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To further our understanding of the pro-viral roles of the host cytosolic heat shock protein 70 (Hsp70) family, we chose the conserved Arabidopsis thaliana Hsp70-2 and the unique Erd2 (early response to dehydration 2), which contain Hsp70 domains. Based on in vitro studies with purified components, we show that AtHsp70-2 and AtErd2 perform pro-viral functions equivalent to that of the yeast Ssa1 Hsp70. These functions include activation of the tombusvirus RdRp, and stimulation of replicase assembly. Yeast-based complementation studies demonstrate that AtHsp70-2 or AtErd2 are present in the purified tombusvirus replicase. RNA silencing and over-expression studies in Nicotiana benthamiana suggest that both Hsp70-2 and Erd2 are co-opted by tomato bushy stunt virus (TBSV). Moreover, we used allosteric inhibitors of Hsp70s to inhibit replication of TBSV and related plant viruses in plants. Altogether, interfering with the functions of the co-opted Hsp70s could be an effective antiviral approach against tombusviruses in plants. • Characterization of the host cytosolic heat shock protein 70 family for TBSV replication. • Pro-viral function of the conserved Arabidopsis Hsp70-2 and the unique Erd2 (early response to dehydration 2). • Allosteric inhibitors of Hsp70s block TBSV replication in vitro , in yeast and in plants. • Interfering with the functions of the co-opted Hsp70s could be a strong antiviral approach. [ABSTRACT FROM AUTHOR]

Rotaviruses (RVs) replicate their segmented, double-stranded RNA genomes in tandem with early virion assembly. In this study, we sought to gain insight into the ultrastructure of RV assembly-replication intermediates (RIs) using transmission electron microscopy (EM). Specifically, we examined a replicase-competent, subcellular fraction that contains all known RV RIs. Three never-before-seen complexes were visualized in this fraction. Using in vitro reconstitution, we showed that ~15-nm doughnut-shaped proteins in strings were nonstructural protein 2 (NSP2) bound to viral RNA transcripts. Moreover, using immunoaffinity-capture EM, we revealed that ~20-nm pebble-shaped complexes contain the viral RNA polymerase (VP1) and RNA capping enzyme (VP3). Finally, using a gel purification method, we demonstrated that ~30–70-nm electron-dense, particle-shaped complexes represent replicase-competent core RIs, containing VP1, VP3, and NSP2 as well as capsid proteins VP2 and VP6. The results of this study raise new questions about the interactions among viral proteins and RNA during the concerted assembly–replicase process. [ABSTRACT FROM AUTHOR]

Abstract: Bamboo mosaic virus (BaMV) has a positive-sense single-stranded RNA genome with a 5′ cap and a 3′ poly(A) tail. To characterize polyadenylation activity in the BaMV replicase complex, we performed the in vitro polyadenylation with various BaMV templates. We conducted a polyadenylation activity assay for BaMV RNA by using a partially purified BaMV replicase complex. The results showed that approximately 200 adenylates at the 3′ end of the RNA were generated on the endogenous RNA templates. Specific fractions derived from uninfected Nicotiana benthamiana plants enhanced the polyadenylation activity, implying that host factors are involved in polyadenylation. Furthermore, polyadenylation can be detected in newly synthesized plus-strand RNA in vitro when using the exogenous BaMV minus-strand minigenome. For polyadenylation on the exogenous plus-strand minigenome, the 3′ end requires at least 4A to reach 22% polyadenylation activity. The results indicate that the BaMV replicase complex recognizes the 3′ end of BaMV for polyadenylation. [Copyright &y& Elsevier]

Abstract: By co-opting host proteins for their replication, plus-stranded RNA viruses can support robust replication and suppress host anti-viral responses. Tomato bushy stunt virus (TBSV) recruit the cellular heat shock protein 70 (Hsp70), an abundant cytosolic chaperone, into the replicase complex. By taking advantage of yeast model host, we demonstrate that the four-member SSA subfamily of HSP70 genes is essential for TBSV replication. The constitutively expressed SSA1 and SSA2, which are resident proteins in the viral replicase, can be complemented by the heat-inducible SSA3 and/or SSA4 for TBSV replication. Using a yeast strain carrying a temperature sensitive ssa1 ts, but lacking functional SSA2/3/4, we show that inactivation of Ssa1pts led to a defect in membrane localization of the viral replication proteins, resulting in cytosolic distribution of the viral proteins and lack of replicase activity. An in vitro replicase assembly assay with Ssa1pts revealed that functional Ssa1p is required during the replicase assembly process, but not during minus- or plus-strand synthesis. Temperature shift experiments from nonpermissive to permissive in ssa1 ts yeast revealed that the re-activated Ssa1pts could promote efficient TBSV replication in the absence of other SSA genes. We also demonstrate that the purified recombinant Ssa3p can facilitate the in vitro assembly of the TBSV replicase on yeast membranes, demonstrating that Ssa3p can fully complement the function of Ssa1p. Taken together, the cytosolic SSA subfamily of Hsp70 proteins play essential and multiple roles in TBSV replication. [Copyright &y& Elsevier]

