Structure of Hibiscus Latent Singapore Virus Determined by X-Ray Fiber Diffraction

Ph.DDOCTOR OF PHILOSOPH

Structure of the NS1 Protein N-Terminal Origin Recognition/Nickase Domain from the Emerging Human Bocavirus

This is the publisher's version, also available electronically from http://www.asm.org/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 single-stranded 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

The human parvovirus B19 non-structural protein 1 N-terminal domain specifically binds to the origin of replication in the viral DNA

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 (Guan et al, 2009, J. Virology, 83, 9541-9553). 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

The human parvovirus B19 non-structural protein 1 N-terminal domain specifically binds to the origin of replication in the viral DNA

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 (Guan et al, 2009, J. Virology, 83, 9541-9553). 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

Structures of minute virus of mice replication initiator protein N-terminal domain: insights into DNA nicking and origin binding

This is the author's accepted manuscript. The original is available at http://www.sciencedirect.com/science/article/pii/S0042682214005236Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45 Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins

Hibiscus Chlorotic Ringspot Virus Coat Protein Is Essential for Cell-to-Cell and Long-Distance Movement but Not for Viral RNA Replication

<div><p><i>Hibiscus chlorotic ringspot virus</i> (HCRSV) is a member of the genus <i>Carmovirus</i> in the family <i>Tombusviridae</i>. In order to study its coat protein (CP) functions on virus replication and movement in kenaf (<i>Hibiscus cannabinus</i> 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. <i>In vitro</i> 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 β-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 β-annulus domain is required in T = 3 assembly <i>in vitro</i>.</p></div

Detection of HCRSV RNA and its CP accumulation in upper leaves at 25 dpi.

<p>(A) Detection of viral RNA in upper leaves by RT-PCR using primer HC-R3, followed by PCR using primers HC-F8 and HC-R3 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113347#pone-0113347-t001" target="_blank">Table 1</a>). (B) Detection of HCRSV CP in upper leaves by western blot. (C) Symptoms of upper leaves of inoculated kenaf plants at 25 dpi.</p

Schematic representations of (A) HCRSV genome and (B) partial HCRSV CP sequence showing mutation sites and deletion.

<p>Rectangles in (A) represent open reading frames. Underlined amino acids in (B) indicate the non-expressed portion as the CP start codon ATG was replaced with GTG in mutant p2590 (A to G) and the boxed proline (CCG) was substituted with alanine (GCG) to remove the kink of CP in mutant p2776 (C to G). The circled methionine residues are the translation initiation sites of CP in wt HCRSV (p223) and mutant p2590 (A to G). Symbol slash (/) represents the cleavage site when swollen HCRSV virions were digested with trypsin.</p

Re-assembly of HCRSV particles after trypsin digestion.

<p>(A) Time-course trypsin digestion of HCRSV virions at room temperature (RT). HCRSV swollen virions were digested with trypsin at RT and harvested at different time points (0, 5, 25 and 60 min, respectively), followed by separation of the digested proteins on 15% SDS-PAGE gel. (B) Limited trypsin digestion. To optimize the digestion in order to obtain the only one smaller size protein band, samples were differentially treated. Symbols “+” and “–” above each lane represent treatment with or without corresponding reagents or temperature conditions. Samples were collected immediately after each denaturation and stored in freezer, followed by separation on 12% SDS-PAGE gel, immediately after the last sample was collected. (C) Observation of trypsin digested HCRSV particles. The limited trypsin-digested swollen HCRSV virions were dialyzed by a two-step method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113347#pone.0113347-Garmann1" target="_blank">[33]</a>, followed by concentration and observation under TEM. Each bar represents 40 nm. (D) Detection of HCRSV RNA in reassembled particles by RT-PCR with primers HC-R3, HC-F5 and HC-R5, HC-F8 and HC-R3 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113347#pone-0113347-t001" target="_blank">Table 1</a>) for detection of its genomic and sgRNA.</p

HCRSV RNA and CP accumulation in transfected kenaf protoplasts.

<p>(A) Schematic representation of mutant 2590 (A to G) and mutant 2776 (C to G). Only region covering sgRNA2 is shown to indicate the mutation sites. p30 is an ORF encoding a putative 30 kDa protein. (B) Northern blot analysis of viral RNA accumulation. <i>In vitro</i> transcripts (10 µg each) were transfected into 9×10⁵ protoplasts and harvested at different time points. Total RNA (2.5 µg each) extracted from protoplasts collected at 24 and 48 h post transfection (hpt), respectively, was used for viral RNA detection. DIG-labeled 425 bp HCRSV PCR product located in the 3′ region of the genome was used as the probe for hybridization. (C) Western blot analysis of HCRSV CP. Total protein was extracted from protoplasts collected at 72 hpt. CBB denotes Coomassie blue staining.</p