lecture16 by HC111126052216

VIEWS: 21 PAGES: 28

									                    Plant Pathology 230
      Lecture 19          17 February 2005               Thursday
               Double-stranded DNA viruses

                              EXAMPLES
                      Banana streak virus (BSV)
                 Cassava vein mosaic virus (CsVMV)
               Cassava common mosaic virus (CsCMV)
                   Cauliflower mosaic virus (CaMV)
               Commelina yellow mottle virus (CYMoV)
                  Petunia vein clearing virus (PVCV)
                 Rice tungro bacilliform virus (RTBV)
               Soybean chlorotic mottle virus (SbCMV)
                 Tobacco vein-clearing virus (TVCV)

Reading: Hull, R. (2002) Mathews’ Plant Virology, pp. 174-180, 339-345
Recent review: M. Haas et al. Molec. Plant Pathology (2002) 3(6):419-429
     DNA and RNA Reverse-transcribing Viruses

DNA reverse-transcribing               RNA reverse-transcribing
viruses (pararetroviruses)             viruses (retroviruses) (have
(generally, no integrase)              integrase; no plant example)


Virion dsDNA (or partially dsDNA)      Virion ssRNA
Transcription to ssRNA                 Reverse transcription to ssDNA
Reverse transcription to ssDNA         Transcription to dsDNA
Transcription to dsDNA                 Transcription to ssRNA

   Examples of RNA Reverse-transcribing Viruses
     Retroviridae
                  Alpharetrovirus     Avian leucosis virus
                     Betaretrovirus     Mouse mammary tumour virus
                     Gammaretrovirus    Murine leukeamia virus
                     Deltaretrovirus    Bovine leukaemia virus
               DNA Reverse-transcribing Viruses
  Family               Genus                     Type Species
  Hepadnaviridae       Orthohepadnavirus         Hepatitis B virus
                       Avihepadnavirus           Duck hepatitis B virus


                                                 Commelina yellow mottle
  Caulimoviridae       Badnavirus
                                                 virus
                       Caulimovirus              Cauliflower mosaic virus
                                                 Rice tungro bacilliform
                       Tungrovirus
                                                 virus
                                                 Soybean chlorotic mottle
                       Soymovirus
                                                 virus
                       Cavemovirus               Cassava vein mosaic virus
                       Petuvirus                 Petunia vein clearing virus
  Unassigned member of Caulimoviridae: Tobacco vein-clearing virus

Tungrovirus, Soymovirus, Cavemovirus and Petuvirus are newly accepted genera
http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/fr-fst-g.htm
                    Cauliflower mosaic virus T=7 capsid




http://icb.usp.br/~mlracz/animations/bock/camv.jpg
http://www.foodsafetynetwork.ca/gmo/camv35s/Image1.gif
                                 Symptoms induced by CaMV




http://www.microbiologyonline.org.uk/graphics/cmv.gif
http://www.bspp.org.uk/publications/pathprofiles/pathprofile19-1th.jpg
http://www.colostate.edu/programs/lifesciences/TransgenicCrops/images/CaMVcanola.jpg
          CaMV genetic and transcription map

   19S RNA                                              -DNA strand

                                              35S RNA

                  35S
                                                         +DNA strand
                                         I
                             VII

                                    II
                    VI
                                        III              ORFs

            19S                    IV
                         V




= single-strand
                                                        Transcripts (RNA)
break
                ORF Map of CaMV 35S RNA
               VII
                     I         III
 cap                                         V                    An
                          II         IV                   VI
       Short
       ORFs

ORF VII: The P7 product has been detected after in vitro translation
and in CaMV-infected protoplasts but not in infected plant tissue.
Results from mutational analysis suggests that P7 is not essential for
infection of protoplast or plant hosts. However, P7 may counter
certain plant defenses.
ORF I: The P1 (=p41) product is found associated with modified
plasmodesmata in infected leaves, has slight sequence similarity to
TMV 30K movement protein, and has nucleic acid binding properties.
Expression of P1 in plant protoplasts or insect results in the
formation of P1-bounded thread-like protuberances on the cells.
   Tubules protruding from the surface of Arabidopsis
protoplasts expressing a CaMV gene 1 protein – GFP fusion




Huang, Z., Han, Y. & Howell, S. H. (2000). Virology 271, 58-64.
                ORF Map of CaMV 35S RNA

               VII
                     I        III
 cap                                        V                   An
                         II         IV                  VI
       Short
       ORFs



ORF II: Analysis of recombinants between aphid transmissible and
non-aphid-transmissible CaMV strains mapped this trait to the P2
(=p18) product. Addition of P2 from a CaMV aphid transmissible strain
to the aphid feed allowed non-aphid-transmissible CaMV to be
transmitted.

ORF III: The P3 (=p15) product is found in CaMV virions. P3 binds
dsDNA without any demonstrated nucleotide sequence specificity.
Recently, P3 was found to be essential for aphid transmission.
Participation of P2 and P3 in CaMV aphid transmission

                                      N     P3     C
                 N        P2      C
                                                  CaMV
                                                  virion


           Postulated
         Aphid Receptor



    Aphid + P2                            Efficient transmission
                           20nM


           CaMV
          CM4-184              P2 does not bind free P3 or virions
           virions 20nM        but binds tightly to pre-formed P3-
              +                virion complexes
              P3
                ORF Map of CaMV 35S RNA

               VII
                     I         III
 cap                                         V                   An
                          II         IV                  VI
       Short
       ORFs



ORF IV: ORF IV gives rise to the coat protein in a process that
involves proteolytic processing of the translation product P4.
Phosphorylation of some coat protein molecules is required for CaMV
infectivity