Abstract: Potato virus X (PVX) infection leads to certain cytopathological modifications of the host endomembrane system. The subcellular location of the PVX replicase was previously unknown while the PVX TGBp3 protein was previously reported to reside in the ER. Using PVX infectious clones expressing the green fluorescent protein reporter, and antisera detecting the PVX replicase and host membrane markers, we examined the subcellular distribution of the PVX replicase in relation to the TGBp3. Confocal and electron microscopic observations revealed that the replicase localizes in membrane bound structures that derive from the ER. A subset of TGBp3 resides in the ER at the same location as the replicase. Sucrose gradient fractionation showed that the PVX replicase and TGBp3 proteins co-fractionate with ER marker proteins. This localization represents a region where both proteins may be synthesized and/or function. There is no evidence to indicate that either PVX protein moves into the Golgi apparatus. Cerulenin, a drug that inhibits de novo membrane synthesis, also inhibited PVX replication. These combined data indicate that PVX replication relies on ER-derived membrane recruitment and membrane proliferation. [Copyright &y& Elsevier]

Abstract: An ideal system to investigate individual determinants of the replication process of (+)-strand RNA viruses is a cell-free extract that supports viral protein and RNA synthesis in a synchronized manner. Here, we applied a translation/replication system based on cytoplasmic extracts of Nicotiana tabacum cells to Tomato bushy stunt virus (TBSV) RNA. In vitro translated TBSV proteins p33 and p92 form viral replicase, which, in the same reaction, accomplishes the entire replication cycle on exogenous TBSV DI or full-length RNA. Tests of mutant TBSV RNAs confirmed the template specificity of the in vitro replication reaction. Complementation experiments ascertained the significance of an earlier identified TBSV host factor. Interestingly, formation of the viral replicase occurs also in the absence of concurrent protein synthesis demonstrating that translation and RNA replication are not functionally linked in this system. Our studies with cell-free extracts of a plant host thus confirmed earlier findings and enabled novel insights into the TBSV RNA replication process. [Copyright &y& Elsevier]

Abstract: Host RNA-binding proteins are likely to play multiple, integral roles during replication of plus-strand RNA viruses. To identify host proteins that bind to viral RNAs, we took a global approach based on the yeast proteome microarray, which contains 4080 purified yeast proteins. The biotin-labeled RNA probes included two distantly related RNA viruses, namely Tomato bushy stunt virus (TBSV) and Brome mosaic virus (BMV). Altogether, we have identified 57 yeast proteins that bound to TBSV RNA and/or BMV RNA. Among the identified host proteins, eleven bound to TBSV RNA and seven bound to BMV RNA with high selectivity, whereas the remaining 39 host proteins bound to both viral RNAs. The interaction between the TBSV replicon RNA and five of the identified host proteins was confirmed via gel-mobility shift and co-purification experiments from yeast. Over-expression of the host proteins in yeast, a model host for TBSV, revealed 4 host proteins that enhanced TBSV replication as well as 14 proteins that inhibited replication. Detailed analysis of one of the identified yeast proteins binding to TBSV RNA, namely translation elongation factor eEF1A, revealed that it is present in the highly purified tombusvirus replicase complex. We also demonstrate binding of eEF1A to the p33 replication protein and a known cis-acting element at the 3′ end of TBSV RNA. Using a functional mutant of eEF1A, we provide evidence on the involvement of eEF1A in TBSV replication. [Copyright &y& Elsevier]

Previously, we localized the β2 interacting portion of the catalytic subunit (α) of DNA polymerase III to the C-terminal half, downstream of the polymerase active site. Since then, two different β2 binding sites within this region have been proposed. An internal site includes amino acid residues 920–924 (QADMF) and an extreme C-terminal site includes amino acid residues 1154–1159 (QVELEF). To permit determination of their relative contributions, we made mutations in both sites and evaluated the biochemical, genetic, and protein binding properties of the mutant α subunits. All purified mutant α subunits retained near wild-type polymerase function, which was measured in non-processive gap-filling assays. Mutations in the internal site abolished the ability of mutant α subunits to participate in processive synthesis. Replacement of the five-residue internal sequence with AAAKK eliminated detectable binding to β2. In addition, mutation of residues required for β2 binding abolished the ability of the resulting polymerase to participate in chromosomal replication in vivo. In contrast, mutations in the C-terminal site exhibited near wild-type phenotypes. α Subunits with the C-terminal site completely removed could participate in processive DNA replication, could bind β2, and, if induced to high level expression, could complement a temperature-sensitive conditional lethal dnaE mutation. C-terminal defects that only partially complemented correlated with a defect in binding to τ, not β2. A C-terminal deletion only reduced β2 binding fourfold; τ binding was decreased ca 400-fold. The context in which the β2 binding site was presented made an enormous difference. Replacement of the internal site with a consensus β2 binding sequence increased the affinity of the resulting α for β2 over 100-fold, whereas the same modification at the C-terminal site did not significantly increase binding. The implications of multiple interactions between a replicase and its processivity factor, including applications to polymerase cycling and interchange with other polymerases and factors at the replication fork, are discussed. [Copyright &y& Elsevier]

