Study of the Assembly of Vesicular Stomatitis Virus N Protein: Role of the P Protein

The nucleocapsid (N) protein of nonsegmented negative-strand (NNS) RNA viruses, when expressed in eukaryotic cells, aggregates and forms nucleocapsid-like complexes with cellular RNAs. The phosphoprotein (P) has been shown to prevent such aggregation by forming a soluble complex with the N protein free from cellular RNAs (designated N 0 ). The N 0 -P complex presumably mediates specific encapsidation of the viral genome RNA. The precise mechanism by which the P protein carries out this function remains unclear. Here, by using a series of deleted and truncated mutant forms of the P protein of vesicular stomatitis virus (VSV), Indiana serotype, we present evidence that the N-terminal 11 to 30 amino acids (aa) of the P protein are essential in keeping the N protein soluble. Furthermore, glutathione S-transferase fused to the N-terminal 40 aa by itself is able to form the N 0 -P complex. Interestingly, the N-terminal 40-aa stretch failed to interact with the viral genome N-RNA template whereas the C-terminal 72 aa of the P protein interacted specifically with the latter. With an in vivo VSV minigenome transcription system, we further show that a deletion mutant form of P (P⌬1-10) lacking the N-terminal 10 aa which is capable of forming the N 0 -P complex was unable to support VSV minigenome transcription, although it efficiently supported transcription in vitro in a transcriptionreconstitution reaction when used as purified protein. However, the same mutant protein complemented minigenome transcription when expressed together with a transcription-defective P deletion mutant protein containing N-terminal aa 1 to 210 (P⌬II؉III). Since the minigenome RNA needs to be encapsidated before transcription ensues, it seems that the entire N-terminal 210 aa are required for efficient genome RNA encapsidation. Taking these results together, we conclude that the N-terminal 11 to 30 aa are required for N 0 -P complex formation but the N-terminal 210 aa are required for genome RNA encapsidation.Vesicular stomatitis virus (VSV) is a negative-strand RNA virus belonging to the family Rhabdoviridae. Its genome RNA is enwrapped in a helical form with the nucleocapsid (N) protein and packaged within the virion together with the RNAdependent RNA polymerase large protein (L) and the phosphoprotein (P). During cell infection, the associated RNA polymerase transcribes the genome RNA into five mRNAs that are subsequently translated into the glycoprotein (G), the matrix (M) protein, and the N, P, and L proteins. The N protein is an RNA-binding protein since it forms a high-molecular-weight complex with cellular RNAs with a quasihelical structure (1, 16) when expressed from plasmids in bacterial, mammalian, or insect cells. In contrast, it specifically encapsidates the viral genome RNA during the course of infection. It has been suggested that the P protein acts as a specific chaperone and forms a soluble complex with the N protein and prevents the latter from concentration-dependent aggregation (11,19,23,25). The N-P complex, once formed, pre...

Section: Resultsmentioning

confidence: 70%

Section: Discussionmentioning

confidence: 99%

Interaction of Vesicular Stomatitis Virus P and N Proteins: Identification of Two Overlapping Domains at the N Terminus of P That Are Involved in N ⁰ -P Complex Formation and Encapsidation of Viral Genome RNA

Chen

Ogino

Banerjee

2007

“…A QuikChange site-directed mutagenesis kit (Stratagene) was used to create mutant N plasmids pET-N (⌬347-352)-P, pET-N (320-324, (Ala) 5 )-P, and pET-N (Ser2903Trp)-P from plasmid pET-N/P (19) following the manufacturer's protocol. For plasmid pET-N (⌬1-22)-P, the coding sequence of N(⌬1-22) was amplified by PCR from pET-N/P with NcoI restriction sites introduced at both its 5Ј and 3Ј ends.…”

Section: Methodsmentioning

confidence: 99%

Role of Intermolecular Interactions of Vesicular Stomatitis Virus Nucleoprotein in RNA Encapsidation

