus-specific CD8+ T cells and B cells in lymphoid tissues from HIV-infected
     human subjects using immunohistochemistry (IHC) and in situ tetramer (IST) staining.
     This analysis revealed that, in situ, HIV-specific CD8+ T cells fail to accumulate inside
     BCF during HIV infection, and instead were found outside of BCF. We are currently
     examining in situ the extent to which virus- producing cells are found in BCF in lymph
     nodes of vaccinated and simian immunodeficiency virus (SIV)- challenged macaques,
     vs. non-vaccinated and SIV-challenged macaques, using IHC, IST, and combined
     IST/in situ hybridization

16   Title: Influenza A matrix protein is necessary but not sufficient for virus-like
     particles production.
     Author(s): Harmon, A.H., D. Wang, Y. Weng, J. Hemeka, J. Christopher-Hennings, E.
     Nelson, and F. Li.

     Abstract: The final stage of the influenza life cycle requires the assembled virus to bud
     from the host cell_s plasma membrane (PM), which is likely mediated through
     interactions of several domains within the Matrix protein (M1) and host proteins. Based
     upon this, it has been speculated that the M1 protein alone in the absence of other viral
     components of influenza can assembly and bud which subsequently lead to production
     of virus like particles (VLPs). Support for this hypothesis has come from two previous
     studies where vaccina virus vector- and baculovirus-based M1 expression systems
     were utilized. We have sought to determine if the expression of M1 alone in the
     absence of viral vectors can produce VLPs in MDCK cells, susceptible to replication of
     influenza virus. We have carried out studies with a western blot-based viral budding
     assay and confocal microscopy. When M1 is expressed alone, we were unable to
     detect any VLP production by the viral budding assay, though cellular accumulation of
     M1 protein was detected. Attempts to enhance yield through addition of post regulatory
     element (PRE) derived from HBV also failed to demonstrate VLPs. Consistent with
     these observations, confocal microscopy studies revealed that the majority of M1
     protein appeared to be predominantly localized in or near the nucleus, not in the PM.
     Slight accumulation of M1 protein at the PM has been observed from M1-eGFP
     constructs, which was enhanced by the incorporation of PRE. The M1 protein contains
     a well-documented nuclear localization signal which traffics the protein into the nucleus,
     so our results were not unexpected. Once in the nucleus, M1 must bind viral RNA
     nucleoproteins (vRNPs) and nuclear export protein (NEP) to escape and begin
     trafficking towards the PM to initiate assembly and budding process. Work is currently
     underway to search for viral components needed to cooperate with M1 protein to form
     VLPs in MDCK cells, with a goal of identifying the minimal budding machinery for
     influenza A virus.

17   Title: A3G expression levels & HIV mutational load: evidence for modulating
     mutagenesis
     Author(s): Holly A. Sadler, Mark D. Stenglein, Reuben S. Harris, and Louis M.
     Mansky

     Abstract: The human protein APOBEC3G (A3G), a DNA cytosine deaminase, has
     recently been discovered to be part of the cellular innate immune response to diverse
     retroviral infections. A3G converts viral cDNA cytosines to uracils, which leads to either26
     incomplete reverse transcription or a loss of infectivity via hypermutation. HIV encodes
     the accessory protein Vif, which can block incorporation of A3G into HIV particles and
     protect the viral genome from the antiviral affect of A3G. We have been investigating
     retroviral infections. A3G converts viral cDNA cytosines to uracils, which leads to either
     incomplete reverse transcription or a loss of infectivity via hypermutation. HIV encodes
     the accessory protein Vif, which can block incorporation of A3G into HIV particles and
     protect the viral genome from the antiviral affect of A3G. We have been investigating
     the relative levels of A3G in virus-producing cells needed to result in a complete loss of
     HIV infectivity and increased mutational load. A panel of stable cell lines expressing a
     250-fold range of A3G was used to analyze their effects on viral infectivity and proviral
     mutational load. This panel includes cells stably expressing A3G at levels that are
     physiologically relevant to that seen in primary cells. In our panel, we observed a 300-
     fold reduction in infectivity with vif (-) virus, and a 5-fold reduction in infectivity with vif
     (+) virus. DNA sequencing analysis of proviral DNAs has revealed the following: 1)
     Increased A3G expression correlates with increased G-to-A hypermutation (and
     decreased infectivity); 2) A3G mutation hotspots were distinct from G-to-A mutations
     created by HIV reverse transcriptase, as determined by experiments done in parental
     control cells in the absence of A3G expression; 3) Single G-to-A mutations at A3G
     hotspots were identified in target sequences of proviruses produced from low level
     A3G-expressing cell lines. Our findings indicate that 1) we can, to some degree,
     differentiate between G-to-A mutations that arise from the action of A3G versus those
     created by reverse transcriptase, and 2) Exposure to low levels of A3G reduces
     mutational load. The implications of our findings for HIV variation will be discussed.