Abstract: A number of nucleotide and non-nucleoside inhibitors of HCV polymerase are currently under investigation as potential antiviral agents to treat HCV-infected patients. HCV polymerase is part of a replicase complex including the polymerase subunit NS5B together with other viral and host proteins and viral RNA. The RNA synthesis activity of the native replicase complex was inhibited by 3′-deoxy-CTP, a chain-terminating nucleotide analog, but not inhibited by non-nucleoside NS5B polymerase inhibitors of three different structural classes. The HCV replicase was also resistant to heparin, a broad-spectrum, RNA-competitive polymerase inhibitor. Prebinding of the recombinant NS5B protein with a RNA template rendered the polymerase largely resistant to the inhibition by heparin and the non-nucleoside inhibitors, but did not affect the inhibitory potency of 3′-deoxy-CTP. Therefore, the HCV replicase showed a similar pattern of inhibitor sensitivity as compared to RNA-bound NS5B. These results suggest that the native HCV replicase complex represents a stable and productive polymerase–RNA complex. The allosteric non-nucleoside NS5B polymerase inhibitors are inactive against established HCV replicase but may function antagonistically with the formation of a productive enzyme–template complex. [Copyright &y& Elsevier]

RNA recombination, which is thought to occur due to replicase errors during viral replication, is one of the major driving forces of virus evolution. In this article, we show evidence that the replicase proteins of Cucumber necrosis virus, a tombusvirus, are directly involved in RNA recombination in vivo. Mutations within the RNA-binding domains of the replicase proteins affected the frequency of recombination observed with a prototypical defective-interfering (DI) RNA, a model template for recombination studies. Five of the 17 replicase mutants tested showed delay in the formation of recombinants when compared to the wild-type helper virus. Interestingly, two replicase mutants accelerated recombinant formation and, in addition, these mutants also increased the level of subgenomic RNA synthesis (Virology 308 (2003), 191–205). A trans-complementation system was used to demonstrate that mutation in the p33 replicase protein resulted in altered recombination rate. Isolated recombinants were mostly imprecise (nonhomologous), with the recombination sites clustered around a replication enhancer region and a putative cis-acting element, respectively. These RNA elements might facilitate the proposed template switching events by the tombusvirus replicase. Together with data in the article cited above, results presented here firmly establish that the conserved RNA-binding motif of the replicase proteins is involved in RNA replication, subgenomic RNA synthesis, and RNA recombination. [Copyright &y& Elsevier]

The genome organization and expression strategy of the newly identified severe acute respiratory syndrome coronavirus (SARS-CoV) were predicted using recently published genome sequences. Fourteen putative open reading frames were identified, 12 of which were predicted to be expressed from a nested set of eight subgenomic mRNAs. The synthesis of these mRNAs in SARS-CoV-infected cells was confirmed experimentally. The 4382- and 7073 amino acid residue SARS-CoV replicase polyproteins are predicted to be cleaved into 16 subunits by two viral proteinases (bringing the total number of SARS-CoV proteins to 28). A phylogenetic analysis of the replicase gene, using a distantly related torovirus as an outgroup, demonstrated that, despite a number of unique features, SARS-CoV is most closely related to group 2 coronaviruses. Distant homologs of cellular RNA processing enzymes were identified in group 2 coronaviruses, with four of them being conserved in SARS-CoV. These newly recognized viral enzymes place the mechanism of coronavirus RNA synthesis in a completely new perspective. Furthermore, together with previously described viral enzymes, they will be important targets for the design of antiviral strategies aimed at controlling the further spread of SARS-CoV. [Copyright &y& Elsevier]

Brevibacterium linens has commercial significance in the dairy industry and potential application in the production of bacteriocins and carotenoids. Strain development of these industrially significant organisms would be facilitated by the use of vectors, yet few are available. In this study we report the isolation of four novel plasmids from the Gram-positive coryneform B. linens, and determine the first complete nucleotide sequence of a native plasmid of B. linens. The cryptic plasmid pLIM is 7610 bp in length, and belongs to a subfamily of θ replicating ColE2-related plasmids. Initial investigation suggests that replication in pLIM requires two replicases, a primase (RepA) and a DNA binding protein (RepB), encoded by a single operon repAB. The origin of replication is located upstream of repAB transcription. [Copyright &y& Elsevier]