RNAs 1 and 2 of Alfalfa mosaic virus, expressed in transgenic plants, start to replicate only after infection of the plants with RNA 3

Thole, Vera; Garcia, M. L.; Rossum, C. M. A. van; Neeleman, Lyda; Brederode, Frans Th.; Linthorst, Huub J. M.; Bol, John F.

doi:10.1099/0022-1317-82-1-25

Cited by 7 publications

(6 citation statements)

References 26 publications

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“…As in R12 plants, the accumulation of plus-strand RNA 1 and 2 could then be the result of protection of the Pol II transcripts from degradation after minusstrand synthesis or of transcription of plus strands by the AMV RdRp on the minus-strand templates (49). In both cases, plusstrand accumulation would be dependent on and coincide with minus-strand accumulation.…”

Section: Resultsmentioning

confidence: 99%

“…However, when RNAs 1 and 2 are expressed from the 35S promoter in transgenic plants (R12 plants), CP is not required for minus-strand RNA synthesis (49). It was proposed that the poly(A) tail of the nuclear AMV transcripts may compensate for a putative role of CP in translation of the inoculum RNAs (49).…”

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“…It was proposed that the poly(A) tail of the nuclear AMV transcripts may compensate for a putative role of CP in translation of the inoculum RNAs (49). When R12 plants were inoculated with RNA 3, RNAs 1 and 2 started to coreplicate with RNA 3 (49). Tobacco plants that express the P1 and P2 proteins from nuclear transgenes that are flanked by incomplete 5Ј-or 3Ј-UTRs (P12 plants) support the replication of AMV RNA 3 but no replication of the transgenic 5Ј-truncated RNA 1 or 3Ј-truncated RNA 2 is observed (47).…”

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Role of the 3′-Untranslated Regions of Alfalfa Mosaic Virus RNAs in the Formation of a Transiently Expressed Replicase in Plants and in the Assembly of Virions

Vlot

Neeleman

Linthorst

et al. 2001

J Virol

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Alfalfa mosaic virus (AMV) RNAs 1 and 2 encode the replicase proteins P1 and P2, respectively, whereas RNA 3 encodes the movement protein and the coat protein (CP). When RNAs 1 and 2 were transiently expressed from a T-DNA vector (R12 construct) by agroinfiltration of Nicotiana benthamiana, the infiltrated leaves accumulated minus-strand RNAs 1 and 2 and relatively small amounts of plus-strand RNAs. In addition, RNA-dependent RNA polymerase (RdRp) activity could be detected in extracts of the infiltrated leaves. After transient expression of RNAs 1 and 2 with the 3-untranslated regions (UTRs) of both RNAs deleted (R1⌬/2⌬ construct), no replication of RNAs 1 and 2 was observed, while the infiltrated leaves supported replication of RNA 3 after inoculation of the leaves with RNA 3 or expression of RNA 3 from a T-DNA vector (R3 construct). No RdRp activity could be isolated from leaves infiltrated with the R1⌬/2⌬ construct, although P1 and P2 sedimented in a region of a glycerol gradient where active RdRp was found in plants infiltrated with R12. RdRp activity could be isolated from leaves infiltrated with constructs R1⌬/2 (3-UTR of RNA 1 deleted), R1/2⌬ (3-UTR of RNA 2 deleted), or R1⌬/2⌬ plus R3. This demonstrates that the 3-UTR of AMV RNAs is required for the formation of a complex with in vitro enzyme activity. RNAs 1 and 2 with the 3-UTRs deleted were encapsidated into virions by CP expressed from RNA 3. This shows that the high-affinity binding site for CP at the 3-termini of AMV RNAs is not required for assembly of virus particles.RNA-dependent RNA polymerase (RdRp) proteins of many plus-strand RNA viruses have been expressed in heterologous systems, including Escherichia coli (15, 23-25, 31, 35, 42, 61), Saccharomyces cerevisiae (17, 36), insect cells (4,14,30,43,63), and mammalian cells (26). The availability of in vitro active RdRp has greatly facilitated research into the mechanisms of plus-strand virus replication. Mutant RNA polymerases can be expressed despite their inability to support viral replication. In this way, insight can be gained into the roles of the various domains of viral replicase proteins.Alfalfa mosaic virus (AMV) (for reviews, see references 6 and 18) is a tripartite plus-strand RNA virus belonging to the family Bromoviridae (Fig. 1A). The AMV replication complex consists of two viral proteins, P1 and P2, and possibly host proteins. P1, encoded by RNA 1, has homologies to known methyltransferase domains in its N-terminal half and homologies to helicase domains in its C-terminal half (12, 21). P2, encoded by RNA 2, contains the RNA polymerase domain characterized by the GDD motif (for a review, see reference 33). AMV RNA 3 codes for the viral movement protein (P3) and the coat protein (CP), which is translated from a subgenomic mRNA 4. All three genomic RNAs of AMV contain 5Ј-and 3Ј-untranslated regions (UTRs), which are highly structured and presumed to contain important cis-acting regulatory elements for viral replication and translation (32,52,54,55,58,59).The 3Ј-terminal 145 bases...

