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Single-amino-acid alterations in a highly conserved central region of vesicular stomatitis virus N protein differentially affect the viral nucleocapsid template functions.

ABSTRACT: The nucleocapsid protein (N) of vesicular stomatitis virus and other rhabdoviruses plays a central role in the assembly and template functions of the viral N-RNA complex. The crystal structure of the viral N-RNA complex suggests that the central region of the N protein interacts with the viral RNA. Sequence alignment of rhabdovirus N proteins revealed several highly conserved regions, one of which spanned residues 282 to 291 (GLSSKSPYSS) in the central region of the molecule. Alanine-scanning mutagenesis of this region suggested that replacement of the tyrosine residue at position 289 (Y289) with alanine resulted in an N-RNA template that is nonfunctional in viral genome replication and transcription. To establish the molecular basis of this defect, our further studies revealed that the Y289A mutant maintained its interaction with other N protein molecules but that its interactions with the P protein or with the viral RNA were defective. Replacement of Y289 with other aromatic, polar, or large amino acids indicated that the hydrophobic and aromatic nature of this position in the N protein is functionally important and that a larger aromatic residue is less favorable. Interestingly, we have observed that several single-amino-acid substitutions in this highly conserved region of the molecule rendered the nucleocapsid template nonfunctional in transcription without adversely affecting the replication functions. These results suggest that the structure of the N protein and the resulting N-RNA complex, in part, regulate the viral template functions in transcription and replication.

SUBMITTER: Nayak D

PROVIDER: S-EPMC2681978 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Single-amino-acid alterations in a highly conserved central region of vesicular stomatitis virus N protein differentially affect the viral nucleocapsid template functions.

Nayak Debasis D Panda Debasis D Das Subash C SC Luo Ming M Pattnaik Asit K AK

Journal of virology 20090325 11

The nucleocapsid protein (N) of vesicular stomatitis virus and other rhabdoviruses plays a central role in the assembly and template functions of the viral N-RNA complex. The crystal structure of the viral N-RNA complex suggests that the central region of the N protein interacts with the viral RNA. Sequence alignment of rhabdovirus N proteins revealed several highly conserved regions, one of which spanned residues 282 to 291 (GLSSKSPYSS) in the central region of the molecule. Alanine-scanning mu ...[more]

PMID: 19321605

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Project description:Polyamides have been shown to bind double-stranded DNA by complementing the curvature of the minor groove and forming various hydrogen bonds with DNA. Several polyamide molecules have been found to have potent antiviral activities against papillomavirus, a double-stranded DNA virus. By analogy, we reason that polyamides may also interact with the structured RNA bound in the nucleocapsid of a negative-strand RNA virus. Vesicular stomatitis virus (VSV) was selected as a prototype virus to test this possibility since its genomic RNA encapsidated in the nucleocapsid forms a structure resembling one strand of an A-form RNA duplex. One polyamide molecule, UMSL1011, was found to inhibit infection of VSV. To confirm that the polyamide targeted the nucleocapsid, a nucleocapsid-like particle (NLP) was incubated with UMSL1011. The encapsidated RNA in the polyamide-treated NLP was protected from thermo-release and digestion by RNase A. UMSL1011 also inhibits viral RNA synthesis in the intracellular activity assay for the viral RNA-dependent RNA polymerase. The crystal structure revealed that UMSL1011 binds the structured RNA in the nucleocapsid. The conclusion of our studies is that the RNA in the nucleocapsid is a viable antiviral target of polyamides. Since the RNA structure in the nucleocapsid is similar in all negative-strand RNA viruses, polyamides may be optimized to target the specific RNA genome of a negative-strand RNA virus, such as respiratory syncytial virus and Ebola virus.IMPORTANCE Negative-strand RNA viruses (NSVs) include several life-threatening pathogens, such as rabies virus, respiratory syncytial virus, and Ebola virus. There are no effective antiviral drugs against these viruses. Polyamides offer an exceptional opportunity because they may be optimized to target each NSV. Our studies on vesicular stomatitis virus, an NSV, demonstrated that a polyamide molecule could specifically target the viral RNA in the nucleocapsid and inhibit viral growth. The target specificity of the polyamide molecule was proved by its inhibition of thermo-release and RNA nuclease digestion of the RNA bound in a model nucleocapsid, and a crystal structure of the polyamide inside the nucleocapsid. This encouraging observation provided the proof-of-concept rationale for designing polyamides as antiviral drugs against NSVs.

Dataset Information

Single-amino-acid alterations in a highly conserved central region of vesicular stomatitis virus N protein differentially affect the viral nucleocapsid template functions.

Publications

Single-amino-acid alterations in a highly conserved central region of vesicular stomatitis virus N protein differentially affect the viral nucleocapsid template functions.

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