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Results for “Viral Structures”

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PubMed1993

Analysis of anti-Tax antibody of HTLV-I carriers in an endemic area in Japan.

Sera from 1197 adult residents in Miyazaki district, an area in Japan endemic for human T-cell leukemia virus type I (HTLV-I), were tested for anti-Tax antibody by the recombinant Tax (r-Tax) Western blot assay. Among HTLV-I-seropositive individuals, including 21.5% of 484 males and 28.6% of 713 females, the prevalence of anti-Tax antibody were 59.6% and 58.3% respectively, with no apparent difference in age. There was a significant 6-fold difference in the prevalence of anti-Tax among seropositive subjects with titer > or = 1:8192 (84.6%) compared with those with the lowest titer of 1:16 (14.3%), suggesting the increased production of antibodies to viral structural proteins in anti-Tax-positive individuals. Furthermore, among those anti-Tax-positive subjects, the intensity of serum reactivity to r-Tax protein in the high antibody titer (1:1024 or higher) group was significantly stronger than that in the lower antibody titer (1:512 or lower) group. We also found that 1.6% (14/889) of individuals without detectable levels of HTLV-I antibody had anti-Tax antibody. HTLV-I pro-viral DNA signals could not be detected in DNA sample from the lymphocytes of these individuals by the nested polymerase chain reaction method. Further evaluation is needed to clarify the significance of an anti-Tax-only status population in which HTLV-I is endemic.

Adult↗

PubMed2002

Bel1-mediated transactivation of the spumaretroviral internal promoter is repressed by nuclear factor I.

Gene expression of the internal and long terminal repeat promoters of the spuma retrovirus is specifically activated by the transactivator Bel1, the key regulator of viral gene expression. Bel1 directly binds to and activates DNA target sites of viral promoters and those of distinct cellular genes. To determine the contribution of cellular transcription factors to viral transactivation, the viral internal promoter (IP) was analyzed by transient expression, electrophoretic mobility shift assays), and supershifts. Here we report that Bel1-mediated transactivation of the full-length and shortened versions of the Bel1 response element (BRE) were repressed by nuclear factor I (NFI). Electrophoretic mobility shift assays using nuclear extracts from transfected 293T cells revealed that different DNA-protein complexes consisting of DNA target sites of NFI and Bel1 proteins were formed. The specificity of the repressor and transactivator DNA binding was shown by NFI- and Bel1-specific antibodies that led to supershifts of the different nuclear protein-oligodeoxynucleotide complexes. The specificity of the complexes was confirmed by using unlabeled, shortened, and mutated IP.BRE oligodeoxynucleotides in competition experiments with the authentic IP.BRE. Cotransfection of the infectious spumavirus DNA genome with a human NFI-X1 expression plasmid into cell cultures greatly reduced the expression of viral structural and Bel1 proteins. These data demonstrate the relevance of NFI-mediated repression of Bel1-driven transactivation in vivo.

Binding, Competitive↗

PubMed1992

Prokaryotic expression of a VP1 polypeptide antigen for diagnosis by a human parvovirus B19 antibody enzyme immunoassay.

To produce parvovirus B19 antigen for diagnostic purposes, partially overlapping segments covering the genes encoding the viral structural proteins VP1 and VP2 were cloned into expression vectors. The constructs were induced in Escherichia coli, resulting in the expression of beta-galactosidase fusion proteins. In immunoblotting experiments with sera from patients with erythema infectiosum, immunoglobulin G (IgG) and IgM antibodies bound to a single polypeptide of 235 amino acids at the N terminus of VP1. The DNA fragment encoding this polypeptide was amplified by the polymerase chain reaction and cloned into an expression vector. The viral capsid antigen expressed in E. coli was purified by preparative agarose gel electrophoresis and used in IgG and IgM solid-phase enzyme immunoassays. Comparison with reference gamma- and mu-capture radioimmunoassays using whole virus antigen showed that these antibody tests are suitable for the serodiagnosis of human infections caused by parvovirus B19.

Antibodies, Viral↗

PubMed1980

Avian myeloblastosis virus proteins in leukemic chicken myeloblasts.

We have analyzed the avian myeloblastosis virus proteins in two types of leukemic myeloblasts: established myeloblastic cell lines (DU 1765 and DU 11157) and leukemic myeloblasts obtained from the peripheral blood of a leukemic C/E Spafas chicken (no. 21957). Using monospecific antisera for immunoprecipitation and polyacrylamide gel electrophoresis, we have detected gag gene-related proteins in the myeloblasts. The DU 1765 and DU 11157 cells contained a p100 protein which possessed antigenic determinants of the viral proteins p27, p19, p15, and p12. The p100 was not found in leukemic myeloblasts from Spafas chickens, and pulse-chase experiments showed that the p100 was not a precursor for the viral proteins. However, the p100 is present in uninfected line 15 chicken embryos. A pr76-like protein was identified in DU 1765 cells but migrated slightly further into gels than the pr76 of Spafas-derived leukemic myeloblasts. The Spafas-derived myeloblasts produced a pr60, whereas the DU 1765 cells contained instead a related protein of 62,000 daltons. Using anti-avian myeloblastosis virus gp85 sera, a glycoprotein of 120,000 daltons (gp120) was detected in all the tested leukemic myeloblasts. The gp120 was also present, in low amounts, in uninfected embyonic spleen and yolk sac cells. The anti-gp85 sera also precipitated a 27,000-dalton protein (h27) in these same cells. Both the gp120 and h27 could not be detected in either uninfected or myeloblastosis-associated virus-infected fibroblasts. Limited peptide hydrolysis revealed that h27 is different from the viral structural protein p27. In conclusion, monospecific antisera for gag and env gene products of avian myeloblastosis virus did not precipitate any unique or aberrant avian myeloblastosis virus protein from leukemic myeloblasts.

