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At least 19 recordsLinked to original sources
PubMed2004

Conservation of the DNA sequences encoding the major structural viral proteins of WSSV.

A cDNA library was constructed from white spot syndrome virus (WSSV)-infected penaeid shrimp tissue. cDNA clones with WSSV inserts were isolated and sequenced. By comparison with DNA sequences in GenBank, cDNA clones containing sequence identical to those of the WSSV envelope protein VP28 and nucleoprotein VP15 were identified. Poly(A) sites in the mRNAs of VP28 and VP15 were identified. Genes encoding the major viral structural proteins VP28, VP26, VP24, VP19 and VP15 of 5 WSSV isolates collected from different shrimp species and/or geographical areas were sequenced and compared with those of 4 other WSSV isolate sequences in GenBank. For each of the viral structural protein genes compared, the nucleotide sequences were 100 to 99% identical among the 9 isolates. Gene probes or PCR primers based on the gene sequences of the WSSV structural proteins can be used for diagnoses and/or detection of WSSV infection.

Animals↗
PubMed1986

Demonstration of a unique viral structure: the molluscum viral colony sac.

Using scanning electron microscopy to study molluscum contagiosum lesions, it has been possible to demonstrate a unique, well defined sac enclosing the virion colony of each infected keratinocyte. This confirms the presence of a structure demonstrated over 50 years ago by micro-dissection, but ignored since it is not seen on either light or transmission electron microscopy. It is postulated that this sac, of unknown origin, favours replication of the virions by providing a site that is both anatomically and immunologically privileged. Such relatively unimpeded growth of the virions results in an enlarged cell in which the cytoplasm and nucleus are compressed into a thin outer shell. In the aggregate, this produces a tumour of virus-packed cells rather than a tumour composed of virus-induced cell hyperplasia.

Adolescent↗
PubMed2005

The unique stacked rings in the nucleocapsid of the white spot syndrome virus virion are formed by the major structural protein VP664, the largest viral structural protein ever found.

One unique feature of the shrimp white spot syndrome virus (WSSV) genome is the presence of a giant open reading frame (ORF) of 18,234 nucleotides that encodes a long polypeptide of 6,077 amino acids with a hitherto unknown function. In the present study, by applying proteomic methodology to analyze the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of purified WSSV virions by liquid chromatography-mass spectrometry (LC-MS/MS), we found that this giant polypeptide, designated VP664, is one of the viral structural proteins. The existence of the corresponding 18-kb transcript was confirmed by sequencing analysis of reverse transcription-PCR products, which also showed that vp664 was intron-less. A time course analysis showed that this transcript was actively transcribed at the late stage, suggesting that this gene product should contribute primarily to the assembly and morphogenesis of the virion. Several polyclonal antisera against this giant protein were prepared, and one of them was successfully used for immunoelectron microscopy analysis to localize the protein in the virion. Immunoelectron microscopy with a gold-labeled secondary antibody showed that the gold particles were regularly distributed around the periphery of the nucleocapsid with a periodicity that matched the characteristic stacked ring subunits that appear as striations. From this and other evidence, we argue that this giant ORF in fact encodes the major WSSV nucleocapsid protein.

Amino Acid Sequence↗
PubMed2005

New insights into viral structure and virus-cell interactions through proteomics.

Although genomics techniques such as DNA microarrays have been widely used in virology, much more limited use has been made of proteomics. Although difficult, proteomics can greatly contribute to an understanding of virus-cell interactions, including the ternary structure of viral receptors at the cell surface, post-translational modifications and isoforms of critical viral and cellular proteins and even to the structure of viruses. Proteomics techniques also offer the potential for discovering markers for diagnostic and prognostic tests of viral infections in vivo. This review describes the use of several proteomic approaches for the analysis of HIV-cellular receptor interactions, the molecular mechanisms of transport of herpes simplex virus within neurons, and the structure of the tegument of herpes simplex virus.

Animals↗
PubMed1990

Dot hybridization detection of plum pox virus using 32P-labeled RNA probes representing non-structural viral protein genes.

