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PubMed1984

Location of DNA-binding proteins and disulfide-linked proteins in vaccinia virus structural elements.

Treatment with sodium dodecyl sulfate (SDS) converted the vaccinia virus strain IHD-J into particles of two types: (i) ghosts which possessed a thin-membrane vesicle derived from basement part of the virus membrane with attached lateral bodies and a membranous structure derived from the core wall and (ii) aggregates of a DNA-nucleoprotein eluted from the core. These particles lacked lipids, and all the viral phospholipids were detected in the SDS-soluble fraction. The viral membrane was composed of an SDS-soluble coat layer and the basement membrane, and the basement membrane was maintained by a mechanism other than the lipid bilayer. By comparisons of protein species in morphologically distinct subviral particles prepared by several solubilizing methods, protein compositions of viral structural elements were suggested as follows: 25,000-molecular-weight viral protein-17,000-molecular-weight viral protein ( VP25K - VP17K ), viral basement membrane; VP13 . 8K , major component of the lateral body; VP70K , VP69K , VP66K , and VP64K , minor components of the lateral body; VP61K , outer layer of core wall; VP57K - VP22K , inner layer of core wall; and VP27K - VP13K , nucleoprotein. These structural elements found in the SDS-insoluble particles dissolved in the same SDS solution under reducing conditions, indicating that the disulfide linkages seem to have a principal role in maintaining their morphological integrity. VP57K , VP27K , VP13 . 8K , and VP13K were revealed to possess affinity for DNA. Denatured calf thymus DNA and viral DNA in double- or single-stranded form associated equally well with these proteins, but RNA did not bind. Therefore, it was strongly suggested that disulfide-linked VP27K - VP13K represented the nucleoproteins of vaccinia virus. A structural model of vaccinia virus is proposed and discussed.

Carcinoma↗

PubMed2002

New targets for antivirals: the ribosomal A-site and the factors that interact with it.

Many viruses use programmed -1 ribosomal frameshifting to ensure the correct ratio of viral structural to enzymatic proteins. Alteration of frameshift efficiencies changes these ratios, in turn inhibiting viral particle assembly and virus propagation. Previous studies determined that anisomycin, a peptidyl transferase inhibitor, specifically inhibited -1 frameshifting and the ability of yeast cells to propagate the L-A and M(1) dsRNA viruses (J. D. Dinman, M. J. Ruiz-Echevarria, K. Czaplinski, and S. W. Peltz, 1997, Proc. Natl. Acad. Sci. USA 94, 6606-6611). Here we show that preussin, a pyrollidine that is structurally similar to anisomycin (R. E. Schwartz, J. Liesch, O. Hensens, L. Zitano, S. Honeycutt, G. Garrity, R. A. Fromtling, J. Onishi, and R. Monaghan, 1988. J. Antibiot. (Tokyo) 41, 1774--1779), also inhibits -1 programmed ribosomal frameshifting and virus propagation by acting at the same site or through the same mechanism as anisomycin. Since anisomycin is known to assert its effect at the ribosomal A-site, we undertook a pharmacogenetic analysis of mutants of trans-acting eukaryotic elongation factors (eEFs) that function at this region of the ribosome. Among mutants of eEF1A, a correlation is observed between resistance/susceptibility profiles to preussin and anisomycin, and these in turn correlate with programmed -1 ribosomal frameshifting efficiencies and killer virus phenotypes. Among mutants of eEF2, the extent of resistance to preussin correlates with resistance to sordarin, an eEF2 inhibitor. These results suggest that structural features associated with the ribosomal A-site and with the trans-acting factors that interact with it may present a new set of molecular targets for the rational design of antiviral compounds.

Anisomycin↗

PubMed1980

Tumor antigen activity in viral DNA-protein complexes from cells productively infected with papovaviruses.

