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PubMed2003

Genomic sequence analyses of segments 1 to 6 of Dendrolimus punctatus cytoplasmic polyhedrosis virus.

The complete nucleotide sequences of genomic segments S1 to S6 from Dendrolimus punctatus cypovirus 1 (DpCPV-1) have been determined. Each segment of S1 to S6 possess a single open reading frame. Conserved motifs 5' (AGUAA) and 3'(GUUAGCC) were found at the ends of each segment. Comparison of the proteins of DpCPV with those of other members in the family Reoviridae lead us to suggest that S1, S3, S4 and S6 encode the viral structural protein VP1, VP2, VP3 and VP4, respectively. S5 encoded viral non-structural protein p100 and S2 encodes an RNA-dependent RNA polymerase (RdRp). Motif analysis shows that VP3 is similar to the methyltransferase of Methanosarcina mazei Goe1, VP4 has motifs for leucine zipper and ATP/GTP-binding sites, and p100 is remarkably similar to foot-and-mouth disease virus 2A protease (FMDV 2Apro). Phylogenetic analysis of RdRps from nine viruses of the family Reoviridae indicates that DpCPV is a type 1 cypovirus, more related to Bombyx mori cypovirus (BmCPV) than to other cypovirus species. DpCPV is more related to Rice ragged stunt virus (RRSV) than to other members of different genera of the family Reoviridae, which seems to confirm the previous hypothesis that plant reoviruses originated from insect reoviruses.

Amino Acid Sequence↗

PubMed1998

Probing viruses with mass spectrometry.

Mass spectrometry offers a new perspective on the solution and gas-phase properties of viruses. Its broad application to local and global viral structure provides unique insights into many biological processes, including viral-antibody binding, protein-protein interactions and protein dynamics. Mass measuring viral proteins is now routine and since viruses are typically well characterized, in that the capsid proteins and DNA (or RNA) sequences are known, identifying a virus based on the mass of the protein and enzymatic digestion fragments is relatively straightforward. Using mass spectrometry, this paper describes the identification of viral protein post-translational modifications such as myristoylation, phosphorylation and disulfide bridging. Furthermore, complementary data obtained with mass spectrometry and x-ray crystallography demonstrate that viruses are highly dynamic particles whose viral capsid's mobility could, until recently, be inferred only from inherently static spectroscopic methods. Lastly, mass spectrometry has been applied on a global scale via the mass measurement of entire intact viruses. Given the general utility of mass spectrometry, its continuing development should further its application to viral dynamics, structure, function and identification.

Animals↗

PubMed2002

Analysis of antigenicity and topology of E2 glycoprotein present on recombinant hepatitis C virus-like particles.

Purification of hepatitis C virus (HCV) from sera of infected patients has proven elusive, hampering efforts to perform structure-function analysis of the viral components. Recombinant forms of the viral glycoproteins have been used instead for functional studies, but uncertainty exists as to whether they closely mimic the virion proteins. Here, we used HCV virus-like particles (VLPs) generated in insect cells infected with a recombinant baculovirus expressing viral structural proteins. Electron microscopic analysis revealed a population of pleomorphic VLPs that were at least partially enveloped with bilayer membranes and had viral glycoprotein spikes protruding from the surface. Immunogold labeling using specific monoclonal antibodies (MAbs) demonstrated these protrusions to be the E1 and E2 glycoproteins. A panel of anti-E2 MAbs was used to probe the surface topology of E2 on the VLPs and to compare the antigenicity of the VLPs with that of truncated E2 (E2(660)) or the full-length (FL) E1E2 complex expressed in mammalian cells. While most MAbs bound to all forms of antigen, a number of others showed striking differences in their abilities to recognize the various E2 forms. All MAbs directed against hypervariable region 1 (HVR-1) recognized both native and denatured E2(660) with comparable affinities, but most bound either weakly or not at all to the FL E1E2 complex or to VLPs. HVR-1 on VLPs was accessible to these MAbs only after denaturation. Importantly, a subset of MAbs specific for amino acids 464 to 475 and 524 to 535 recognized E2(660) but not VLPs or FL E1E2 complex. The antigenic differences between E2(660,) FL E1E2, and VLPs strongly point to the existence of structural differences, which may have functional relevance. Trypsin treatment of VLPs removed the N-terminal part of E2, resulting in a 42-kDa fragment. In the presence of detergent, this was further reduced to a trypsin-resistant 25-kDa fragment, which could be useful for structural studies.

Amino Acid Sequence↗

PubMed2003

Molecular characterization of segments 7-10 of Dendrolimus punctatus cytoplasmic polyhedrosis virus provides the complete genome.