A cDNA library covering the complete genome of plum pox virus strain D (PPV D) has been obtained, and an endonuclease restriction map derived from it. This map was superposed on the PPV genomic organisation map, established for a nonaphid transmissible strain of PPV (Maiss et al., 1989). This allowed us to select seven probes, corresponding to different regions on the PPV genome. These probes were tested in a dot-blot hybridization assay for the detection of PPV. Probes of various lengths (0.25 to 1.5 kb) were tested and those measuring at least 0.8 kb (4 of the 7 probes selected) proved to be the most sensitive. The detection limit was of about 5 pg of purified virus per assay. Probes representing non-structural viral protein genes were equally sensitive in detecting both serotypes D and M of PPV. The previously described probe pBPPV1 (Varveri et al., 1988), covering the coat protein gene of strain D, was less sensitive, when compared to the above probes, in detecting heterologous strains of PPV. The polyvalence of probes transcribed from non-structural viral protein genes was confirmed by screening isolates of PPV, collected in infected orchards in several Mediterranean countries.

Autoradiography↗
PubMed1998

Human MxA protein confers resistance to Semliki Forest virus and inhibits the amplification of a Semliki Forest virus-based replicon in the absence of viral structural proteins.

Mx proteins form a small family of interferon (IFN)-induced GTPases with potent antiviral activity against various negative-strand RNA viruses. To examine the antiviral spectrum of human MxA in homologous cells, we stably transfected HEp-2 cells with a plasmid directing the expression of MxA cDNA. HEp-2 cells are permissive for many viruses and are unable to express endogenous MxA in response to IFN. Experimental infection with various RNA and DNA viruses revealed that MxA-expressing HEp-2 cells were protected not only against influenza virus and vesicular stomatitis virus (VSV) but also against Semliki Forest virus (SFV), a togavirus with a single-stranded RNA genome of positive polarity. In MxA-transfected cells, viral yields were reduced up to 1,700-fold, and the degree of inhibition correlated well with the expression level of MxA. Furthermore, expression of MxA prevented the accumulation of 49S RNA and 26S RNA, indicating that SFV was inhibited early in its replication cycle. Very similar results were obtained with MxA-transfected cells of the human monocytic cell line U937. The results demonstrate that the antiviral spectrum of MxA is not restricted to negative-strand RNA viruses but also includes SFV, which contains an RNA genome of positive polarity. To test whether MxA protein exerts its inhibitory activity against SFV in the absence of viral structural proteins, we took advantage of a recombinant vector based on the SFV replicon. The vector contains only the coding sequence for the viral nonstructural proteins and the bacterial LacZ gene, which was cloned in place of the viral structural genes. Upon transfection of vector-derived recombinant RNA, expression of the beta-galactosidase reporter gene was strongly reduced in the presence of MxA. This finding indicates that viral components other than the structural proteins are the target of MxA action.

3T3 Cells↗
PubMed2000

Nucleotide sequences of genome segments 6 and 7 of Bombyx mori cypovirus 1, encoding the viral structural proteins V4 and V5, respectively.

Nucleotide sequence analyses of cDNAs derived from the double-stranded RNA genome segments 6 and 7 (S6 and S7) of Bombyx mori cypovirus 1 (BmCPV-1) have revealed that they consist of 1796 and 1501 nucleotides encoding putative proteins of 561 and 448 amino acids with molecular masses of 63604 and 49875 (p64 and p50), respectively. The amino acid sequence of p64, which has a high leucine residue content (10%), contains a leucine zipper motif. Antiserum raised against p64 specifically bound to a viral structural protein of ca. 68 kDa (V4), while antiserum against p50, which specifically bound to a protein of ca. 56 kDa in BmN4 cells infected with BmCPV-1, reacted with a cluster of four viral structural proteins ranging from ca. 34 to 40 kDa (V5). These observations indicate that p50 might be cleaved to V5 during the formation of virus particles.

Amino Acid Sequence↗
PubMed1984

Subcellular distribution of viral structural proteins during simian virus 40 infection.

