SUPPLEMENTARY MATERIAL by xL11v3Q5

VIEWS: 2 PAGES: 6

									SUPPLEMENTARY MATERIAL



MATERIAL AND METHODS



Construction of tagged forms of LCMVGP, LFVGP, Junin GP, DG, and LARGE



The C-terminally flag-tagged LCMV and LFVGP variants LCMVGP-flag and LFVGP-

flag consist of full-length LCMVGP (ARM53b and clone-13) and LFVGP (Josiah) in

which the normal Stop codon has been replaced by a spacer-sequence (GGGS) followed

by the flag-tag (DYKDDDDK). For the construction of the C-terminally flag-tagged

LCMV and LFVGP variants, LCMVGP and LFVGP cDNA fragment were amplified by

PCR using the primer pairs LCMf/LCMVflag and LFVf/LFVflag. The resulting

fragments were cut with KpnI and XhoI and inserted into the eukaryotic expression

vector pc-LCMVGP containing full-length GPs derived from LCMV ARM53b and

clone-13 and pC-LFVGP containing the full-length cDNA of LFVGP Josiah. Sequences

were verified by double-strand DNA sequencing. The following primers were used:

LCMf: 5’-AAC CAC TGC ACA TAT GCA GGT-3’

LCMflag: 5’-TAA CTC GAG TCA TTT ATC GTC ATC GTC TTT GTA GTC AGA

TCC GCC ACC GCG TCT TTT CCA GAC GGT-3’

LFVf: 5’-ACA TCA TGG GAA TTC CAT ACT-3’

LFVflag: 5’-TAA CTC GAG TCA TTT ATC GTC ATC GTC TTT GTA GTC AGA

TCC GCC ACC TCT CTT CCA TTT CAC AGG-3’




                                           1
       For the construction of C-terminally tagged Junin GP, a 3’ cDNA fragment of

Junin GP cDNA (XJ13) was amplified using the primers JUNS3 5’-CCC TAA ATG

ATG AAA CTA AGA-3’ and JFLA 5’-ACA GTT TGG CGT AGA GGA CAC GGT

GGC GGA TCT GAC TAC AAA GAC GAT GAC GAT AAA TGA CTC GAG TTA-3’.

After digestion with EcoRI and XhoI, the fragment was inserted into the eukaryotic

expression vector pC-JUNGP containing full-length Junin virus GP.

       For the construction of C-terminally myc-tagged LARGE, a 3’ cDNA fragment of

human LARGE1 was amplified by PCR using the forward primer LARf 5’-GCT GGA

GGC CAT CTG CAA GCA-3’ and the backward primer LAMY 5’-ATA TAT AGC

GGC CGC CTA CAG ATC CTC TTC TGA GAT GAG TTT TTG TTC GCT GTT GTT

CTC GGC TGT GAG ATA-3’. The resulting PCR fragment was digested with ApaI and

NotI and inserted into the expression construct pIRES3-LARGE-puro containing the full-

length cDNA of human LARGE1, resulting in the construct pIRES3-LARGE-myc-puro.

       The recombinant DG variant DGHA consists of full-length rabbit DG cDNA in

which the normal Stop codon has been replaced by a spacer-sequence (GGGS) followed

by the influenza HA-tag. For the construction of DGHA, a rabbit DG cDNA fragment

was amplified by PCR using the primers SK1df 5’-CGC GGG GGA GAA CCC AAC

CAG CGC-3’and DGHA 5’-CCG CTC GCG TTA AGC ATA ATC TGG AAC ATC

ATA TGG ATA GCT ACC TCC CCC AGG GGG AAC ATA AGG AGG GGG-3’. The

resulting fragment was cut with SacII and XhoI and inserted into the eukaryotic

expression vector DG pcDNA3 containing full-length DG (Kunz et al., 2001).

       The DG variant HADG contains an N-terminal HA tag, separated from the

putative N-terminus (H30) by the intervening sequence GAQPARSPG derived from the




                                            2
vector pDisplay. A 5’ fragment of the rabbit DG cDNA was amplified using the primers

SK1uf 5’-GCC GTG GCT CCC GGG CAT TGG CCC AGC GAA CCC TCG-3’ AND

SK1ub 5’-GTT ATT CAC CAC CGG CAC CAA CTT-3’. The resulting PCR fragment

was cut with XmaI and AccI and inserted into the eukaryotic expression vector DG

pcDNA3 containing full-length DG (Kunz et al., 2001).

       The LCMVGP variant HALCMVGP contains an N-terminal HA tag, separated

from the mature N-terminus (M59) of LCMVGP by the intervening sequence

GAQPARSPG derived from the vector pDisplay (Invitrogen). The stable signal peptide

(SSP) of LCMVGP has been deleted from the construct and replaced by the signal

peptide of Igk light chain also derived from pDisplay. A 5’ fragment of the cDNA of

LCMVGP clone-13 was amplified by PCR using the forward primer GPHAf 5’-AAT

TCC CGG GAT GTA CGG TCT TAA GGG ACC C-3’ and the backward primer

GPHAb 5’-TTA GTC GCA GCA TGT CAC-3’. The resulting PCR fragment was cut

with XmaI and NdeI and into the eukaryotic expression vector pcAGGS containing full-

length LCMVGP cl-13 (Kunz et al., 2003a). All constructs were verified by sequencing

of both DNA strands.