A polypeptide with a molecular weight in the range from 70,000 to 90,000 daltons, previously identified in DNA-protein complex isolated from polyoma virus- and SV40-infected cells, was isolated after deoxyribonuclease treatment of the complex followed by extensive dialysis and hydroxylapatite chromatography. The nonstructural protein was specifically retained by columns of protein A-Sepharose CL-4B onto which antibodies directed against SV40 T-antigen had been bound. T-antigen was unable to react with SV40-transformed cells in an immunofluorescence test. Finally the same antiserum reacted positively with the partially purified protein in the radioimmunoassay. These data are interpreted as indicating that, in productively infected cells, some of the nascent papovavirus DNA is complexed with T-antigen and viral structural polypeptides.

Animals↗

PubMed2002

Identification and characterization of a conserved baculoviral structural protein ODVP-6E/ODV-E56 from Choristoneura fumiferana granulovirus.

A gene that encodes a homologue to baculoviral ODVP-6E/ODV-E56, a baculoviral envelope-associated viral structural protein, has been identified and sequenced on the genome of Choristoneura fumiferana granulovirus (ChfuGV). The ChfuGV odvp-6e/odv-e56 gene was located on an 11-kb BamHI subgenomic fragment using different sets of degenerated primers, which were designed using the results of the protein sequencing of a major 39 kDa structural protein that is associated with the occlusion-derived virus (ODV). The gene has a 1062 nucleotide (nt) open-reading frame (ORF) that encodes a protein with 353 amino acids with a predicted molecular mass of 38.5 kDa. The amino acid sequence data that was derived from the nucleotide sequence in ChfuGV was compared to those of other baculoviruses. ChfuGV ODVP-6E/ODV-E56, along with other baculoviral ODVP-6E/ODV-E56 proteins, all contained two putative transmembrane domains at their C-terminus. Several putative N- and O-glycosylation, N-myristoylation, and phosphorylation sites were detected in the ChfuGV ODVP-6E/ODV-E56 protein. A similar pattern was detected when a hydrophobicity-plots comparison was performed on ChfuGV ODVP-6E/ODV-E56 with other baculoviral homologue proteins. At the nucleotide level, a late promoter motif (GTAAG) was located at -14 nt upstream to the start codon of the ChfuGV odvp-6e/odv-e56 gene. A slight variant of the polyadenylation signal, AATAAT, was detected at the position +10 nt that is downstream from the termination signal. A phylogenetic tree for baculoviral ODVP-6E/ODV-E56 was constructed using a maximum parsimony analysis. The phylogenetic estimation demonstrated that ChfuGV ODVP-6E/ODV-E56 is most closely related to those of Cydia pomonella granulovirus (CpGV) and Plutella xylostella granulovirus (PxGV).

Amino Acid Sequence↗

PubMed2001

Targeted site-specific cleavage of HIV-1 viral Rev responsive element by copper aminoglycosides.

Site-specific cleavage of the HIV-1 viral Rev responsive element by copper aminoglycosides is reported under physiological conditions. This bubble and stem-loop RNA structure is efficiently targeted at micromolar concentrations of complex. The specificity of cleavage of structured viral RNA relative to a non-cognate tRNAPhe of well-defined secondary and tertiary structure is demonstrated. Cleavage products from simpler substrates [diribonucleotide (ApA) and 2',3'-cyclic monophosphate ester (cAMP)] were analyzed by 31P NMR and demonstrate a hydrolytic mechanism in the absence of external redox agents. These results demonstrate copper aminoglycosides to be highly efficient chemical nucleases with a targeting capability for viral RNA and suggest a novel methodology to counter RNA viruses.

Aminoglycosides↗

PubMed2000

Seroprevalence of parvovirus B19 NS1-specific IgG in B19-infected and uninfected individuals and in infected pregnant women.