18   Title: Immune cell infiltration into the brain during herpes encephalitis
     Author(s): Stina L. Urban, Cristina P. Marques, Rajesh Ravindran, Thomas Bakken,
     Maxim C-J Cheeran, and James R. Lokensgard

     Abstract: Herpes simplex virus (HSV)-1 infection of the central nervous system (CNS)
     initiates vigorous yet non-protective neuroimmune responses. Previous in vitro studies
     have demonstrated that murine microglial cells initiate a cascade of proinflammatory
     immune responses to HSV. These host responses are characterized by robust cytokine
     and chemokine production, as well as immune cell infiltration which peaks between 7-9
     d post infection (p.i.). Production of proinflammatory cytokines and chemokines
     precedes immune cell infiltration into the brain and is correlated with survival. The type
     of immune cell infiltration and their persistence in the brain has yet to be determined. In
     the present study, we investigated the kinetics and type of immune cells infiltrating the
     brain during herpes encephalitis. Flow cytometry was performed on immune cells
     isolated from the brains of infected mice at 7 and 14 d p.i. Real-time bioluminescence
     imaging, using splenocytes obtained from _-actin-luciferase transgenic mice, was used
     to analyze trafficking patterns and persistence of both primed and unprimed
     splenocytes following adoptive transfer into MHC-matched recipients. Despite the
     transfer of HSV-primed splenocytes, these brain-infiltrating immune cells were not
     protective, leading to the high mortality rates observed. Viral titers in the cerebellum,
     cortex, and subcortex as well as the heart, liver, lungs, and spleen, were also
     measured. Results from these experiments will help elucidate beneficial as well as
     immunopathologic roles of immune cell infiltration into the brain in the pathogenesis of
     herpes encephalitis.

19   Title: HSV-Induced Epithelial Damage and Increased Susceptibility to HIV
     Infection in Human Cervicovaginal Organ Cultures
     Author(s): J Horbul, B Miller, D Maher, X Wu, T Schacker, S Rice, P Southern

     Abstract: In the continuing global pandemic, HIV infection is spread primarily by sexual
     transmission. More than half of all new HIV infections occur in young women.
     Epidemiological studies have established a correlation between pre-existing sexually
     transmitted infections and increased susceptibility to primary HIV infection. Many
     details relating to HIV transmission remain unresolved but increased HIV susceptibility 27
     is probably linked to physical disruption of mucosal surfaces and local inflammation.
     We have developed ex vivo human organ cultures to simulate biologically relevant
     routes of HIV transmission and are now examining sequential infections with HSV,
     details relating to HIV transmission remain unresolved but increased HIV susceptibility
     is probably linked to physical disruption of mucosal surfaces and local inflammation.
     We have developed ex vivo human organ cultures to simulate biologically relevant
     routes of HIV transmission and are now examining sequential infections with HSV,
     followed by HIV. HSV replicates extensively in cervicovaginal epithelial cells and HSV-
     induced damage is readily apparent in columnar epithelial cells at the endocervical
     surface. These lesions represent sites of vulnerability in mucosal surfaces that may
     facilitate primary HIV infection. Insight into fundamental mechanisms underlying
     sexually transmitted infections, including HIV, is essential for the development of new
     protective strategies.

20   Title: Inflammation and pre-existing virus infection as cofactors in primary HIV
     infection
     Author(s): BRL Miller, JE Horbul, IH Huntley, DM Maher and PJ Southern

     Abstract: Most new HIV infections occur by heterosexual contacts. In the case of a
     male donor, HIV in semen is deposited onto mucosal surfaces in the female
     reproductive tract. CD4+ T cells appear to be the principal target for initial HIV infection
     but mechanisms for virus uptake across mucosal surfaces remain controversial. In
     order to develop a more complete understanding of HIV transmission, we are
     examining normal human pre-menopausal cervicovaginal tissues for anatomical
     characteristics and target cell populations that might indicate a particular susceptibility
     to HIV infection. We have observed extensive variability in the macro- and microscopic
     organization of epithelial surfaces and in the numbers and distribution of CD4+ T cells,
     both across the surface of any tissue piece and between different tissue donors.
     Unexpectedly, 1-10% of lymphocytes within inflammatory foci were found to be actively
     infected with other viruses, including human herpes virus 6 (HHV6). HHV6 is not
     normally considered an STI as the virus is shed in saliva and infection is widespread in
     elementary school children. These findings suggest that the circulation and
     accumulation of infected lymphocytes may contribute to localized changes in mucosal
     barriers and thereby facilitate HIV transmission.

