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PubMed1969

Kinetics of incorporation of structural proteins into Sindbis virions.

The morphogenesis of Sindbis virus was studied by determining the kinetics with which newly synthesized nucleocapsid and envelope proteins appeared in virions released into the extracellular medium. Assembly of the nucleocapsid was more rapid than modification of the cellular membrane by the addition of the viral envelope protein. However, both viral structural proteins were efficiently incorporated into virions; a 0.5-hr pulse-labeling period resulted in the release of maximally labeled virus during the next hour. When protein synthesis was inhibited, release of virus soon declined even though large amounts of both viral structural proteins were present within the cell and ribonucleic acid replication was unaffected.

Animals↗

PubMed2006

Does rapid oligomerization of hepatitis B envelope proteins play a role in resistance to proteasome degradation and enhance chronicity?

This review discusses the nature of hepatitis B and C chronicity from a virological perspective. Work described in the literature and our in vitro studies of HBV polypeptide morphogenesis lead us to speculate about a role for HBsAg complex formation in immune evasion that may be especially important during the initial period of infection. Briefly, although viral structural proteins do eventually provide epitopes recognized by the host, we suggest that these HBs Ag complexes, which may themselves be refractory to proteasomal degradation, are an important way by which the virus shields its epitopes and evades early recognition by the cellular immune system. This suggests a central strategy by which the virus has evolved, structurally, to enable the establishment of persistent infection of its host. The concept offers an explanation for the nearly unidirectional and rapid kinetics whereby HBV proteins form multimers and generate a surplus of viral structures that have not been thought to serve any useful structural purpose.

Animals↗

PubMed1992

Human papillomavirus DNA in cervix. In-situ hybridization with biotinylated probes on Bouin's fixed paraffin embedded specimens.

We examined retrospectively a series of 65 Bouin's fixed, paraffin-embedded tissue specimens from 8 condylomatous lesions, 16 condylomas associated with cervical intraepithelial neoplasia (CIN), and 12 neoplasia without condylomatous signs, for histological characteristics, the detection of viral structural antigen, the presence and typing of HPV DNA by molecular in situ hybridization with biotinylated probes types 6, 11, 16 and 18 under stringent conditions (Tm - 12 degrees C). HPV DNA was present in 34/65 (52%) specimens. Detection of viral structural antigen was positive in only 14% (3/22) specimens. HPV DNA were identified in 9/9 (100%) condylomatous lesions (with HPV type 6, 11, 18). Three condylomas were coinfected with both HPV type 6 or 11 and type 18; viral antigen was found in two specimens. HPV DNA were detected in 18/31 (58%) low grade and advanced CIN associated with condylomatous changes (type 6 = 5 specimens, type 11 = 3 specimens, type 16 = 4 specimens, type 18 = 6 specimens). Four of these cases were coinfected with both HPV type 6/11 and HPV type 16/18. Viral antigen was negative in all specimens. HPV DNA were detected in 7/25 (28%) advanced intra-cervical neoplasia (CIN III) without anatomopathological condylomatous changes (type 6 = 1 specimen, type 16 = 3 specimens, type 18 = 3 specimens). One of these specimens contained both HPV types 6 and 18. Viral antigen was found in one case. Our data confirm the association of HPV types 6 and 11 with condyloma and low grade neoplasia; HPV types 16 and 18 were associated with advanced cervical neoplasia.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetates↗

PubMed1987

[Electrophoretic characteristics of the soluble glycoproteins of the A-2 and MK-25 strains of Aujeszky's disease virus].

Studied were soluble glycoproteins of the Aujeszky's disease virus strains--the virulent A-2 one and its IUDR-resistant MK-25 mutant, following purification via affinity chromatography. Two protein bands were found through polyacrylamide gel. The molecule weight of the glycoproteins isolated from A-2 strain was 67,000 daltons, and that of the MK-25 ones--42,000 daltons. It was shown to be close to the molecule weight of two structural viral proteins. These properties of theirs as well as the capacity of the antisera to neutralize the Aujeszky's disease virus made it reasonable to believe that the isolated glycoproteins were in fact soluble structural viral glycoproteins.

Chromatography, Affinity↗

PubMed1989

Selective separation of virus proteins and double-stranded RNAs by SDS-KCl precipitation.