Parvovirus B19 is the causative agent of erythema infectiosum in children, but the virus is associated with an increasing range of different diseases. These include acute and chronic arthritis, hydrops fetalis in pregnant women, aplastic anemia, and thrombocytopenia. The host's immune response is directed against the viral structural proteins VP1 and VP2. This study investigated the presence of IgG against the viral nonstructural protein NS1 using Western blot. Serum panels from healthy individuals, B19-infected pregnant women, and various disease groups were tested. The disease groups included patients with symptoms that may be linked to parvovirus B19 infection. The results showed that IgG against the NS1 protein was present in 22% of healthy individuals with past B19 infection. In cases of persistent or prolonged B19 infections, the prevalence of NS1-specific antibodies was as high as 80%. It is concluded that NS1-specific IgG may be used as an indicator of chronic or more severe courses of parvovirus B19 infections.

Adolescent↗

PubMed2004

Parvovirus H-1 infection of human glioma cells leads to complete viral replication and efficient cell killing.

The extremely poor prognosis of malignant gliomas requires the investigation of other than standard therapies, i.e., the application of oncolytic viruses. In our study, we evaluated the effects of the oncosuppressive parvovirus H-1 on different established glioblastoma cell lines of rat and human origin and on short-term/low-passage cultures of human glioblastoma cells. We observed an efficient and dose-dependent killing of all glioma cell cultures at low multiplicities of infectious particles (MOI) per cell. Southern blot analysis of viral DNA amplification, RT-PCR analysis of viral RNA expression and Western blot analysis of the expression of viral structural (VP-1/VP-2) and nonstructural (NS-1) proteins demonstrated the biosynthesis of these viral macromolecular components in all of the cultures. Moreover, all the glioma cells were proficient for the production of infectious H-1 virus particles. The amount of virus production differed between a several fold increase of the input virus titer in most of the short-term/low-passage cultures up to 1,000-fold in one short-term glioma and in the rat cells. Glioma cells lines and, more importantly, short-term/low-passage cultures of human glioblastomas were found to be highly susceptible target cells for H-1 virus mediated cytotoxicity. The formation of fully infectious progeny particles in infected glioma cells offers the chance for the induction of secondary rounds of infection resulting in an advanced cytotoxic effect. These advantageous characteristics of H-1 virus infection of glioma cells, combined with the known low toxicity of H-1 virus in nontransformed cells, make parvovirus H-1 a promising candidate for oncolytic glioma therapy.

Animals↗

PubMed1997

Methyltransferase activity of the insect orbivirus JKT-7400: photoaffinity labeling of a minor virion protein, VP4, with S-adenosylmethionine.

JKT-7400 virus is an orbivirus originally isolated from Culex mosquitoes. In earlier work we had described the viral structural proteins and presented evidence suggesting that a minor protein, VP6, located in the viral core was the viral guanylyltransferase. We now show that gradient-purified JKT-7400 virions possess a methyltransferase (MTase) activity which can use GTP or GDP as the methyl acceptor. The apparent Km of the MTase for S-adenosylmethionine (AdoMet) was 25 microM. Photoaffinity labeling experiments in which 3H-[methyl]-AdoMet was incubated with virions or viral cores demonstrated labeling of VP4, a minor protein present in the viral core, suggesting that this protein is the viral MTase. Labeling of VP4 was inhibited by addition of unlabeled AdoMet or S-adenosylhomocysteine (AdoHcy).

Aedes↗

PubMed2001

High amplification of a densovirus-derived vector in larval and adult tissues of Drosophila.

The Lepidopteran densovirus-derived vector, pJlacZDeltaNS3, is a defective virus genome with an insertion of lacZ DNA in the viral structural protein coding sequence, and a deletion of the sequence coding the non-structural polypeptide NS3. pJlacZDeltaNS3 was injected into Drosophila eggs and the maintenance of the viral genome was monitored by expression of beta-galactosidase and by Southern blot hybridizations. Intense beta-galactosidase activity was observed in many somatic tissues of third-instar larvae and adult flies, in more than 60% of the injected animals. DNA analyses showed that staining in adult tissues correlated with the amplification of the vector. Together, these results suggest the occurrence of early events of integration of the vector into the Drosophila host genome.

Animals↗

PubMed1997

Protection of primary human T cells from HIV infection by Trev: a transdominant fusion gene.