21   Title: Congenital Cytomegalovirus Detection in Newborn Blood Spots who failed
     Newborn Hearing Screening by Real time PCR
     Author(s): K. Yeon Choi1, Cindy Khan1, Lisa A. Schimmenti1, Mark McCann2, Mark
     R. Schleiss1 1 University of Minnesota, Division of Pediatric Infectious Diseases,
     Center for Infectious Diseases and Microbiology Translational Research

     Abstract: Background/Objective: CMV is a major cause of congenital infection in
     newborns leading to disabilities such as sensorineural hearing loss (SNHL) and
     developmental delay. Approximately 15% of congenitally infected newborns will
     develop SNHL; however, the majority are asymptomatic at birth and will not present to
     clinicians until later in childhood. Therefore, early diagnosis of CMV infection in
     newborns is important for the identification of asymptomatic infected newborns.
     Functional newborn hearing screening (NHS) does not provide information about
     potential etiologies of SNHL, and delays in definitive diagnosis impair the ability to
     intervene with specific therapies, including antiviral agents. These studies sought to
     examine newborn blood spots (NBS) from an anonymized group of infants who had
     failed NHS for the presence of CMV DNA by real-time PCR. Methods: Following
     Institutional Review Board approval, DBS were obtained from infants who had failed
     NHS from the Minnesota Department of Health. Nucleic acid was extracted from blood
     spots using the X-tractor Gene" system. For CMV PCR, Roche CMV UL54
     hybridization probes were utilized and PCR was performed on the Lightcycler®
     instrument. Results: The real-time PCR assay was highly sensitive, with a lower limit of
     detection of 2.5 genomes/reaction. Of 335 children referred for further evaluation
     because of a failed NHS, 9 had CMV DNAemia (2.7%). This compared to only 1/335 28
     positive results from a control group of infants who passed the NHS (0.3%; p=0.02,
     Fisher_s exact test). The mean viral load among positive samples was 1.4 x 103
     genomes/microgram of total DNA (SD, 792). Conclusion: Real-time PCR is a sensitive
     because of a failed NHS, 9 had CMV DNAemia (2.7%). This compared to only 1/335
     positive results from a control group of infants who passed the NHS (0.3%; p=0.02,
     Fisher_s exact test). The mean viral load among positive samples was 1.4 x 103
     genomes/microgram of total DNA (SD, 792). Conclusion: Real-time PCR is a sensitive
     technique for identification of CMV DNA from NBS. Examination of newborns who
     failed NHS suggests that approximately 3% of these infants have congenital CMV
     infection. NBS screening may be a useful and rapid adjunct to functional NHS and
     enable more rapid etiologic diagnosis of SNHL in newborns.

22   Title: Survival of HIV-1 in Whole Saliva
     Author(s): Elizabeth A. Dietrich, Kristin H. Gebhard, Claudine E. Fasching, Karen F.
     Ross, and Mark C. Herzberg