The total viral structural polypeptides and the double-stranded genomic RNAs of bluetongue virus can be selectively separated by a single SDS-KCl precipitation step. This simple, rapid and highly reproducible method enables greater than 95% recovery and purity of both viral proteins and dsRNAs within 30 min. The serotypic identity of the separated dsRNAs can be analyzed by SDS-PAGE electrophorogram immediately. After a single phenol/chloroform extraction, the dsRNA can also be used as hybridization probes, templates for molecular cloning and direct RNA sequencing. The SDS-KCl-precipitated viral proteins could be used readily for peptide mapping and as immunogens. Polyclonal and monoclonal antibodies raised against SDS-KCl-precipitated viral structural polypeptides were useful in Western immunoblots.

Blotting, Northern↗

PubMed1986

Genomic diversity of the acquired immunodeficiency syndrome retroviruses is reflected in alteration of its translational products.

We have isolated retroviruses from six acquired immunodeficiency syndrome (AIDS) and three lymphadenopathy syndrome (LAS) patients by cocultivation of patients' lymphocytes with phytohemagglutinin-stimulated normal T cells. In an effort to address the extent to which these viruses have identical genetic information or there is divergence in their genomic sequences, we have compared the nine retrovirus isolates by the following criteria: (i) antigenic cross-reactivity by highly specific and sensitive competition radioimmunoassay for the major internal antigen; (ii) restriction site mapping analysis; and (iii) immunoblot analysis and metabolic labeling of viral structural proteins and their analysis by polyacrylamide gel electrophoresis. The data indicate that individual retroviruses have significant restriction site polymorphism in their genome even though they were isolated from patients residing at one geographic location. Furthermore, this polymorphism is reflected in the variation of the apparent size of the gag and env gene-encoded structural proteins. The heterogeneity in AIDS retrovirus-encoded proteins may be due to either substitutions in the primary amino acid sequence of the protein or deletions or additions in the coding regions of proteins. The alterations in viral structural proteins among various AIDS retroviruses could have important implications in antigenic properties and/or pathogenicity in development of the disease, its detection, and ultimately its eradication.

Acquired Immunodeficiency Syndrome↗

PubMed1977

Human mammary gland associated antigens. Lack of D-type retravirus-induced antigens.

Antisera against tumor-associated and organ-specific antigens of human mammary glands, as well as mammary carcinoma patient sera were used in cross-reactions with the test-system for a nucleoid antigen of Hep-2 retravirus. None of the antigens, revealed in mammary carcinomas and human milk seems to be either viral structural, or virus-induced cellular antigen. No cross-reactivity was revealed between preparations, containing A-type particles, isolated from Hep-2 cells and human milk and mammary carcinomas. Cell surface antigen, nonidentical to viral structural antigens, was detected in Hep-2 cells. It proved to be shared by E16b and some other stable cell lines, however it cannot be detected in mammary fibroadenomas, positive for the D-type nucleoid antigen, and in mammary carcinomas. Only gastric carcinoma homogenates exhibited reaction of identity with the cell surface antigen, shared by Hep-2 and E16b cells. It suggests that some common stem cells might be involved in its synthesis, rather than D-type retravirus genome. This question is worth a special investigation. The findings obtained show the genome of D-type retravirus is repressed in mammary carcinomas in contrast with fibroadenomas, when synthesis of nucleoid antigens was revealed by ILYIN [7].

Antigens↗

PubMed1990

Identification of virus-specific polypeptides and translatable mRNAs in the isolated pupal abdomens of the silkworm, Bombyx mori, infected with nuclear polyhedrosis virus.

Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that infection of the isolated pupal abdomens of the silkworm, Bombyx mori, with B. mori nuclear polyhedrosis virus (BmNPV) caused generation of a number of polypeptides with a concomitant decrease of cellular polypeptides. These generated polypeptides were not identified as viral structural polypeptides, but were characterized as the degradation products of cellular polypeptides, as evidenced by the reaction with specific antiserum against storage proteins 1 and 2. Immunoblot analysis using anti-BmNPV serum identified at least 14 virus-specific polypeptides in the BmNPV-structural polypeptides of the virus. In vitro translation and subsequent immunoprecipitation with anti-BmNPV serum showed that translation yielded at least 15 polypeptides at the expense of cellular polypeptides. Time-course experiments showed that the viral structural polypeptides and virus-specific translatable mRNAs were not detectable until 3 days postinoculation. On the basis of the fact that the isolated pupal abdomens are in an arrested state of development, the delayed onset of virus-specific macromolecule production in the infected isolated pupal abdomens, as compared with that in the developing intact pupae, implies that some cellular function associated with the pupal-adult development is an important prerequisite for the efficient commencement of BmNPV replication.