The nucleotide sequences of genomic segments S7-S10 from Dendrolimus punctatus cypovirus strain Hunan (DpCPV-Hn) have been determined. This provides the complete genome sequences of DpCPV-Hn. Each segment of S7-S10 possess a single segment each. Homology searches showed that the nucleotide sequences and the deduced amino acid sequences of DpCPV S7-10 had high level of identities with those of Bombyx mori cypovirus (BmCPV) S7-10, respectively. While the amino acid sequences of the proteins encoded by DpCPV S7 and S8 have low identities with those of the proteins encoded by type 14 Lymantria dispar cypovirus S7 and S8, respectively. DpCPV S7 encodes viral structural protein VP5, S8 and S9 encode viral non-structural proteins, and S10 encodes polyhedrin gene, according to the function of the genome segments of BmCPV. There are glutamic-acid-rich and proline-rich domains in the central region of DpCPV S8 encoded protein. A nuclear localization signal was found in the protein encoded by DpCPV S9. Phylogenetic analysis of RNA-dependent RNA polymerases from nine viruses of the family Reoviridae and polyhedrin from eight viruses of the genus Cypovirus indicate that DpCPV is a type 1 cypovirus, more closely related to BmCPV than to other cypovirus species. These results also support the classification of CPV groups based on the electrophoretic migration of genomic dsRNA.

Animals↗

PubMed1994

Inhibition of type 5 adenovirus infectivity by periodate oxidation.

Periodate oxidation of purified type 5 Adenovirus (Ad5) led to a mean loss of infectivity of 6.84 logs. There were no significant differences in adsorption and penetration between oxidized and mock-oxidized virus. However, after infection with oxidized virus, no synthesis of viral structural proteins could be detected and a 78.5% inhibition of viral DNA synthesis was observed. Labelling experiments performed by treating oxidized and mock-oxidized virus with tritiated sodium borohydride revealed that the fiber glycoprotein was one of the proteins labelled in oxidized virus whereas no labelled proteins were detected in non oxidized virus. In addition, it was found that one mol of formaldehyde generated during oxidation of sugar residues was bound per 500 base pairs in oxidized virus. One consequence of this in situ generation of formaldehyde is the formation of DNA-protein crosslinks. The DNA so crosslinked showed different patterns of restriction fragments with endonucleases such as Hpa I, Hind III and Kpn I but not with Xho I.

Adenoviruses, Human↗

PubMed1993

Neutralizing antibody to vesicular stomatitis virus (HTLV-I) pseudotype in infants born to seropositive mothers.

Breast feeding is the major route of mother-to-child transmission of human T-cell leukemia virus type I (HTLV-I). Our experiments with rabbits have shown that passive immunization is capable of blocking cell-to-cell infection of HTLV-I by blood transfusion or breast feeding. In this study, sera were collected serially from 3 infants born to seropositive mothers and were tested for the presence of neutralizing antibody to vesicular stomatitis virus (HTLV-I) pseudotype as well as antibodies to viral structural proteins. There was a good correlation between neutralizing and viral antibody titers, both of which were detectable until 3-6 months after birth. Whether maternally transmitted neutralizing antibody is protective against perinatal infection of HTLV-I remains to be studied.

Adult↗

PubMed1991

Protein p22 of African swine fever virus: an early structural protein that is incorporated into the membrane of infected cells.

The open reading frame K'177, located at the left end of the African swine fever virus genome, codes for an early induced structural protein of 22,000 Da (p22), which is released from the viral particle by the nonionic detergent n-octyl-beta-D-glucopyranoside under conditions that solubilize external viral structural proteins. The predicted amino acid sequence of the protein contains a hydrophobic region at its N-terminus with characteristics of a signal peptide and, at early times after virus infection of Vero cells, the protein can be detected in the cell membrane by immunolabeling.

Amino Acid Sequence↗

PubMed2001

Post-translational modification of the hepatitis C virus core protein by tissue transglutaminase.