The amounts of simian virus 40 structural polypeptides Vp1, Vp2, and Vp3 in different subcellular fractions at various times after lytic infection were determined by a quantitative immunoblotting procedure. Simian virus 40-infected cells were lysed with a buffer containing Nonidet P-40 to yield a soluble fraction. The Nonidet P-40-insoluble fraction was further fractionated in the presence of deoxycholate and Tween 40 to yield a soluble fraction (cytoskeletal) and an insoluble fraction (Nuc), which is primarily cell nuclei. At 33 h postinfection, the majority of viral structural proteins was found in the cell nucleus, whereas, at 48 to 65 h postinfection, Vp1 was distributed evenly among all cell fractions and Vp2 and Vp3 were found predominantly in the cytoskeletal and Nuc fractions. Thus, not all of the viral polypeptides synthesized in the cytoplasm migrated into the cell nucleus. Throughout infection, the molar ratio (Vp3/Vp2) was rather constant in all subcellular fractions, indicating that the synthesis or processing or both of Vp2 and Vp3 are coordinately regulated. The molar ratio of Vp1/(Vp2 + Vp3) varied among the fractions. The Vp1/(Vp2 + Vp3) molar ratio in the soluble fraction varied during the course of infection; however, constant ratios were maintained in the cytoskeletal and Nuc fractions. Thus, the mechanism which controls the movement of Vp1 to different compartments of the cell appears to be different from that of Vp2 and Vp3. The Vp1/(Vp2 + Vp3) value in the Nuc fraction was similar to the ratio found in virus particles. The constant molar distribution of Vp1, Vp2, and Vp3 in the Nuc fraction throughout infection suggests that there is a specific mechanism which regulates the transport of viral structural proteins. These results support the hypothesis that the structural proteins of simian virus 40 are transported into the cell nucleus in precise proportions.

Animals↗
PubMed1982

Localization of viral structural proteins in the cytoplasm and nucleus of Rous-associated virus-2-infected chicken embryo fibroblasts.

The cellular location of viral structural proteins was carried out by immunohistochemistry and by cell fractionation. Antibody against the structural protein p27 was used in immunohistochemical reactions to demonstrate the presence of viral proteins in the cytoplasm and nucleus of Rous-associated virus 2-infected chicken cells. Localization in the nucleus was found over heterochromatic regions; in the cytoplasm it was found in discrete particulate structures. These observations were extended in cell fractionation studies in which cytoplasmic and nuclear fractions were immunoprecipitated with antibody against the viral structural proteins.

Animals↗
PubMed1995

Replication and amplification of novel vesicular stomatitis virus minigenomes encoding viral structural proteins.

We have developed a system in which vesicular stomatitis virus (VSV) minigenomes encoding viral structural proteins can be expressed from plasmids. These RNAs can be replicated, transcribed, and packaged into infectious particles when coexpressed with the other VSV proteins. The minigenomes contain either the glycoprotein (G protein) gene (GMG [stands for G minigenome]) or both the G and matrix (M) protein genes (GMMG [stands for G/M minigenome]) from the Indiana serotype of VSV flanked by the trailer and leader regions from the wild-type VSV genome. Northern (RNA) blot analysis showed that the minigenome RNAs were replicated and that a positive-sense replicative intermediate was synthesized when coexpressed with the nucleocapsid (N) protein and the two VSV polymerase proteins (phosphoprotein [P] and the large catalytic subunit [L]) in vivo. In addition, functional mRNAs were transcribed from the minigenome templates, and the appropriate encoded proteins were expressed. Expression of the G and M proteins from GMMG resulted in the assembly and release of infectious particles that could be passaged on cells expressing the N, P, and L proteins only. Amplification occurred during successive passages, and after four passages approximately 30% of the cells expressed both the G and M proteins. Analysis of the RNAs produced in the GMMG-infected cells also showed that the minigenomes accurately reproduced all of the replicative and transcriptional events that normally occur in a VSV-infected cell. GMMG is therefore a novel type of defective particle which encodes functional viral proteins critical to its own propagation.

Animals↗
PubMed1991

Antibody response to the negative regulatory factor (nef) in experimentally infected macaques: correlation with viremia, disease progression, and seroconversion to structural viral proteins.