Zhang

Green

Tsao

et al. 2008

Self Cite

The crystal structure of the vesicular stomatitis virus nucleoprotein (N) in complex with RNA reveals extensive and specific intermolecular interactions among the N molecules in the 10-member oligomer. What roles these interactions play in encapsidating RNA was studied by mutagenesis of the N protein. Three N mutants intended for disruption of the intermolecular interactions were designed and coexpressed with the phosphoprotein (P) in an Escherichia coli system previously described (T. J. Green et al., J. Virol. 74:9515-9524, 2000). Mutants N (⌬1-22), N (⌬347-352), and N (320-324, (Ala) 5 ) lost RNA encapsidation and oligomerization but still bound with P. Another mutant, N (Ser2903Trp), was able to form a stable ring-like N oligomer and bind with the P protein but was no longer able to encapsidate RNA. The crystal structure of N (Ser2903Trp) at 2.8 Å resolution showed that this mutant can maintain all the same intermolecular interactions as the wild-type N except for a slight unwinding of the N-terminal lobe. These results suggest that the intermolecular contacts among the N molecules are required for encapsidation of the viral RNA.All negative-strand RNA viruses contain a ribonucleoprotein (RNP) complex that consists of the viral genome RNA completely enwrapped by the nucleoprotein (N). Vesicular stomatitis virus (VSV) is a nonsegmented negative-strand RNA virus that belongs to the rhabdovirus family. The genome of VSV encodes five proteins: the nucleoprotein (N), the phosphoprotien (P), the matrix protein (M), the glycoprotein (G), and the large subunit of the polymerase (L). The RNP of VSV is packaged in a bullet-shaped virion by M, the protein that condenses the RNP during virion assembly, and G, the surface protein that is embedded in the viral envelope (14,16,22,29). P and L, which are other viral proteins, are packaged through their association with the RNP. After entry into the cytoplasm via membrane fusion mediated by G, the RNP is released from the virion and serves as the active template with which the copackaged polymerase proteins transcribe mRNAs from the five viral genes in the RNP. In the later stage of the virus replication cycle, a positive strand of the viral genome (cRNA) is produced in the form of an RNP. The cRNA-containing RNP then serves as the template for replication that also generates the viral genomic RNA in the form of an RNP ready to be packaged in the virion. Throughout the entire virus replication cycle of a negative-strand RNA virus, the genomelength viral RNA (cRNA or viral genomic RNA) is only present in the form of an RNP that is either serving as a template for RNA synthesis or being packaged in the virion. The assembly of the RNP is therefore a critical step in the replication of negative-strand RNA viruses.Functions of the N protein require it to have two essential properties. First, the N protein needs to bind a single-strand RNA. Second, the N protein has to polymerize in order to cover the entire length of the viral genome RNA. A number of experiments have attempted to...

“…In rhabdovirus-infected cells, P is present in the form of oligomers (20). Binding of P to N is thought to chaperone N such that it specifically encapsidates viral RNA (9,23,26), making it a suitable template for RNA synthesis by the viral polymerase complex. In addition, P is an essential cofactor of the polymerase complex itself and directly binds the catalytic L protein (6).…”

mentioning

confidence: 99%

Tracking Fluorescence-Labeled Rabies Virus: Enhanced Green Fluorescent Protein-Tagged Phosphoprotein P Supports Virus Gene Expression and Formation of Infectious Particles

Finke

Brzózka

Conzelmann

2004

Rhabdoviruses such as rabies virus (RV) encode only five multifunctional proteins accomplishing viral gene expression and virus formation. The viral phosphoprotein, P, is a structural component of the viral ribonucleoprotein (RNP) complex and an essential cofactor for the viral RNA-dependent RNA polymerase. We show here that RV P fused to enhanced green fluorescent protein (eGFP) can substitute for P throughout the viral life cycle, allowing fluorescence labeling and tracking of RV RNPs under live cell conditions. To first assess the functions of P fusion constructs, a recombinant RV lacking the P gene, SAD ⌬P, was complemented in cell lines constitutively expressing eGFP-P or P-eGFP fusion proteins. P-eGFP supported the rapid accumulation of viral mRNAs but led to low infectious-virus titers, suggesting impairment of virus formation. In contrast, complementation with eGFP-P resulted in slower accumulation of mRNAs but similar infectious titers, suggesting interference with polymerase activity rather than with virus formation. Fluorescence microscopy allowed the detection of eGFP-P-labeled extracellular virus particles and tracking of cell binding and temperature-dependent internalization into intracellular vesicles. Recombinant RVs expressing eGFP-P or an eGFP-P mutant lacking the binding site for dynein light chain 1 (DLC1) instead of P were used to track interaction with cellular proteins. In cells expressing a DsRed-labeled DLC1, colocalization of DLC1 with eGFP-P but not with the mutant P was observed. Fluorescent labeling of RV RNPs will allow further dissection of virus entry, replication, and egress under live-cell conditions as well as cell interactions.Rabies virus (RV) of the Lyssavirus genus and related members of the Rhabdoviridae, such as vesicular stomatitis virus (VSV, Vesiculovirus genus), are composed of only five multifunctional viral proteins, namely, nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and a large (L) RNA-dependent RNA polymerase. The viral RNA is enwrapped with N and associated with P and L to form a typical helical ribonucleoprotein (RNP) complex, which is active in RNA synthesis. During RV assembly, highly condensed RNPs of the typical rhabdovirus or bullet shape are enwrapped into an envelope containing the M and G proteins (31, 32). Entry of rhabdoviruses into cells involves receptor-mediated endocytosis, pH-dependent fusion of the viral and endosomal membranes (21), release into the cytoplasm, and uncoating of RNPs from M (35) such that gene expression can resume. Although these basic principles of the rhabdovirus infection pathway have been known for some time, details of many particular steps during entry, uncoating, gene expression, and virus egress await illumination. The possibility of real-time visualization of the entire infection pathway of rhabdoviruses and of tracking virus components such as RNPs in living cells is one highly desirable tool for the study of the rhabdovirus life cycle. A widely used approach in imaging of proteins or v...