The hepatitis C virus (HCV) core protein is a structural protein that packages the viral genomic RNA. In this study, we demonstrate that a stable core protein dimer could be produced in liver cells. The production of this protein could be enhanced by calphostin C and serum deprivation. This protein was determined to be the core protein dimer because of its reactivity with the anti-core antibody, its similar electrophoretic mobility compared with that of the core protein dimer generated by cross-linking with glutaraldehyde, and its increase in size by a hemagglutinin tag fused to the core protein sequence. This core protein dimer was highly stable and resistant to SDS and beta-mercaptoethanol. The enzyme that mediated the formation of this stable core protein dimer was determined to be the tissue transglutaminase (tTG) because, first, tTG could be activated by calphostin C and serum deprivation; second, the formation of this dimer was suppressed by monodansylcadaverine, a tTG inhibitor; and third, the core protein could be cross-linked by tTG in vitro. Thus, the HCV core protein represents the first known viral structural protein substrate of tTG. The post-translational modification by tTG reduced the RNA binding activity of the core protein, raising the possibility that tTG may regulate the biological functions of the HCV core protein.

Cell Line↗

PubMed2004

Homology between two different Salmonella phages: Salmonella enterica serovar Typhimurium phage P22 and Salmonella enterica serovar Anatum var. 15 + phageepsilon34.

A distinguishing feature of many microorganisms, belonging to the Gram negative group of bacteria, is the presence of the lipopolysaccharide on their cell surface. Salmonella is a prominent member of this group of bacteria. Many Salmonella phages use the LPS as the initial receptor in the infection process and they can distinguish subtle changes in the LPS molecules. The phage protein that is responsible for recognition of these cells is the tail or tailspike protein (TSP). Those TSPs, which use LPS as a receptor, are prokaryotic LPS-binding proteins. As an initial step in using phage TSPs as model systems for a detailed molecular genetic analysis of protein-LPS interactions, a comparison of two phages and their TSPs from two different Salmonella bacterial viruses (phages), Salmonella enterica serovar Typhimurium phage P22 and Salmonella enterica serovar Anatum var. 15 + phage epsilon34, is being carried out. This present study shows significant viral protein homology between many viral structural proteins from these two phages including their TSPs. Significantly this report suggests a general structural motif for part of the TSP of phages and suggests that a more detailed comparative analysis of these TSPs is warranted.

Animals↗

PubMed1989

Functional dissection of the HIV-1 Rev trans-activator--derivation of a trans-dominant repressor of Rev function.

Human immunodeficiency virus type 1 (HIV-1) encodes a nuclear trans-activator, termed Rev, that is required for the expression of the viral structural proteins and, hence, for viral replication. The Rev protein acts posttranscriptionally to induce the sequence-specific nuclear export of unspliced HIV-1 mRNA species that are otherwise excluded from the cell cytoplasm. We have used site-directed mutagenesis to identify two distinct regions of the HIV-1 Rev protein that are required for in vivo biological activity. The larger and more N-terminal of these two regions includes, but extends beyond, an arginine-rich sequence element required for nuclear localization. Mutation of a second, more C-terminal Rev protein sequence element was found to yield defective Rev proteins that act as trans-dominant inhibitors of Rev function. These Rev mutants are shown to inhibit HIV-1 replication when expressed in transfected cells and may have potential application in the treatment of HIV-1 related disease.

Amino Acid Sequence↗

PubMed2001

Ty1 retrotransposition and programmed +1 ribosomal frameshifting require the integrity of the protein synthetic translocation step.

Programmed ribosomal frameshifting is utilized by a number of RNA viruses to ensure the correct ratio of viral structural to enzymatic proteins for viral particle assembly. Altering frameshifting efficiencies upsets this ratio, inhibiting virus propagation. Two yeast viruses that induce host cell ribosomes to shift translational reading frame were used as tools to explore the interactions between viruses and host cellular protein synthetic machinery. Previous studies showed that the ribosome-inactivating protein pokeweed antiviral protein specifically inhibited propagation of the Ty1 retrotransposable element of yeast as a consequence of inhibition of programmed +1 ribosomal frameshifting. Here, complementary genetic and pharmacological approaches were employed to test whether inhibition of Ty1 retrotransposition is a general feature of alterations in the translocation step of elongation and +1 frameshifting. The results demonstrate that cells harboring a variety of mutant alleles of two host-encoded proteins that are involved in translocation, eukaryotic elongation factor-2 and the ribosome-associated protein RPP0, have Ty1 propagation defects. We also show that sordarin, a fungus-specific inhibitor of eEF-2 function, specifically inhibits programmed +1 ribosomal frameshifting and Ty1 retrotransposition. These findings serve to link inhibition of Ty1 retrotransposition and +1 frameshifting to changes in the translocation step of elongation.

Frameshifting, Ribosomal↗

PubMed1988

Direct adverse effects of Sendai virus DI particles on virus budding and on M protein fate and stability.