Abdomen↗

PubMed2005

Pre-organized structure of viral DNA at the binding-processing site of HIV-1 integrase.

The integration of the human immunodeficiency virus type 1 DNA into the host cell genome is catalysed by the viral integrase (IN). The reaction consists of a 3'-processing [dinucleotide released from each 3' end of the viral long terminal repeat (LTR)] followed by a strand transfer (insertion of the viral genome into the human chromosome). A 17 base pair oligonucleotide d(GGAAAATCTCTAGCAGT), d(ACTGCTAGAGATTTTCC) reproducing the U5-LTR extremity of viral DNA that contains the IN attachment site was analysed by NMR using the classical NOEs and scalar coupling constants in conjunction with a small set of residual dipolar coupling constants (RDCs) measured at the 13C/15N natural abundance. The combination of these two types of parameters in calculations significantly improved the DNA structure determination. The well-known features of A-tracts were clearly identified by RDCs in the first part of the molecule. The binding/cleavage site at the viral DNA end is distinguishable by a loss of regular base stacking and a distorted minor groove that can aid its specific recognition by IN.

Binding Sites↗

PubMed1993

Establishment of HTLV-I-infected cell lines from French, Guianese and West Indian patients and isolation of a proviral clone producing viral particles.

Human T-cell leukemia virus (HTLV-I) induces adult T cell leukemia/lymphoma (ATL) and a chronic neurological disease named either tropical spastic paraparesis (TSP) or HTLV-I associated myelopathy (HAM). We report here the establishment and characterization of eight HTLV-I-infected lymphoid cell lines derived either from patients with TSP (5) or from asymptomatic carriers (1). Southern blot analysis of T cell beta chain gene rearrangements indicates that all cell lines are composed of clonal populations. The same type of analysis performed with HTLV-I-specific probes showed that they harbor 1 to 5 copies of full length proviruses often associated with deleted proviruses with a restriction map for BamHI, HindIII, PstI and SacI restriction enzymes resembling those of HTLV-I previously isolated from Japan and Caribbean area. One of the cell lines, 2060, derived from a TSP patient was shown to express a relative large amount of virus easily transmissible to fresh peripheral and cord blood lymphocytes. The full length proviral genome contained in this cell line was cloned and used in transient expression experiments. We showed that the cloned provirus was able to direct the synthesis of the major structural viral proteins, the protease and the tax and rex regulatory proteins. The structural viral proteins could be assembled into free particles detected in the culture medium of transfected cells. Although the infectivity of these viral particles remains to be determined, this new clone can be employed to examine the cell types in which this TSP-derived provirus directs viral protein synthesis and eventually replicates. It should also prove of value in studies on the early cellular events induced by viral products.

Animals↗

PubMed1997

Chronic ethanol effects on cellular immune responses to hepatitis B virus envelope protein: an immunologic mechanism for induction of persistent viral infection in alcoholics.

Hepatitis B virus (HBV) is common in alcoholics and may result in chronic infection. Persistence of HBV infection could be partially caused by the effects of ethanol on the cellular and humoral immune response to viral structural proteins. The DNA-based immunization approach was used to experimentally assess the effects of chronic ethanol feeding on immune responses directed against the middle envelope protein (MHBs) of HBV. Mice were fed an ethanol or isocaloric, pair-fed control liquid diet for 8 weeks, followed by immunization with a plasmid construct containing the pre-S2/S gene that encodes for MHBs. Chronic ethanol consumption marginally reduced the levels of the antibody to hepatitis B surface proteins (anti-HBs) generated by the DNA-based immunization approach. Initially, cytotoxic lymphocyte (CTL) activity was higher in ethanol-fed mice but progressively declined following the second and third immunizations as compared with control mice. In addition, CTL and CD4+ T helper (TH) cells responded poorly to increasing concentrations of envelope protein and peptides in vitro with respect to generation of CTL activity and proliferative responses. Finally, proliferating CD4+ T cells derived from ethanol-fed animals had substantial changes in the levels of cytokines secreted into the culture supernatants as compared with control mice. These studies show that chronic ethanol consumption substantially alters the cellular immune responses to a human viral structural protein, and that these effects may contribute to the persistence of viral infection.