Gene therapy is one of several approaches that are being tested in the search for an effective anti-human immunodeficiency virus (HIV) treatment. In this strategy, a "protective" gene would be introduced into target cells, rendering them relatively resistance to the virus-induced cytopathicity. Tat and Rev are viral proteins essential for HIV gene expression. Tat increases viral gene transcription and Rev is responsible for the nuclear export of mRNA encoding structural viral proteins. A fusion protein (Trev) was constructed, joining Tat and Rev transdominant mutant gene sequences. Previously, we showed that Trev inhibits both Tat and Rev activities in Jurkat T cells. To determine whether Trev could inhibit HIV replication in primary cells, we transferred the trev gene to peripheral blood lymphocytes and challenged them with different HIV strains. Levels of HIV p24 antigen (Ag) were reduced 4- to 15-fold in cultures of Trev-CD4+ T cells infected with two HIV primary clinical isolates and were not detectable in cultures infected with HIV strains NL4-3 and SF2. In contrast, cultures of nontransduced CD4+ T cells infected with the same viruses had levels of HIV p24 Ag up to 10 ng/ml. Trev-transduced CD4+ T cells demonstrated increased survival following HIV challenge for the length of the experiments (30 days). We did not observe rapid emergence of Trev-resistant HIV in our cultures. Following HIV challenge, cell-associated Trev protein was increased, supporting the hypothesis that cells surviving Trev expression provided a cell survival advantage. This work showed that Trev was able to inhibit HIV replication in primary CD4+ T cells, and, therefore the trev gene could be a candidate for gene therapy against HIV.

CD4-Positive T-Lymphocytes↗

PubMed1988

Virus-lymphocyte interactions. II. Expression of viral sequences during the course of persistent lymphocytic choriomeningitis virus infection and their localization to the L3T4 lymphocyte subset.

Viruses that cause in vivo persistent infections need to selectively compromise the host's immunologic surveillance machinery in order to survive. To understand the molecular basis of how this is accomplished we have analyzed persistent virus infection by lymphocytic choriomeningitis in its normal host, the mouse. Earlier we noted by infectious center analysis that five in 10(4) lymphocytes carried by persistently infected mice contained infectious materials throughout the course of infection. A previous publication extended these results, in BALB mice by showing that the L3T4+ lymphocyte subset in lymph nodes and spleens was predominantly involved. Using cDNA labeled probes to the viral genome and in situ hybridization we report that 1 to 2% of circulating lymphocytes from several mouse strains contain viral RNA sequences for the three viral structural genes. By FACS analysis, the Thy-1.2+, L3T4+ subset primarily harbors virus while viral sequences are usually not detected in the Lyt-2+ subset as early as 6 days after initiating infection in newborns and throughout the course of the persistence. These findings suggest that incomplete, presumably defective, virus is generated in a subset of Th lymphocytes during persistent infection and that during this time infection of cytotoxic T cell subsets is minimal.

Animals↗

PubMed1977

Frog oocytes synthesize and completely process the precursor polypeptide to virion structural proteins after microinjection of avian myeloblastosis virus RNA.

After microinjection of Xenopus laevis oocytes with RNA from avian myeloblastosis virus, viral structural proteins p27, p19, p15, and p12 are formed by a sequence of posttranslational cleavages of a high-molecular-weight precursor polypeptide. The 60-70S RNA aggregate or its 30-40S RNA subunits obtained by heat or formamide treatment possess the same ability to serve as template in X. laevis oocytes. The processing pattern of virus-specific precursor polypeptides is the same in X. laevis oocytes as in chick embryo fibroblasts infected with avian myeloblastosis virus, but the processing takes place at a much slower rate.

Animals↗

PubMed2003

Functional domain structure of human T-cell leukemia virus type 2 rex.