Upon infections of BHK cells with a mixture of Sendai standard and defective interfering (DI) viruses (mixed virus infection), viral budding was found to be restricted by factors ranging from 5 to more than 20. The reduced viral budding correlated with a high intracellular M protein turnover. M appeared to be degraded shortly after its synthesis, and seemed not to be able to self-associate in a stable way under the plasma membrane as it did in St virus-infected cells. These data, added to the previous findings that infection with DI particles allowed infected cell survival and favored the cell-surface turnover of the hemagglutinin-neuraminidase protein, led to the hypothesis that DI genomes directly act by preventing the stable formation inside the cells of a viral structure composed of M/HN/nucleocapsids. When involved in this structure M would be protected from degradation and HN would be stably anchored in the plasma membrane. Formation of this structure would be necessary for viral budding and would be damaging for the cells. Comparison with results published by other authors shows that such a model is consistent with other data. It can integrate, as well, data obtained in the analysis of mutant viruses involved in persistence.

Defective Viruses↗

PubMed1967

Ultraviolet sensitivity of the biological activity of phi-X-174 virus, single-stranded DNA, and RF DNA.

Action spectra for inactivation of varphiX virus, free varphiX single-stranded DNA, and double-stranded varphiX DNA (RF) have been measured using light of wavelength 225-302 mmu. The sensitivity of RF has been determined using bacterial hosts both capable and incapable of reactivation of UV damage. The inactivation of varphiX virus is due, at all wavelengths, to damage to its DNA; it appears that, below 240 mmu, energy absorbed by viral structural protein may inactivate the viral DNA. The variation of the probability of inactivation by an absorbed quantum (quantum yield) with wavelength, in the case of free-single-stranded varphiX DNA, suggests that energy absorbed by pyrimidine residues is more likely to yield inactivation than absorption by purines. This implies that energy transfer is not so extensive as to make all absorbed energy available to pyrimidines.

Coliphages↗

PubMedDate not supplied

Recent contributions of molecular biology to the clinical virology of myxoviruses.

Recent advances in the clinical virology of influenza are based on non-pragmatically oriented research on the genetics and biochemistry of the influenza virus. Antigenically hybrid recombinant viruses can be tailored to provide monospecific reagents for serological studies. Basic research on viral structure and the mechanism of viral replication has directly influenced the establishment of a cell culture system suitable for the isolation of most influenza viruses. Identification of viral genotype by RNA gel electrophoresis and mapping of oligonucleotides of viral RNA has already facilitated epidemiologic investigations. The clinical virologist of the future must have an understanding of the potential limitations of these techniques for specific strain identification.

Genes, Viral↗

PubMed1993

Sindbis virus expression vectors: packaging of RNA replicons by using defective helper RNAs.

Since the recovery of infectious RNA transcripts from full-length cDNA clones, alphavirus genome RNAs have been engineered to allow expression of heterologous RNAs and proteins. The highest levels of expression of heterologous products are achieved when the viral structural genes are replaced by the heterologous coding sequences. Such recombinant RNAs are self-replicating (replicons) and can be introduced into cells as naked RNA, but they require trans complementation to be packaged and released from cells as infectious virion particles. In this report, we describe a series of defective Sindbis virus helper RNAs which can be used for packaging Sindbis virus RNA replicons. The defective helper RNAs contain the cis-acting sequences required for replication as well as the subgenomic RNA promoter which drives expression of the structural protein genes. In cells cotransfected with both the replicon and defective helper RNAs, viral nonstructural proteins translated from the replicon RNA allow replication and transcription of the defective helper RNA to produce the virion structural proteins. A series of defective helper RNAs were compared for the ability to package the replicon RNA as well as for the ability to be replicated and packaged. One defective helper RNA not only packaged the replicon but also was itself encapsidated and would be useful under conditions in which extensive amplification is advantageous. Other defective helper RNAs were able to package the replicon efficiently but were packaged very poorly themselves. These helpers should be useful for applications in which expression of the viral structural proteins or virus spread is not desired.

Animals↗

PubMed2005

Persistence of HIV-1 structural proteins and glycoproteins in lymph nodes of patients under highly active antiretroviral therapy.