Incorporation of arenavirus GPs into retroviral pseudotypes



Recombinant Moloney murine leukemia virus (MLV) pseudotypes were produced as

described (Rojek et al., 2006). The package cell line GP2-293 (BD Biosciences) was

transfected with the packable MLV genome pLZRs-Luc-gfp, which containins a

luciferase reporter gene and a green fluorescent protein (GFP) reporter (Yang et al.,




                                             3
1998), kindly provided by Dr. Gary Nabel. The C-terminally flagged and wild-type GPs

of LCMV (ARM53b and clone-13) and LFV, as well as vesiculat stomatitis virus (VSV)

were provided in trans by co-transfection with expression plasmids containing their full-

length cDNAs. Briefly, 4 x 10 GP2-293 cells were plated in poly-L-lysine coated M6

tissue culture plates. After 16 hours, cells were co-transfected with 2 µg each of pLZRs-

Luc-gfp and the GP expression plasmid using calcium phosphate. Forty hours after

transfection, cell supernatants were harvested and cleared by centrifugation for 15 min at

3, 000 rpm.

       For pseudotype infection, VeroE6 cells were plated in 96-well plates in a density

of 104 cells/well. After 24 hours, conditioned supernatants containing the retroviral

pseudotypes were added and incubated for 1 hour at 37oC. The viral particles were

removed, cells washed twice with DMEM, and fresh medium added. For luciferase assay,

the Steady-Glo® luciferase assay from Promega was used according to the

manufacturer’s recommendations.



FIGURE LEGENDS



Figure S1. DGHA shows functional glycosylation and virus binding. DGHA and wild-

type DG were transiently transfected into human lung epithelial WI-26 VA4 cells (ATCC

CCL-95.1) using Superfect®. After 48 hours, cells were lyzed and the -DG complex

immunoprecipitated (IP) with either polyclonal antibody AP83 (anti--DG) or Y11 (anti-

HA). Immunocomplexes were separated by SDS-PAGE, transferred to nitrocellulose and

probed in Western-blot with mAb IIH6 anti--DG. Virus binding was assessed by virus



                                             4
overlay protein binding assay (VOPBA). Blots were probed with 107 PFU/ml LFV

Josiah, which was inactivated as described (Kunz et al., 2005a). Bound inactivated virus

was detected with mAb 83.6 anti-GP using HRP-conjugated secondary antibody and

ECL. Note that the C-terminal HA tag present in DGHA does not affect functional

glycosylation or virus binding to -DG.



Figure S2. The DG variant HADG is proteolytically cleaved. (A) DG (-/-) ES cells were

transfected with wild-type DG, HADG, or GFP using Amaxa Nucleofection®. After 48

hours, cells were subjected to cell surface biotinlyation as described (Kunz et al., 2003b).

Cell lysates were prepared and subjected to either pull-down with avidine-agarose

(Avidine) or IP with polyclonal antibody Y11 to HA (IP HA). Precipitated proteins were

separated by SDS-PAGE, transferred to nitrocellulose and probed in Western-blot with

mAb IIH6 anti--DG or mouse mAb F7 to HA (Anti-HA). To assure specificity of the

cell surface biotinylation, avidine-bound proteins and 10% of the corresponding total cell

lysate were probed with an antibody to the intracellular control protein -tubulin. Note

that the N-terminal HA tag on HADG is only detected in total lysates after IP with anti-

HA antibody, but not in the biotinylated cell surface protein fraction. This indicates that

the N-terminal HA tag on HADG is cleaved during biosynthesis, as expected based on

previous studies that showed proteolytic processing of the N-terminal domain of -DG

by a furin-related protease (Kanagawa et al., 2004).



Figure S3. Functional characterization of C-terminally flag-tagged viral GPs. (A) Normal

cell surface of LCMVGP, LFVGP, and Junin GP in presence of the C-terminal flag tag.



                                              5
HEK293T cells were transfected with wild-type or C-terminally flag tagged GPs of

LCMV cl-13, LFV, and Junin GP. Surface expression of LCMV and LFV GP was

determined using mAb 83.6. The GP of Junin virus was detected with mAb BE08 anti-

Junin GP1 (Sanchez et al., 1989). FITC labeled secondary antibody were used.

Fluorescence intensity is shown in the x axis and relative cell counts in the y axis. (B) C-

terminally flag tagged GPs of LCMV cl-13, LFV, and Junin is biologically functional:

Recombinant retroviruses pseudotyped with wild-type or C-terminally flag tagged GPs of

LCMV cl-13, LFV, and Junin GP, as well as VSVGP were generated as described in

Supplementary M&M. No GP corresponds to cells transfected with empty vector.

Conditioned culture supernatants containing pseudotypes were used to infect A549 cells

and infection assessed after 48 hours using Steady Glo® luciferase assay. Luminescence

is given as fold-increase over background of uninfected cells (n = 3 + SD).




                                             6

								
To top