The antibody response to structural and regulatory viral proteins was studied in 14 rhesus (Macaca mulatta) and 6 cynomolgus (Macaca fascicularis) macaques experimentally infected with HIV-2 or SIVMAC. To investigate the humoral antibody response to the negative regulatory factor (nef), the recombinant protein was expressed to high levels with recombinant vaccinia virus (VV). nef-specific antibodies were detected in 14 of 20 infected macaques (70%). In sera of all infected monkeys antibodies directed to the structural proteins gp120, p56, and p24 appeared 2 to 6 weeks postinfection. In contrast, the extent and the appearance of nef-specific antibodies during the course of infection varied considerably between individual animals. However, only in sera of four animals (20%) were nef-specific antibodies detectable as early as those against the core proteins p24 and p56. In SIVMAC-infected rhesus macaques at different clinical stages, the antibody response towards nef neither correlated with the development of viral latency nor to disease progression or viremia. Our data indicate that in macaques experimentally infected with SIV or HIV-2 antibody formation against nef is not a useful diagnostic marker either for early detection of viral infection or of disease progression.

AIDS-Related Complex↗
PubMed1990

Visna virus encodes a post-transcriptional regulator of viral structural gene expression.

Visna virus is an ungulate lentivirus that is distantly related to the primate lentiviruses, including human immunodeficiency virus type 1 (HIV-1). Replication of HIV-1 and of other complex primate retroviruses, including human T-cell leukemia virus type I (HTLV-I), requires the expression in trans of a virally encoded post-transcriptional activator of viral structural gene expression termed Rev (HIV-1) or Rex (HTLV-I). We demonstrate that the previously defined L open reading frame of visna virus encodes a protein, here termed Rev-V, that is required for the cytoplasmic expression of the incompletely spliced RNA that encodes the viral envelope protein. Transactivation by Rev-V was shown to require a cis-acting target sequence that coincides with a predicted RNA secondary structure located within the visna virus env gene. However, Rev-V was unable to function by using the structurally similar RNA target sequences previously defined for Rev or Rex and, therefore, displays a distinct sequence specificity. Remarkably, substitution of this visna virus target sequence in place of the HIV-1 Rev response element permitted the Rev-V protein to efficiently rescue the expression of HIV-1 structural proteins, including Gag, from a Rev- proviral clone. These results suggest that the post-transcriptional regulation of viral structural gene expression may be a characteristic feature of complex retroviruses.

Animals↗
PubMed2006

Viral structural transitions: an all-atom multiscale theory.

An all-atom theory of viral structural transitions (STs) is developed based on a multiscale analysis of the N-atom Liouville equation. The approach yields an understanding of viral STs from first principles and a calibrated interatomic force field. To carry out the multiscale analysis, we introduce slow variables characterizing the whole-virus dynamics. Use of the "nanocanonical ensemble" technique and the fundamental hypothesis of statistical mechanics (i.e., the equivalence of long-time and ensemble averages) is shown to imply a Fokker-Planck equation yielding the coarse-grained evolution of the slow variables. As viral STs occur on long time scales, transition state theory is used to estimate the energy barrier of transition between free energy wells implied by observed hysteresis in viral STs. Its application to Nudaurelia capensis omega virus provides an upper bound on the free energy barrier when a single dilatational order parameter is used. The long time scale of viral STs is shown to follow from the aggregate effect of inertia, energy barrier, and entropic effects. Our formulation can be generalized for multiple order parameter models to account for lower free energy barrier pathways for transition. The theory with its all-atom description can be applied to nonviral nanoparticles as well.

Computer Simulation↗
PubMed1988

Nucleotide sequence of dengue type 3 virus genomic RNA encoding viral structural proteins.

Complementary DNAs to the 5' proximal region of the dengue virus type 3 RNA were cloned into bacterial plasmids and the nucleotide sequence of 3,000 bases from the 5' terminus of the genome were determined by DNA and RNA sequencing methods using dideoxy chain-termination reactions. Comparison of the nucleotide sequence thus obtained with those of other flavivirus genomes revealed significant homology existing in nucleotide sequence of the flavivirus genomes. When we compared amino acid sequence deduced from the nucleotide sequence with those of other flaviviruses, this genome region was found to include sequences encoding three viral structural proteins C, M, and E and a part of the viral nonstructural protein NS1 in this order in addition to the 5'-noncoding sequence. The characteristics and functions of these proteins were discussed based on the deduced amino acid sequences and their hydrophobic profiles. The genetic relationship of flaviviruses was also discussed based on the genetic variation observed in their genomes.

