The termini of VSV DI particle RNAs are sufficient to signal RNA encapsidation, replication, and budding to generate infectious particles

bThe phosphoprotein (P) of vesicular stomatitis virus (VSV) plays essential roles in viral RNA synthesis. It associates with nascent nucleoprotein (N) to form N 0 -P (free of RNAs), thereby preventing the N from binding to cellular RNAs and maintaining the N in a viral genomic RNA encapsidation-competent form for transcription and replication. The contributions of phosphorylation of P to transcription and replication have been studied intensively, but a concrete mechanism of action still remains unclear. In this study, using a VSV minigenome system, we demonstrated that a mutant of P lacking N-terminal phosphorylation (P3A), in which the N-terminal phosphate acceptor sites are replaced with alanines (S60/A, T62/A, and S64/A), does not support transcription and replication. However, results from protein interaction assays showed that P3A self-associates and interacts with N and the large protein (L) as efficiently as P does. Furthermore, purified recombinant P3A from Sf21 cells supported transcription in an in vitro transcription reconstitution assay. We also proved that P3A is not distributed intranuclearly in vivo. CsCl gradient centrifugation showed that P3A is incapable of preventing N from binding to cellular RNAs and therefore prevents functional template formation. Taken together, our results demonstrate that N-terminal phosphorylation is indispensable for P to prevent N from binding to nonviral RNAs and to maintain the N-specific encapsidation of viral genomic RNA for functional template formation. V esicular stomatitis virus (VSV) is a nonsegmented negativestrand RNA virus that serves as the prototype of the rhabdovirus group. The active RNA polymerase complex of VSV is composed of the large protein (L) and its cofactor, the phosphoprotein (P) (1), and both are required for the transcription and replication of the viral negative-sense single-stranded RNA genome, which is tightly encapsidated by nucleoprotein (N) (2, 3). P is a multifunctional protein: it associates with newly synthesized N during infection to prevent N from binding to cellular RNAs and maintains the replication-competent form of N by specifically encapsidating the nascent viral genomic/antigenomic RNA (4-6). On the other hand, it may also help to protect the L protein from proteolytic degradation (7). In addition, P also forms a tripartite replicase complex with L and N (L-N-P) to convert the N-RNA template to positive-sense genomic RNA in vivo. Via mutational and biochemical studies, P of the VSV Indiana serotype has been divided arbitrarily into three domains, i.e., N-terminal domain I (residues 1 to 137), domain II (residues 211 to 244), and domain III (residues 245 to 265), and a hypervariable hinge region (residues 138 to 210).Like the P's of many other negative-strand RNA viruses, the P of VSV is phosphorylated in infected cells and virions (8-10). The P of the VSV Indiana serotype has been shown to be phosphorylated in two different domains: N-terminal domain I, in which phosphorylation sites were mapped to S60, T62, and S64, and ...

Section: Discussionmentioning

confidence: 99%

N-Terminal Phosphorylation of Phosphoprotein of Vesicular Stomatitis Virus Is Required for Preventing Nucleoprotein from Binding to Cellular RNAs and for Functional Template Formation

Chen

Zhang

Banerjee

et al. 2013

“…Furthermore, these viruses have a bipartite replication promoter, in which two cis-acting elements lie on the same face of the helical nucleocapsid (17,24,25,35). In rhabdo-and pneumoviruses, there is no evidence that relative phasing of the N subunits is significant, or that the replication promoter extends beyond the Le region (5,7,22,27,31). Therefore it is possible that the combination of RNA and protein signatures in Sendai virus results in a high-affinity binding site for the polymerase, which can be recognized as an internal element, whereas in RSV and vesicular stomatitis virus, the polymerase relies more heavily on the 3Ј terminus for promoter recognition.…”

Section: Discussionmentioning

confidence: 99%

Evidence that the Respiratory Syncytial Virus Polymerase Is Recruited to Nucleotides 1 to 11 at the 3′ End of the Nucleocapsid and Can Scan To Access Internal Signals