ORF V: The P5 product develops reverse transcriptase activity when
expressed in yeast and is found in viroplasms of CaMV-infected plant
cells. The amino end of P5 has protease activity. P5 has ribonuclease
H activity.
                ORF Map of CaMV 35S RNA

               VII
                     I          III
 cap                                           V                    An
                           II         IV                    VI
       Short
       ORFs



ORF VI: P6 is a multifunctional protein. P6 is the matrix protein of the
CaMV inclusion body (viroplasm). This structure also has all of the
other CaMV proteins (except any product of ORF VII). P6 also is the
transactivator of translation.
Synthesis of CaMV DNA from 35S pre-genomic RNA template
                       tRNA primer for RTase

                                               3’

Primer extension
 Initiating (-)DNA
         synthesis

RNAase H action 3’


      Transfer to
      RNA 3’ end

    Extension to
 complete (-)DNA




                                                      (+)DNA
                                                    synthesis
CaMV replication




                      19S
              35S     RNA
   Nucleus    RNA


  Cytoplasm




                    35S RNA
Evidence for ribosome shunting in CaMV 35S RNA translation



                    CAT                                 CAT
   cap                                         cap

          Moderate CAT synthesis               Extensive CAT synthesis
          Enhanced in vivo in the              Not enhanced by P6
          presence of P6

                      Stable stem-loop


                    CAT                                  CAT
    cap                                  cap


          No CAT synthesis                       Moderate CAT synthesis
          with or without P6                     Enhanced in vivo in the
                                                 presence of P6
      Secondary structure of 35S RNA 5’ untranslated region
       Short
       ORFs
               VII      I           III
cap                                                         V          An
                               II         IV                      VI

                                            Ψ = coat protein binding
                                          Ψ      (packaging signal)

                                          sORF D

                                          sORF E

                                              Splice
                                              donor
                              sORF C          site 1

                                              Primer
                       sORF B                 binding
                                              site
                                          F
                        cap                            ORF VII
                            sORF A
               Ribosome shunt from end of sORF A to landing
                  site at 548-562 (cf ribosome covers ~30nt)
              Requirements for shunting

Base stem must be more
stable (more negative) than
-50Kcal/mole (sequence not
important)

sORF A 2-10 codons
(sequence not important)

5-10 nt between sORF A stop
codon and base of stem

Changing sORF A initiation
codon from AUG to UUG          cap        ORF VII
prevents translation of ORF      sORF A
VII
P6 (TAV) facilitates translation of 3’ ORFs



                        AUGA
     cap        GUS                     An
                                  CAT



                  GUS           CAT
                -P6   +P6   -P6   +P6
                175   190   0     105
          Detection of eIF3 and CaMV P6 (TAV) in association with
                   polyribosomes of CaMV-infected cells




Park, et al. Cell, Vol. 106, 723–733 (2001)
            Model for re-initiation of protein synthesis at
                     3’ ORFs of CaMV 35S RNA




Initiation factors remain associated with the translating ribosomes,
       allowing efficient re-initiation of translation at the 3’ ORF
     Functional dissection of CaMV P6 protein = TAV




  The central portion of P6 is required for synthesis of P4 and P5
CaMV mutated in central P6 can replicate in cells expressing P4 and
                  P5 from separate constructions
               Splicing of CaMV 35S RNA

         VII                                                    30% of
                     I          III                           transcripts
cap                                                V                        An
                           II             IV                      VI
                             One
                            accep              Does splicing result in mRNAs
  Four                                         that can direct the synthesis of
 donor                       -tor
                             site              proteins from ORFs III through
 sites                                         V?

                                               Replace ORF III with
                            70% of             thioredoxin, eliminating P3
                          Transcripts          production and splicing acceptor
                          are spliced          site. Result – viable virus!!


               III                                              Example of
cap                                   V                    An
                     IV                          VI             spliced RNA
             Cauliflower mosaic virus              Cacao swollen shoot virus




www.ncbi.nlm.nih.gov/ICTVdb/ ICTVdB/15000000.htm
Rice tungro disease
The causal agent of rice tungro disease is a complex of two
viruses, Rice tungro bacilliform virus (RTBV) and Rice tungro
spherical virus (RTSV). RTBV is restricted to phloem cells in
the infected host.


RTSV has a 12.5 kb single-stranded, polyadenylated genomic
RNA.

RTBV is responsible for most of the symptoms, and RTSV is
almost symptomless in singular infections. However, RTBV is
not leafhopper transmitted from plants infected only with
RTBV.

RTSV confers leafhopper transmission on both viruses in the
mixed infections
 Endogenous plant pararetrovirus DNA sequences



                   Petunia PVCV
                   Banana BSV
                   Tobacco TVCV
             (1000 segments in genome)


       Each can be activated to episomal virus

Conditions conducive to production of episomal virus:
                 Wide genetic cross
         Abiotic stress, including wounding
             Tissue culture propagation
Endogenous RTBV sequences in the rice genome




      No intact ORF for RTBV exists in rice, so
       formation of episomal virus is unlikely
Comparison of ORF maps: CaMV, other Caulimoviridae,
                Copia of Drosophila
                                      Pr          R       H
        VII       I
                           III                    V                          CaMV
                      II         IV                                    VI


                                                          Pr       R     H
                  I                         III                              BSV
                           II
                                      Pr          R       H
                  I                   III
                                                                             RTBV
                           II                                      IV



              I                                   Pr           R         H
                                                                             PVCV


              G       Pr         I     R              H            (E)
                                                                             Copia virus

								
To top