Alcoholism↗

PubMed2005

Crystal structure of viral macrophage inflammatory protein I encoded by Kaposi's sarcoma-associated herpesvirus at 1.7A.

Viral macrophage inflammatory protein I (vMIP-I) is a chemokine encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) that selectively activates the CC chemokine receptor 8 (CCR8), for which the endogenous ligand is CCL1. The crystal structure of vMIP-I was determined at 1.7A for comparison with other chemokines, especially those that bind CCR8, such as vMIP-II from KSHV, a CCR8 antagonist and the closest homolog (40% identical). vMIP-I has a typical chemokine fold consisting of an extended N-terminal loop, followed by a three-stranded antiparallel beta-sheet and a C-terminal alpha-helix. The four molecules in the asymmetric unit comprise two MIP-1beta-like dimers. Electrostatic surface representations of CCR8-binding chemokines reveal only minor areas of correlating surface potential, which must be reconciled with promiscuity in receptor and glycosaminoglycan (GAG) binding. In addition, the biological relevance of chemokine oligomerization is examined by comparing the oligomeric states of all chemokine structures deposited to date in the RCSB PDB.

Amino Acid Sequence↗

PubMed1986

Cloning and expression of a viral phosphoprotein: structure suggests vesicular stomatitis virus NS may function by mimicking an RNA template.

The phosphoprotein (NS) gene from the Indiana serotype of vesicular stomatitis virus (VSV; Mudd-Summers strain) was cloned and sequenced. The NS gene encodes a protein of 265 amino acids which was expressed from a simian virus 40 vector in COS cells. The post-translational modification characteristic of viral NS, the extensive phosphorylation of a cluster of serine and threonine residues, was also evident in recombinant NS protein. The NS gene displays a property common to the phosphoprotein genes of negative-strand RNA viruses: the phosphoprotein mRNA has a second open reading frame (ORF) which could encode a small (7500 mol. wt.) protein. Both measles virus and Sendai virus employ the second ORF of their phosphoprotein gene, and the resultant proteins have an amino acid composition similar to that predicted for the VSV ORF. Comparison of phosphoproteins from different VSV strains revealed two conserved domains that we propose are critical for the function of NS in transcription and replication.

Animals↗

PubMed1991

[Current etiological structure of acute viral hepatitis in children].

The etiological structure of viral hepatitides (VH) was defined in 345 children admitted to the clinic during one year with a diagnosis of acute viral hepatitis. It was established on the basis of clinico-laboratory studies and identification of serological markers of viral hepatitides A, B and delta. It has been demonstrated that in the structure of viral hepatitides in children living in Moscow, VHA accounts for 83.1%, acute VHB for 11.1%, HBV and HDV coinfection for 2.6%, and VH non A non B for 1.7%. In children of the first year of life, acute hepatitis was etiologically related to HBV-infection or to HBV and HDV coinfection. In a negligible part of the patients, VHA was diagnosed in the presence of chronic HBV-infection (1.2%) or exacerbation of chronic hepatitis B (0.3%). These data should be taken into consideration in planning and carrying out diagnostic, treatment and prophylactic measures aimed at solving VH problem in childhood.

Acute Disease↗

PubMed1988

Effect of actinomycin D and cycloheximide on replication of Sindbis virus in Aedes albopictus (mosquito) cells.