Cowton

Fearns

2005

The 3-terminal end of the respiratory syncytial virus genomic RNA contains a 44-nucleotide leader (Le) region adjoining the gene start signal of the first gene. Previous mapping studies demonstrated that there is a promoter located at the 3 end of Le, which can signal initiation of antigenome synthesis. The aim of this study was to investigate the role of the 3 terminus of the RNA template in (i) promoter recognition and (ii) determining the initiation site for antigenome synthesis. A panel of minigenomes containing additional sequence at the 3 end of the Le were analyzed for their ability to direct antigenome and mRNA synthesis. Minigenomes containing heterologous extensions of 6 nucleotides or more were unable to support efficient RNA synthesis. However, the activity of a minigenome with a 56-nucleotide extension could be restored by insertion of Le nucleotides 1 to 11 or 1 to 13 at the 3 end, indicating that these nucleotides, in conjunction with the 3 terminus, are sufficient to recruit polymerase to the template. Northern blot and 5 rapid amplification of cDNA ends analysis of antigenome RNA indicated that antigenome initiation occurred at the first position of Le, irrespective of the terminal extension. This finding demonstrates that the 3 terminus of the RNA is not necessary for determining the antigenome initiation site. Data are presented which suggest that following recruitment to a promoter at the 3 end of Le, the polymerase is able to scan and respond to a promoter signal embedded within the RNA template.

“…1B]) have been predicted to represent promoters for viral RNA synthesis. However, the Paramyxovirus promoters outflank these regions, in contrast with those of the Rhabdoviruses (vesicular stomatitis virus [VSV]) (12,46,60). They extend over 96 nucleotides, overlapping the first (N gene) and last (L gene) transcription units (Fig.…”

mentioning

confidence: 99%

“Rule of Six”: How Does the Sendai Virus RNA Polymerase Keep Count?

Vulliémoz

Roux

2001

The "rule of six" stipulates that the Paramyxovirus RNA polymerase efficiently replicates only viral genomes counting 6n ؉ 0 nucleotides. Because the nucleocapsid proteins (N) interact with 6 nucleotides, an exact nucleotide-N match at the RNA 3-OH end (3-OH congruence) may be required for recognition of an active replication promoter. Alternatively, assuming that the six positions for the interaction of N with the nucleotides are not equivalent, the nucleotide position relative to N may be critical (N phase context). The replication abilities of various minireplicons, designed so that the 3-OH congruence could be discriminated from the N phase context, were studied. The results strongly suggest that the application of the rule of six depends on the recognition of nucleotides positioned in the proper N phase context.The active genome of the negative-stranded RNA viruses is found in the form of a nucleocapsid. In the nucleocapsid, the single-stranded RNA is tightly associated with the nucleocapsid proteins (N) in a rod-shaped helical structure. The interaction between the RNA and the N subunits can be so stable that the nucleocapsid structure does not disintegrate in high salt concentrations, even upon density equilibrium centrifugation (35). In such cases, the sugar-phosphate backbone may not be accessible to solvent (29) and the RNA chain may be protected from RNase attack (37). This suggests that the RNA is inside the nucleocapsid structure. However, its exact position is not known, because the RNA-N three-dimensional structure has not been resolved at atomic resolution.For the Sendai virus (SeV), a member of the Paramyxoviridae family, Respirovirus genus (other genera of the same family include Morbillivirus, Rubulavirus, and Pneumovirus), the nucleocapsid rod, containing an RNA of 15,384 nucleotides (54, 55), is ϳ1.0 m long and has a diameter of ϳ20.0 nm (19,28). A three-dimensional reconstruction at 2.4-nm resolution has been obtained from electron microscopy images (17). The lefthanded helix can coexist in three different pitch conformations: 5.3, 6.8, and 37.5 nm. In the most prevalent pitch state, 5.3 nm, there are 13 N subunits per helix turn and each N subunit is proposed to interact with 6 nucleotides.The RNA genome is linearly organized in six successive transcription units separated by a few nucleotides (see Fig. 1A). This coding region is flanked, at the 3Ј and 5Ј ends, by short extracistronic sequences, of 55 and 57 nucleotides, respectively, for SeV. These sequences (called leader ϩ and leader Ϫ or trailer [see Fig. 1B]) have been predicted to represent promoters for viral RNA synthesis. However, the Paramyxovirus promoters outflank these regions, in contrast with those of the Rhabdoviruses (vesicular stomatitis virus [VSV]) (12, 46, 60). They extend over 96 nucleotides, overlapping the first (N gene) and last (L gene) transcription units (Fig. 1B) (41, 58). At the genome 3Ј end, the genomic promoter (GP) operates in transcription and replication, while at the antigenome 3Ј end, the antigenomic ...