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

Abstract

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 ...

Common Mechanism for RNA Encapsidation by Negative-Strand RNA Viruses

“…This complex is essential to prevent premature N oligomerization rather than to prevent RNA binding. This concept is supported by results reported by Chen et al, who showed that a phosphorylation-deficient triple mutant of P (P3A) that is mutated outside the cavity binding region could not prevent encapsidation of cellular RNA by VSVN (37).…”

“…The N terminus of P interacts with the back of the C-terminal half (C-lobe) of N. An ␣-helix corresponding to residues 17 to 31 of P was shown to occupy the RNA cavity in the structure. Subsequent studies by Chen et al (37), using a P3A mutant (triple mutations of Ser or Thr to Ala), showed that phosphorylation of P at Ser60, Thr62, and Ser64 is required to prevent N 0 from encapsidating cellular RNA, which leads to a dead-end product of N and diminishes viral replication. In the SAXS model, Ser60, Thr62, and Ser64 were notably positioned to form charge interactions with Lys398, Arg399, Lys414, and Lys417 of N (here named the "PO binding site").…”

Several residues within the N-terminal arm of vesicular stomatitis virus nucleoprotein play a critical role in protecting viral RNA from nuclease digestion

“…Our previous study showed that the CAT expression in the VSV miningenome system was regulated by both transcription and replication; that is, defects in either transcription or replication result in the loss of CAT expression (Chen et al, 2013). Having observed that N TVK4-6A3 , N RII7-9A3 , N VIV13-15A3, and N R7A did not support CAT expression, we next sought to determine whether these mutants are transcription or replication defective or both in RNA synthesis by real-time RT-PCR.…”

“…Thus, we assumed that these mutants had an effect on both transcription and replication, to greater degree on transcription. Because replication defects also influence transcription, and to confirm that transcription is also defective independent of effect of replication, we used a replication-deficient VSV minigenome (ΔTr minigenome) (Chen et al, 2013), and we repeated the aforementioned experiment using the same mutants. The results showed that the CAT expression levels supported by N TVK4-6A3 , N RII7-9A3 , N VIV13-15A3, and N R7A were 10% less than the levels supported by N wt (Fig.…”

“…BHK-21 cells in six-well plates were infected with vTF7-3 and transfected with either the plasmid BD-N wt or plasmids encoding mutant N proteins (BD-mutant N) plus pBS-L, pGADT7-P (AD-P), and pVSV-CAT2 encoding either a VSV minigenome or the replication-defective minigenome (ΔTr) (Chen et al, 2013). At 40 h post-transfection, cell lysates were subjected to a chloramphenicol acetyltrans-ferase (CAT) enzyme-linked immunosorbent assay (ELISA) for the detection of CAT expression levels as described by Chen et al (2006).…”