     Abstract: HIV-1 infection through the oral cavity is an important low-frequency event,
     occurring during breast-feeding by an HIV-positive mother and during oral intercourse.
     Since saliva of an HIV-infected individual is not infectious, and saliva contains
     numerous antiviral factors, it has generally been assumed that infection occurs in the
     gut rather than at the oral mucosa. Yet, the kinetics of HIV-1 infection of oral
     keratinocytes relative to inactivation in crude, whole saliva has not been studied in
     detail. We hypothesize that HIV-1 enters oral keratinocytes before being inactivated by
     saliva. To test this hypothesis, confocal microscopy was used to examine HIV uptake
     into TERT-2 cells, an immortalized oral epithelial cell line. Fluorescently labeled HIV-1
     envelope pseudovirus enters oral keratinocytes within 30 minutes. Next the rate of HIV-
     1 inactivation in crude, whole saliva was determined. Crude whole saliva was collected
     by expectoration from three healthy adults. To model the presentation of virus in a
     bolus of semen or breast milk, saliva samples were pooled, immediately mixed with X4-
     or R5-type HIV-1 at volume ratios of saliva to virus ranging from 10:1 to 2:1, dispersed
     by repeated gentle inversion, and incubated for up to 60 min. At various times, saliva-
     HIV mixtures were inoculated into cultures of TZM-bl reporter cells, which are
     permissive to HIV-1 and express beta-galactosidase under the control of the HIV
     promoter. The infectivity of the virus decreased over time. This decrease was more
     marked at a 10:1 or 4:1: ratio of saliva to virus than a 2:1 ratio. At a 10:1 ratio, the half-
     life of X4 and R5 HIV-1 was about 4 min. Up to 20% of the original inocula was still
     infectious in all conditions after 30 minutes of incubation with saliva. These data
     suggest, therefore, that HIV-1 may be capable of entering oral keratinocytes before it is
     completely inactivated by saliva. If the virus is protected from inactivation within a
     keratinocyte, this pathway may be a novel mechanism that enables HIV-1 to cross the
     mucosal barrier and infect a host. Supported by NIH/NIDCR R01DE015503.

23   Title: MECHANISTIC STUDIES OF HIV-1 NC_S CHAPERONE ACTIVITY: MG2+-
     DEPENDENCE AND COMPARISON TO CHAPERONE ACTIVITY OF GAG
     PRECURSOR PROTEIN VARIANTS

     Author(s): My-Nuong Vo1, Ioulia Rouzina2, Meng Sun3, Siddhartha A. Datta4,
     George Barany1, Alan Rein4, Karin Musier-Forsyth1,3. 1Department of Chemistry and
     2Department of Biochemistry, Molecular Biology and Biophysics, University of
     Minnesota, Minneapolis, MN 55455; 3Department of Chemistry, The Ohio State
     University, Columbus, OH 43210; 4HIV Drug Resistance Program, National Cancer
     Institute at Frederick, Frederick, MD 21702

     Abstract: HIV-1 nucleocapsid protein (NC) is a chaperone protein that facilitates
     nucleic acid remodeling during the reverse transcription process through a combination
     of aggregation and duplex destabilization activities. Optimal nucleic acid chaperone
     activity in vitro requires saturation with NC at a nucleotide to protein ratio of
     approximately 6:1. The cytoplasm contains high and variable concentrations of cations
     such as Mg2+, which presumably compete with NC for electrostatic binding to nucleic 29
     acids. In this work, we systematically explore the effects of varying NC and Mg2+
     concentration on NC_s annealing activity using complementary RNA and DNA hairpins
     derived from the TAR sequence of the HIV-1 genome (NL4-3 isolate). This reaction
such as Mg2+, which presumably compete with NC for electrostatic binding to nucleic
acids. In this work, we systematically explore the effects of varying NC and Mg2+
concentration on NC_s annealing activity using complementary RNA and DNA hairpins
derived from the TAR sequence of the HIV-1 genome (NL4-3 isolate). This reaction
mimics the annealing step of minus-strand transfer. We find that subsaturating levels of
NC, either due to the presence of low NC concentrations or high Mg2+, which
effectively displaces NC from nucleic acids, lead to reduced nucleic acid aggregation
and duplex destabilization activity. In addition, the annealing pathway changes with
increasing NC concentration. Whereas initiation via hairpin loop-loop interactions is
favored at low NC, initiation through the hairpin stem ends is the preferred pathway
under conditions of high NC binding. Several previous studies showed that HIV-1 Gag
protein is also an effective nucleic acid chaperone. Here, we quantitatively compare the
TAR hairpin annealing activity of ”p6-Gag to that of NC, and observe that less Gag is
required to achieve the same chaperone activity. We hypothesize that better chaperone
function of Gag is a result of stronger nucleic acid binding due to either cooperative
Gag-Gag interactions, and/or additional nucleic acid interactions via the Matrix (MA)
domain. Results of TAR annealing studies in the presence of WM-”p6-Gag, a variant
containing mutations that disrupt Gag dimerization (Datta et al., 2007, J. Mol. Biol.
365:799), are consistent with the conclusion that Gag-Gag interactions enhance its
chaperone function. We also show that the MA protein alone demonstrates chaperone
function, albeit much weaker than NC. Studies are underway to examine the chaperone
function of additional Gag truncation constructs. This research was supported [in part]
by the Intramural Research Program of the NIH, National Cancer Institute, Center for
Cancer Research.




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