Here we report a long-term persistence of HIV-1 structural proteins and glycoproteins in germinal centers (GCs) of lymph nodes (LNs) in the absence of detectable virus replication in patients under highly active antiretroviral therapy (HAART). The persistence of viral structural proteins and glycoproteins in GCs was accompanied by specific antibody responses to HIV-1. Seven patients during the chronic phase of HIV-1 infection were analyzed for the presence of the capsid protein (HIV-1p24), matrix protein (HIV-1p17), and envelope glycoproteins (HIV-1gp120/gp41), as well as for viral RNA (vRNA) in biopsy specimens from LNs obtained before initiation of therapy and during HAART that lasted from 5 to 13 months. In parallel, these patients were also monitored for viremia and specific anti-HIV-1 antibody responses to structural proteins and glycoproteins both before and during treatment. Before-therapy viral levels, as determined by RT-PCR, ranged from 3 x 10(3) to 6.3 x 10(5) copies of vRNA per ml, whereas during treatment, vRNA was under detectable levels (<25 copies per ml). The pattern of vRNA detection in peripheral blood was concordant with in situ hybridization results of LN specimens. Before treatment, vRNA associated with follicular dendritic cells (FDCs) was readily detected in GCs of LNs of the patients, whereas during therapy, vRNA was consistently absent in the GCs of LN biopsies of treated patients. In contrast to vRNA hybridization results, viral structural proteins and glycoproteins, evaluated by immunohistochemical staining, were present and persisted in the GC light zone of LNs in abundant amounts not only before initiation of therapy but also during HAART, when no vRNA was detected in GCs. Consistent with immunohistochemical findings, specific antibody responses to HIV-1p17, -p24, and -gp120/gp41, as evaluated by ELISA and virus neutralization, persisted in patients under therapy for up to 13 months of follow-up. The implications of these findings are discussed in relation to HIV-1 persistence in infected individuals and the potential role of chronic antigenic stimulation by the deposited structural proteins in GCs for AIDS-associated B cell malignancies.

Antiretroviral Therapy, Highly Active↗

PubMed2001

Induction of a protective response in swine vaccinated with DNA encoding foot-and-mouth disease virus empty capsid proteins and the 3D RNA polymerase.

This work focuses on the development of a potential recombinant DNA vaccine against foot-and-mouth disease virus (FMDV). Such a vaccine would have significant advantages over the conventional inactivated virus vaccine, in particular having none of the risks associated with the high security requirements for working with live virus. The principal aim of this strategy was to stimulate an antibody response to native, neutralizing epitopes of empty FMDV capsids generated in vivo. Thus, a plasmid (pcDNA3.1/P1-2A3C3D) was constructed containing FMDV cDNA sequences encoding the viral structural protein precursor P1-2A and the non-structural proteins 3C and 3D. The 3C protein was included to ensure cleavage of the P1-2A precursor to VP0, VP1 and VP3, the components of self-assembling empty capsids. The non-structural protein 3D was also included in the construct in order to provide additional stimulation of CD4(+) T cells. When swine were immunized with this plasmid, antibodies to FMDV and the 3D polymerase were synthesized. Furthermore, neutralizing antibodies were detected and, after three sequential vaccinations with DNA, some of the animals were protected against challenge with live virus. Additional experiments suggested that the antibody response to FMDV proteins was improved by the co-administration of a plasmid encoding porcine granulocyte-macrophage colony-stimulating factor. Although still not as effective as the conventional virus vaccine, the results encourage further work towards the development of a DNA vaccine against FMDV.

3C Viral Proteases↗

PubMed1998

Antiviral agent blocks breathing of the common cold virus.

A dynamic capsid is critical to the events that shape the viral life cycle; events such as cell attachment, cell entry, and nucleic acid release demand a highly mobile viral surface. Protein mass mapping of the common cold virus, human rhinovirus 14 (HRV14), revealed both viral structural dynamics and the inhibition of such dynamics with an antiviral agent, WIN 52084. Viral capsid digestion fragments resulting from proteolytic time-course experiments provided structural information in good agreement with the HRV14 three-dimensional crystal structure. As expected, initial digestion fragments included peptides from the capsid protein VP1. This observation was expected because VP1 is the most external viral protein. Initial digestion fragments also included peptides belonging to VP4, the most internal capsid protein. The mass spectral results together with x-ray crystallography data provide information consistent with a "breathing" model of the viral capsid. Whereas the crystal structure of HRV14 shows VP4 to be the most internal capsid protein, mass spectral results show VP4 fragments to be among the first digestion fragments observed. Taken together this information demonstrates that VP4 is transiently exposed to the viral surface via viral breathing. Comparative digests of HRV14 in the presence and absence of WIN 52084 revealed a dramatic inhibition of digestion. These results indicate that the binding of the antiviral agent not only causes local conformational changes in the drug binding pocket but actually stabilizes the entire viral capsid against enzymatic degradation. Viral capsid mass mapping provides a fast and sensitive method for probing viral structural dynamics as well as providing a means for investigating antiviral drug efficacy.

Antiviral Agents↗