The Rex protein of human T-cell leukemia virus (HTLV) acts posttranscriptionally to induce the cytoplasmic expression of the unspliced and incompletely spliced viral RNAs encoding the viral structural and enzymatic proteins and is therefore essential for efficient viral replication. Rex function requires nuclear import, RNA binding, multimerization, and nuclear export. In addition, it has been demonstrated that the phosphorylation status of HTLV-2 Rex (Rex-2) correlates with RNA binding and inhibition of splicing in vitro. Recent mutational analyses of Rex-2 revealed that the phosphorylation of serine residues 151 and 153 within a novel carboxy-terminal domain is critical for function in vivo. To further define the functional domain structure of Rex-2, we evaluated a panel of Rex-2 mutants for subcellular localization, RNA binding capacity, multimerization and trans-dominant properties, and the ability to shuttle between the nucleus and the cytoplasm. Rex-2 mutant S151A,S153A, which is defective in phosphorylation and function, showed diffuse cytoplasmic staining, whereas mutant S151D,S153D, previously shown to be functional and in a conformation corresponding to constitutive phosphorylation, displayed increased intense speckled staining in the nucleoli. In vivo RNA binding analyses indicated that mutant S151A,S153A failed to efficiently bind target RNA, while its phosphomimetic counterpart, S151D,S153D, bound twofold more RNA than wild-type Rex-2. Taken together, these findings provide direct evidence that the phosphorylation status of Rex-2 is linked to cellular trafficking and RNA binding capacity. Mutants with substitutions in either of the two putative multimerization domains or in the putative activation domain-nuclear export signal displayed a dominant negative phenotype as well as defects in multimerization and nucleocytoplasmic shuttling. Several carboxy-terminal mutants that displayed wild-type levels of phosphorylation and localized to the nucleolus were also partially impaired in shuttling. This is consistent with the hypothesis that the carboxy terminus of Rex-2 contains a novel domain that is required for efficient shuttling. This work thus provides a more detailed functional domain map of Rex-2 and further insight into its regulation of HTLV replication.

Base Sequence↗

PubMed1995

The crystal structure of bluetongue virus VP7.

Bluetongue virus (BTV), a representative of the orbivirus genus of the Reoviridae, is considerably larger (at 80 nm across), and structurally more complex, than any virus for which we have comprehensive structural information. Orbiviruses infect mammalian hosts through insect vectors and cause economically important diseases of domesticated animals. They possess a segmented double-stranded RNA genome within a capsid composed of four major types of polypeptide chains. An outer layer of VP2 and VP5 is removed as the virus enters the target cell, to leave an intact core within the cell. This core is 70 nm across and composed of 780 copies of VP7 (M(r) 38K) that, as trimers, form 260 'bristly' capsomeres clothing an inner scaffold constructed from VP3 (M(r) 103K). We report here the crystal structure of VP7 from BTV serotype 10, which reveals a molecular architecture not seen previously in viral structural proteins. Each subunit consists of two domains, one a beta-sandwich, the other a bundle of alpha-helices, and a short carboxy-terminal arm which might tie trimers together during capsid formation. A concentration of methionine residues at the core of the molecule could provide plasticity, relieving structural mismatches during assembly.

Amino Acid Sequence↗

PubMed1999

Crystal structure of Escherichia coli uracil DNA glycosylase and its complexes with uracil and glycerol: structure and glycosylase mechanism revisited.

The DNA repair enzyme uracil DNA glycosylase (UDG) catalyzes the hydrolysis of premutagenic uracil residues from single-stranded or duplex DNA, producing free uracil and abasic DNA. Here we report the high-resolution crystal structures of free UDG from Escherichia coli strain B (1.60 A), its complex with uracil (1.50 A), and a second active-site complex with glycerol (1.43 A). These represent the first high-resolution structures of a prokaryotic UDG to be reported. The overall structure of the E. coli enzyme is more similar to the human UDG than the herpes virus enzyme. Significant differences between the bacterial and viral structures are seen in the side-chain positions of the putative general-acid (His187) and base (Asp64), similar to differences previously observed between the viral and human enzymes. In general, the active-site loop that contains His187 appears preorganized in comparison with the viral and human enzymes, requiring smaller substrate-induced conformational changes to bring active-site groups into catalytic position. These structural differences may be related to the large differences in the mechanism of uracil recognition used by the E. coli and viral enzymes. The pH dependence of k(cat) for wild-type UDG and the D64N and H187Q mutant enzymes is consistent with general-base catalysis by Asp64, but provides no evidence for a general-acid catalyst. The catalytic mechanism of UDG is critically discussed with respect to these results.

