Sunil Tewary | National University of Singapore (original) (raw)

Papers by Sunil Tewary

Members ofthe Parvoviridae family allencodeanon-structuralprotein1(NS1)thatdirectsreplicationof s... more Members ofthe Parvoviridae family allencodeanon-structuralprotein1(NS1)thatdirectsreplicationof
single-stranded viralDNA,packagesviralDNAintocapsid,andservesasapotenttranscriptional
activator.HerewereporttheX-raystructureoftheminutevirusofmice(MVM)NS1N-terminaldomain
at 1.45Åresolution,showingthatsitesfordsDNAbinding,ssDNAbindingandcleavage,nuclear
localization, andotherfunctionsareintegratedonacanonicalfoldofthehistidine-hydrophobic-
histidine superfamilyofnucleases,includingelementsspecific forthis Protoparvovirus but distinctfrom
its Bocaparvovirus or Dependoparvovirus orthologs. Highresolutionstructuralanalysisrevealsanickase
activesitewithanarchitecturethatallowshighlyversatilemetalligandbinding.Thestructuressupport
a unified mechanismofreplicationoriginrecognitionforhomotelomericandheterotelomericparvo-
viruses, mediatedbyabasic-residue-richhairpinandanadjacenthelixintheinitiatorproteinsandby
tandem tetranucleotidemotifsinthereplicationorigins.
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Hibiscus chlorotic ringspot virus (HCRSV) is a member of the genus Carmovirus in the family Tombu... more Hibiscus chlorotic ringspot virus (HCRSV) is a member of the genus Carmovirus in the family Tombusviridae. In order to study
its coat protein (CP) functions on virus replication and movement in kenaf (Hibiscus cannabinus L.), two HCRSV mutants,
designated as p2590 (A to G) in which the first start codon ATG was replaced with GTG and p2776 (C to G) in which proline
63 was replaced with alanine, were constructed. In vitro transcripts of p2590 (A to G) were able to replicate to a similar level
as wild type without CP expression in kenaf protoplasts. However, its cell-to-cell movement was not detected in the
inoculated kenaf cotyledons. Structurally the proline 63 in subunit C acts as a kink for b-annulus formation during virion
assembly. Progeny of transcripts derived from p2776 (C to G) was able to move from cell-to-cell in inoculated cotyledons
but its long-distance movement was not detected. Virions were not observed in partially purified mutant virus samples
isolated from 2776 (C to G) inoculated cotyledons. Removal of the N-terminal 77 amino acids of HCRSV CP by trypsin
digestion of purified wild type HCRSV virions resulted in only T = 1 empty virus-like particles. Taken together, HCRSV CP is
dispensable for viral RNA replication but essential for cell-to-cell movement, and virion is required for the virus systemic
movement. The proline 63 is crucial for HCRSV virion assembly in kenaf plants and the N-terminal 77 amino acids including
the b-annulus domain is required in T = 3 assembly in vitro.

The non-structural protein 1 (NS1) of human parvovirus B19 plays a critical role in viral DNA rep... more The non-structural protein 1 (NS1) of human parvovirus B19 plays a critical role in viral DNA replication. Previous studies identified the origin of replication in the viral DNA, which contains four DNA elements, namely NSBE1 to NSBE4, that are required for optimal viral replication ). Here we have demonstrated in vitro that the NS1 N-terminal domain (NS1N) binds to the origin of replication in a sequence-specific, length-dependent manner that requires NSBE1 and NSBE2, while NSBE3 and NSBE4 are dispensable. Mutagenesis analysis has identified nucleotides in NSBE1 and NSBE2 that are critical for NS1N binding. These results suggest that NS1 binds to the NSBE1-NSBE2 region in the origin of replication, while NSBE3 and NSBE4 may provide binding sites for potential cellular factors. Such a specialized nucleoprotein complex may enable NS1 to nick the terminal resolution site and separate DNA strands during replication.

Human bocavirus is a newly identified, globally prevalent, parvovirus that is associated with res... more Human bocavirus is a newly identified, globally prevalent, parvovirus that is associated with respiratory infection in infants and young children. Parvoviruses encode a large nonstructural protein 1 (NS1) that is essential for replication of the viral singlestranded DNA genome and DNA packaging and may play versatile roles in virus-host interactions. Here, we report the structure of the human bocavirus NS1 N-terminal domain, the first for any autonomous parvovirus. The structure shows an overall fold that is canonical to the histidine-hydrophobic-histidine superfamily of nucleases, which integrates two distinct DNA-binding sites: (i) a positively charged region mediated by a surface hairpin (residues 190 to 198) that is responsible for recognition of the viral origin of replication of the double-stranded DNA nature and (ii) the nickase active site that binds to the single-stranded DNA substrate for site-specific cleavage. The structure reveals an acidic-residue-rich subdomain that is present in bocavirus NS1 proteins but not in the NS1 orthologs in erythrovirus or dependovirus, which may mediate bocavirus-specific interaction with DNA or potential host factors. These results provide insights into recognition of the origin of replication and nicking of DNA during bocavirus genome replication. Mapping of variable amino acid residues of NS1s from four human bocavirus species onto the structure shows a scattered pattern, but the origin recognition site and the nuclease active site are invariable, suggesting potential targets for antivirals against this clade of highly diverse human viruses.

$Research paper thumbnail of Structure of hibiscus latent singapore virus by fiber diffraction: a nonconserved his122 contributes to coat protein stability.$

Tobamoviruses, the HLSV genome contains a unique poly(A) tract in its 3′ untranslated region. The... more Tobamoviruses, the HLSV genome contains a unique poly(A) tract in its 3′ untranslated region. The virion is composed of a monomeric coat protein (CP) unit of 18 kDa, arranged as a right-handed helix around the virus axis. We have determined the structure of HLSV at 3.5 Å by X-ray fiber diffraction and refined it to an R-factor of 0.096. While the overall structure of the HLSV CP resembles that of other Tobamoviruses, there are a few unique differences. There is a kink in the LR helix due to the presence of His122. Also, the adjacent Lys123 may further destabilize the helix by positive charge repulsion, making the kink more pronounced. The His122-Asp88 salt bridge provides significant stability to the loop adjacent to the RR helix. Carboxyl-carboxylate interactions that drive viral disassembly are also different in HLSV. The nucleotide recognition mechanisms for virus assembly between HLSV and ribgrass mosaic virus are similar, but different between tobacco mosaic virus and cucumber green mottle mosaic virus.

Conference Presentations by Sunil Tewary

Thesis Chapters by Sunil Tewary

$Research paper thumbnail of Structure of hibiscus latent singapore virus by fiber diffraction: a nonconserved his122 contributes to coat protein stability.$