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

Abstract

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...

Structure of the paramyxovirus parainfluenza virus 5 nucleoprotein–RNA complex

“…RNA binding in the groove then induces the closed conformation of N by bringing together the CTD and NTD domains supported by the finding that VSV nucleocapsid rings, which were defective in RNA binding, were in a slightly open conformation. The closed conformation of the nucleocapsid ring is then stabilized by the lateral arm connections between adjacent N-protomers (18), because the removal of RNA from already formed nucleocapsid rings doesn't result in the opening of the RNA groove (21).…”

“…This assumption was based on the fact that NTD made minimal lateral contacts in the RSV and VSV nucleocapsid rings (5,6,9,18). The NTD domain in the PIV5-N ring contributes significantly to the lateral interface (Table 2).…”

“…In both PIV5-N and RSV-N, the RNA pocket could be divided into two subpockets: pocket 1 and pocket 2 consisting of highly conserved residues from NTD and CTD, respectively. Although, pocket 1-RNA interactions are through the amino acid side chains and those in pocket 2 are through backbone amide atoms, the side chain identities of pocket 2 residues still play an important role as mutations in VSV targeting Ser290 (A351 in PIV5-N) resulted in ring formations unable to encapsidate RNA (18), and mutating VSV-Y289 (Y350 in PIV5-N) affects RNA encapsidation and P binding, and consequently viral transcription and replication (19).…”

Kernel energy method applied to vesicular stomatitis virus nucleoprotein

“…This article focuses on calculations of total energy for the entire molecule and the hydrogen bond interaction energies between the chains of VSV which make up the full nucleoprotein, whose PDB ID code is 2QVJ (1).…”

Le virus de la stomatite vésiculaire dans la lutte contre le cancer

“…Des études de cristallographie ont permis de déterminer que P agit comme une chaperonne en conduisant N au site de réplication virale et en la stabilisant avec la polymérase associée à l'ARN. Puis, la protéine N se lie directement à l'ARN pour former un complexe oligomérique de haut poids moléculaire en forme d'anneau encapsidant le génome en son centre chargé positivement, afin qu'il résiste à la dégradation par les RNases [9]. …”