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Role of the 3′-Untranslated Regions of Alfalfa Mosaic Virus RNAs in the Formation of a Transiently Expressed Replicase in Plants and in the Assembly of Virions

Vlot

Neeleman

Linthorst

et al. 2001

J Virol

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“…1, sequences with apparent similarity to the SIN Pro180-Thr-X-Arg-Ser184 nsP4 sequence also have upstream sequences rich in Lys and other basic amino acids. The SIN nsP4 Arg183 region did not resemble the ilarvirus and alfalfamovirus coat protein motif's role in plus-strand synthesis (50). The Arg residue in the poliovirus VPg sequence is proposed to stabilize a UTP moiety for transfer to the terminal Tyr residue of VPg (30) and to function during plus-and minus-strand initiation (14,32).…”

Section: Discussionmentioning

confidence: 99%

Alphavirus Minus-Strand RNA Synthesis: Identification of a Role for Arg183 of the nsP4 Polymerase

Fata

Sawicki

2002

J Virol

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A partially conserved region spanning amino acids 142 to 191 of the Sindbis virus (SIN) nsP4 core polymerase is implicated in host restriction, elongation, and promoter recognition. We extended the analysis of this region by substituting Ser, Ala, or Lys for a highly conserved Arg183 residue immediately preceding its absolutely conserved Ser184-Ala-Val-Pro-Ser188 sequence. In chicken cells, the nsP4 Arg183 mutants had a nonconditionally lethal, temperature-sensitive (ts) growth phenotype caused by a ts defect in minus-strand synthesis whose extent varied with the particular amino acid substituted (Ser>Ala>Lys). Plus-strand synthesis by nsP4 Arg183 mutant polymerases was unaffected when corrected for minus-strand numbers, although 26S mRNA synthesis was enhanced at the elevated temperature compared to wild type. The ts defect was not due to a failure to form or accumulate nsP4 at 40°C. In contrast to their growth in chicken cells, the nsP4 Arg183 mutants replicated equally poorly, if at all, in mosquito cells. We conclude that Arg183 within the Pro180-Asn-Ile-Arg-Ser184 sequence of the SIN nsP4 polymerase contributes to the efficient initiation of minus strands or the formation of its replicase and that a host factor(s) participates in this event.After the genome of an alphavirus is released into the cytoplasm of an infected cell, it is translated into the four nonstructural proteins (nsPs) that form the viral RNA-dependent RNA polymerase activities (47). The viral polymerase, probably also with host factors, forms a replication complex with the genome RNA and copies it into a minus-strand template. The minus strand, in turn, is used as a template to produce both the genome and the subgenomic 26S mRNA, the latter by recognition of an internal promoter in the minus strand. The 26S subgenomic mRNA is translated to produce the capsid and envelope proteins. Therefore, three polymerase activities are formed during the replication cycle as a result of the stepwise and regulated cleavage of the nsP polyprotein precursors (21,23,24,36,42,51). The four nsPs are numbered according to their gene order and are translated initially as polyproteins P123 and P1234, both of which are subsequently cleaved by the viral papain-like protease activity present in nsP2. Briefly, cleavage of nsP4 from nascent P1234 occurs first and likely cotranslationally, activating the polymerase and forming an initial P123ϩnsP4 complex that is active in minus-strand synthesis. Second, cleavage at the 1/2 site produces nsP1ϩP23ϩ nsP4 that is active in both minus-strand and genome synthesis. Finally, intermediate polyprotein P23 is cleaved, giving stable (fully processed) nsP1ϩnsP2ϩnsP3ϩnsP4 complexes that are active in the synthesis of genome and 26S subgenomic mRNA but not minus-strand RNA.Each of the alphavirus nsPs has been assigned specific roles in viral replication based on the mapping of temperaturesensitive (ts) mutants, biochemical analyses, and protein homology comparisons (36,47). How the nsP4 core polymerase functions in the formation of ...