Amino Acid Sequence↗

PubMed1983

Nucleotide sequence to the v-myc oncogene of avian retrovirus MC29.

Avian myelocytomatosis viruses are retroviruses whose oncogene (v-myc) induces an unusually wide variety of tumors, including carcinomas, endotheliomas, sarcomas, and myelocytomatoses. The viral gene v-myc arose by transduction of an undetermined portion of a cellular gene known as c-myc. In order to facilitate further studies of the functions of v-myc and c-myc and to permit detailed comparisons between the two genes, we have determined the nucleotide sequence of v-myc in the genome of the MC29 strain of myelocytomatosis virus. The v-myc domain in MC29 virus encodes a hydrophilic polypeptide with a molecular weight of 47,000, fused to a portion of the polyprotein encoded by the viral structural gene gag. The carboxyl-terminal half of the v-myc polypeptide is rich in basic amino acid residues. This feature may account for the DNA-binding properties of the hybrid gag-myc-encoded protein which would have a molecular weight of approximately 100,000, in accord with results from previous studies of the protein encoded by v-myc. The junctions between v-myc and the genome of the transducing virus are apparent but reveal no clues to the mechanism by which transduction might occur.

Amino Acid Sequence↗

PubMed1969

Filamentous structures of type 2 Herpesvirus hominis infection of the chorioallantoic membrane.

In view of recent recognition of the existence of two Herpesvirus hominis (HVH) types with antigenic and biological differences, an electron microscopic study was undertaken of pocks produced on the choriallantoic membrane of embryonated eggs after infection with type 1 and type 2 HVH strains. Besides the typical morphological features of herpesvirus infection noted by several investigators, it was observed that type 2 HVH also produced microtubules measuring approximately 19 nm in both nucleus and cytoplasm. Although the nature of these filamentous structures is still unclear, consideration is given in this paper to the possibility that they may represent viral structural subunits, aberrant forms or neoantigens.

Animals↗

PubMed1999

Modulation of the RNA binding and protein processing activities of poliovirus polypeptide 3CD by the viral RNA polymerase domain.

To study the role of the RNA polymerase domain (3D) in the proteinase substrate recognition and RNA binding properties of poliovirus polypeptide 3CD, we generated recombinant 3C and 3CD polypeptides and purified them to near homogeneity. By using these purified proteins in in vitro cleavage assays with structural and non-structural viral polyprotein substrates, we found that 3CD processes the poliovirus structural polyprotein precursor (P1) 100 to 1000 times more efficiently than 3C processes P1. We also found that trans-cleavage of other 3CD molecules and sites within the non-structural P3 precursor is more efficiently mediated by 3CD than 3C. However, 3C and 3CD appear to be equally efficient in the processing of a non-structural polyprotein precursor, 2C3AB. Four mutated 3CD polyproteins with site-directed lesions in the 3D domain of the proteinase were analyzed for their ability to process viral polyprotein precursors and to form a ternary complex with RNA sequences encoded in the 5' terminus of the viral genome. Analysis of mutated 3CD polypeptides revealed that specific mutations within the 3D amino acid sequences of 3CD confer differential effects on 3CD activity. All four mutated 3CD proteins tested were able to process the P1 structural precursor with wild type or near wild type efficiency. However, three of the mutated enzymes demonstrated an impaired ability to process some sites within the P3 non-structural precursor, relative to wild type 3CD. One of the mutant 3CD polypeptides, 3CD-3DK127A, also displayed a defect in its ability to form a ternary ribonucleoprotein complex with poliovirus 5' RNA sequences.

3C Viral Proteases↗