Production of Sindbis virus in the presence of transcription and translation inhibitors was examined in three Aedes albopictus cell lines. Addition of cycloheximide to heat-resistant Sindbis virus (SVHR)-infected mosquito cells arrested viral RNA synthesis completely, in contrast to the effects of this drug on virus-infected vertebrate cells. Production of mature virus by both SVHR (a variant commonly used as a wild-type virus) and SBamr (a mutant which is resistant to the effects of 18 h of pretreatment of vertebrate cells with actinomycin D) in mosquito u4.4, C6-36, and C7-10 cells was inhibited by 2 h of pretreatment with actinomycin D. Pretreatment with this drug for 2 h slightly enhances virus production in vertebrate cells. Treatment of mosquito cells with actinomycin D resulted in shutoff of SVHR RNA synthesis. The mutant SBamr was able to overcome the effects of actinomycin D on viral RNA synthesis and produced both 26S and 49S RNAs, even though no viral structural proteins or mature particles were produced in the presence of the drug. This result suggests that, in the presence of actinomycin, the nonstructural genes of SBamr are translated sufficiently to allow for RNA synthesis but that 26S RNA may not be translated to an extent that allows significant virus production. These data demonstrate that host components are involved in at least two distinct steps in the production of Sindbis virus in mosquito cells: (i) production of viral RNA and (ii) synthesis of viral structural polypeptides.

Aedes↗

PubMed1983

Immunohistological demonstration of serum proteins and structural and viral antigens in paraffin sections of nervous tissues.

A brief outline is given of applications of immunohistological techniques to the study of normal and diseased nervous tissue. Protease treatment of paraffin sections usually enhances sensitivity and reliability both of IF and PAP techniques. Sensitivity of immunohistological examination of paraffin sections is comparable to that of virus detection by normal virological techniques in animal rabies and slightly superior to EM search for virions in SSPE and PML. Immunostaining for MBP appears to be the most sensitive method for myelin, especially for demonstration of very thin myelin sheaths, which are important in studies of myelogenesis and cortical myeloarchitecture. Prolonged fixation in formalin clearly diminishes or abolishes immunoreactivity. Compacted myelin stains less well for MBP than preparative myelin artefacts and the surface of myelinated fibers. GFAP production is enhanced when glioma cells invade surrounding mesenchymal structures. The chance finding of GFAP-like immunoreactivity in a cancer metastasis casts doubt on the astroglial specificity of GFAP.

Animals↗

PubMed1979

The isolation of avian viral RNA and polypeptides.

From the same batch of virus, the four major avian viral structural proteins p27, p19, p15, and p12, the reverse transcriptase, the envelope glycoprotein gp85, and the high molecular weight 70 S RNA have been recovered. All proteins, except for gp85, have been purified by use of column chromatography procedures to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gels and isoelectric focusing. A new isolation procedure for p12 by affinity column chromatography takes advantage of its nucleic acid binding properties. The recovery of nondenatured viral structural proteins is demonstrated by the proteolytic activity revealed by p15. The purified proteins were used for the production of monospecific antibodies. The 70 S RNA served as source for the isolation of 35 S RNA subunits.

Avian Leukosis Virus↗

PubMed1988

pp60v-src tyrosine kinase is expressed and active in sarcoma-free avian embryos microinjected with Rous sarcoma virus.

Early embryonic avian tissue is resistant to transformation by Rous sarcoma virus. To determine the nature of this resistance, we examined the expression and properties of the Rous sarcoma virus transforming protein pp60v-src, in infected embryonic chicken limbs in ovo. Lysates from Rous sarcoma virus-infected limbs contained the viral structural protein p19gag, as detected by immunoblot analysis, and showed pp60v-src kinase activity in vitro. Immunoblot analysis of lysates with anti-phosphotyrosine antibodies revealed a number of phosphotyrosine-containing proteins present in lysates of Rous sarcoma virus-infected embryos but not in lysates of control, uninfected embryos. Anti-phosphotyrosine immunoreactivity was observed in frozen sections in the same cell types that expressed pp60v-src and p19gag. These studies demonstrate that pp60v-src is co-expressed with viral structural determinants in infected embryonic avian tissue. Furthermore, pp60v-src is active in ovo as a tyrosine-specific phosphotransferase, despite the apparent lack of sarcoma induction. The localization pattern of the major src gene substrate p36 (calpactin I) was compared with that of p19gag by double-label immunofluorescence and found to be generally nonoverlapping. These observations are consistent with the concept that the induction of tumors in ovo requires complementation between viral determinants and host factors. These host factors, which may be critical substrates of pp60v-src, are subject to developmental regulation in the avian embryo.

Animals↗