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“…CP is required to initiate minus-strand RNA synthesis in protoplasts inoculated with T7 RNA polymerase transcripts corresponding to the genomic RNAs (18). However, CP is not required to initiate minus-strand RNA synthesis when the genomic RNAs are expressed from cDNAs flanked by the cauliflower mosaic virus 35S promoter and nopaline synthase terminator from nuclear transgenes in transgenic plants (19,20), from a T-DNA vector in agroinfiltrated leaves (21), or from plasmids used to inoculate plants (22). We have suggested that in a natural infection, CP is required in a step before the initiation of AMV minus-strand RNA synthesis, probably the translation of the viral RNAs (15, 18).…”

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Translation of a nonpolyadenylated viral RNA is enhanced by binding of viral coat protein or polyadenylation of the RNA

Neeleman

Olsthoorn²,

Linthorst³

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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C ircularization of the genome of RNA viruses plays an essential role in the replication and translation of viral RNAs. Formation of panhandle structures by base pairing between complementary sequences at the 5Ј and 3Јtermini of several genera of minus-strand RNA viruses has been known already for some time (1). Recently, it became clear that cyclization by RNA-RNA interactions may also play an essential role in the replication of positive-strand RNA viruses such as f laviviruses (2, 3). However, for a growing number of positive-strand RNA viruses, circularization seems to be mediated by protein bridges between the 5Ј and 3Ј termini of the viral RNA. On entry into the host cell, the genome of positive-strand RNA viruses has to serve as messenger for the translation of viral proteins including replication factors. A number of viral RNAs resemble cellular mRNAs by having a 5Ј-cap structure (m 7 GpppN) and 3Ј-poly(A) tail. In cellular mRNAs, these structures permit the formation of a protein bridge by interactions between eIF4G and eIF4E from the cap-binding complex of translation initiation factors and the poly(A) binding protein (PABP) associated with the poly(A) tail (4, 5). Picornaviruses do not have a cap structure, but the internal ribosome entry site in the 5Ј untranslated region (UTR) of the RNA binds initiation factors and a poly(rC) binding protein that mediate the interaction with PABP. Circularization of Poliovirus RNA seems to play a role both in translation and replication of the viral RNA (6, 7).The genomic RNAs of many viruses lack a poly(A) tail, and elements in the 5Ј and͞or 3Ј UTR are believed to compensate for the absence of a poly(A) tail (8-12). Recently, the rotavirus (family Reoviridae) NSP3 protein was shown to interact with eIF-4G and with the 3Ј UTR of the nonpolyadenylated viral mRNAs. Both interactions were required for efficient translation of the viral mRNA, and NSP3 was suggested to mimic the function of PABP (13). Here, we report evidence that the coat protein (CP) of alfalfa mosaic virus (AMV; genus Alfamovirus, family Bromoviridae) also may act as a functional equivalent of PABP.RNAs 1 and 2 of the tripartite AMV genome encode the replicase proteins P1 and P2, respectively. P1 contains methyltransferase-and helicase-like motifs, whereas P2 contains polymerase-like motifs. RNA 3 encodes the movement protein and CP, which is translated from a subgenomic messenger, RNA 4. The RNAs contain a 5Ј-cap structure but the 3Ј ends are not polyadenylated (14). At their 3Ј termini, RNAs 1-4 contain a homologous sequence of 145 nucleotides that can be folded into two alternative conformations: a linear array of hairpins with flanking AUGC sequences that represents a strong CP binding site or a tRNA-like structure that represents the promoter for minus-strand RNA synthesis (15). A mixture of the three genomic RNAs becomes infectious only after addition of CP or its messenger, RNA 4, to the inoculum (16). Initiation of infection requires binding of a few molecules of CP to the 3Ј termini of the g...

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RNAs 1 and 2 of Alfalfa mosaic virus, expressed in transgenic plants, start to replicate only after infection of the plants with RNA 3

Cited by 7 publications

References 26 publications

Role of the 3′-Untranslated Regions of Alfalfa Mosaic Virus RNAs in the Formation of a Transiently Expressed Replicase in Plants and in the Assembly of Virions

Role of the 3′-Untranslated Regions of Alfalfa Mosaic Virus RNAs in the Formation of a Transiently Expressed Replicase in Plants and in the Assembly of Virions

Alphavirus Minus-Strand RNA Synthesis: Identification of a Role for Arg183 of the nsP4 Polymerase

Translation of a nonpolyadenylated viral RNA is enhanced by binding of viral coat protein or polyadenylation of the RNA

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