Amino Acid Sequence↗
PubMed1996

Functions of the 5' leader of murine leukemia virus genomic RNA in virion structure, viral replication and pathogenesis, and MLV-derived vectors.

Retroviruses are a family of widespread small animal viruses that can cause a variety of neoplastic and immunosuppressive diseases. Murine leukemia viruses (MuLV) have been used as model systems to investigate virion and genomic RNA structure, viral replication and variability, and pathogenesis. Detailed knowledge of the genetic structure of MuLV and of the viral life cycle has led to the development of MuLV-derived retroviral vectors for gene transfer with potential applications in human gene therapy. In this review we have summarized the properties and functions of the 5' domain, called the leader, of MuLV genomic RNA. The 5' leader is formed of small interspersed and superimposed genetic elements involved in every step of the viral life cycle. In addition, the 3' domain of the leader encodes the N-terminal part of glycosylated forms of the GAG polyprotein, also named Gross cell surface antigen (GCSA or glycoGAG) which is essential for full spreading and pathogenic abilities of the virus in the animal. Therefore, the 5' leader of MuLV genomic RNA appears to be a very attractive model to study structure-function relationships of a small and multifunctional genetic domain in vitro, in cell culture and in the animal.

Gene Transfer Techniques↗
PubMed2004

Analysis of baculovirus IE1 in living cells: dynamics and spatial relationships to viral structural proteins.

IE1, a principal transcriptional activator of the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV), is an essential factor for viral DNA replication. During viral infection, IE1 accumulates in discrete subnuclear structures where viral DNA replication occurs. To analyse the dynamic properties of IE1, we monitored green fluorescent protein-tagged IE1 (IE1-GFP) in BmNPV-infected B. mori cells by live-cell microscopy. Time-lapse imaging showed that IE1-associated structures gradually expanded and occasionally fused with one another, while photobleaching experiments revealed that IE1-GFP was relatively immobile inside the IE1-associated structures. To investigate the spatial relationships between IE1 and viral structural proteins in infected cells, three GFP-tagged viral components were expressed together with DsRed-tagged IE1. Two structural proteins that constitute the occlusion-derived virus (ODV), P91-GFP and GFP-ODV-E25, localized to the periphery of the IE1-associated structures. While local accumulations of these proteins were often in contact with the IE1-associated structures, they did not extend beyond the boundaries of the structures. In contrast, the major capsid protein VP39-GFP predominantly accumulated within the IE1-associated structures. These data indicated, in conjunction with the finding of a high DNA content in the structures, that IE1 localizes to the virogenic stroma and therefore support the prediction previously proposed that the virogenic stroma is a site for viral DNA replication as well as for the assembly of nucleocapsids.

Animals↗
PubMed2002

Aura virus structure suggests that the T=4 organization is a fundamental property of viral structural proteins.

Aura and Sindbis viruses are closely related alphaviruses. Unlike other alphaviruses, Aura virus efficiently encapsidates both genomic RNA (11.8 kb) and subgenomic RNA (4.2 kb) to form virus particles. Previous studies on negatively stained Aura virus particles predicted that there were two major size classes with potential T=3 and T=4 capsid structures. We have used cryoelectron microscopy and three-dimensional image reconstruction techniques to examine the native morphology of different classes of Aura virus particles produced in BHK cells. Purified particles separated into two components in a sucrose gradient. Reconstructions of particles in the top and bottom components were computed to resolutions of 17 and 21 A, respectively, and compared with reconstructions of Sindbis virus and Ross River virus particles. Aura virus particles of both top and bottom components have similar, T=4 structures that resemble those of other alphaviruses. The morphology of Aura virus glycoprotein spikes closely resembles that of Sindbis virus spikes and is detectably different from that of Ross River virus spikes. Thus, some aspects of the surface structure of members of the Sindbis virus lineage have been conserved, but other aspects have diverged from the Semliki Forest/Ross River virus lineage.

Alphavirus↗