Animals↗

PubMed1997

Reovirus variants selected during persistent infections of L cells contain mutations in the viral S1 and S4 genes and are altered in viral disassembly.

Reoviruses isolated from persistently infected cultures (PI viruses) can grow in the presence of ammonium chloride, a weak base that blocks acid-dependent proteolysis of viral outer-capsid proteins during viral entry into cells. We used reassortant viruses isolated from crosses of wild-type (wt) reovirus strain, type 1 Lang, and three independent PI viruses, L/C, PI 2A1, and PI 3-1, to identify viral genes that segregate with the capacity of PI viruses to grow in cells treated with ammonium chloride. Growth of reassortant viruses in ammonium chloride-treated cells segregated with the S1 gene of L/C and the S4 gene of PI 2A1 and PI 3-1. The S1 gene encodes viral attachment protein sigma1, and the S4 gene encodes outer-capsid protein sigma3. To identify mutations in sigma3 selected during persistent reovirus infection, we determined the S4 gene nucleotide sequences of L/C, PI 2A1, PI 3-1, and four additional PI viruses. The deduced amino acid sequences of sigma3 protein of six of these PI viruses contained a tyrosine-to-histidine substitution at residue 354. To determine whether mutations selected during persistent infection alter cleavage of the viral outer capsid, the fate of viral structural proteins was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after treatment of virions of wt and PI viruses with chymotrypsin in vitro. Proteolysis of PI virus outer-capsid proteins sigma3 and mu1C occurred with faster kinetics than proteolysis of wt virus outer-capsid proteins. These results demonstrate that mutations in either the S1 or S4 gene alter acid-dependent disassembly of the reovirus outer capsid and suggest that increased efficiency of proteolysis of viral outer-capsid proteins is important for maintenance of persistent reovirus infections of cultured cells.

Animals↗

PubMed1977

Expression of Mason-Pfizer and simian type C viruses in the presence of 5-iododeoxyuridine and dexamethasone.

Production of infectious Mason-Pfizer monkey virus (M-PMV) was enhanced after treatment of the CMMT cell line with 2.5 x 10(-5) M dexamethasone phosphate (DXM). The reverse transcriptase (RT) activity and infectivity titers of treated culture fluids were enhanced by five- and tenfold, respectively. Along with stimulation of M-PMV synthesis, a simian type C virus (SCV) was also detected by electron microscopic and RT analyses. The SCV was serologically related to the endogenous baboon type C virus. 5-iododeoxyuridine (IUDR) also activated the SCV in the CMMT cell line while significantly inhibiting the production of infectious M-PMV. The activation of endogenous SCV by IUDR or DXM was transitory, since removal of these compounds from the growth medium resulted in the disappearance of SCV buds and the related RT activity; however, low levels of specific viral structural proteins continued to be synthesized intracellularly. Similarly, the enhancement of M-PMV production seen with DXM was lost when the treated cells were subcultured for 2 weeks in the absence of the hormone.

Cell Line↗

PubMed2001

In vivo adenoviral transduction of the neonatal rat cochlea and middle ear.

Virally mediated gene transfer to the adult mammalian ear appears to be a powerful strategy to investigate gene function in the auditory system and to develop new therapeutic treatment for hearing impaired patients. However, there has been little work done in the neonatal middle and inner ear. In this study, a recombinant adenoviral (AdV) vector was used for gene transfer of a beta-galactosidase (beta-gal) reporter gene to the neonatal middle ear and cochlea of 5 day old rats. For transduction of middle ear, AdV was injected through the tympanic membrane into the tympanic cavity. Three and 7 days later, strong expression of beta-gal was observed in epithelial cells of the mucosa, but not in the underlying stroma or mesenchyme. There was little or no infiltration of leukocytes. No expression of beta-gal was detected inside the cochlea or vestibular system. When AdV was injected into the basal turn of the cochlea, high levels of beta-gal expression were observed in cells lining the perilymphatic space and in parts of the spiral ligament 3, 7 and 21 days later. Spiral ganglion cells did not express beta-gal. However, virally mediated gene transfer was observed in some cells of the organ of Corti. A moderate infiltration of leukocytes into the labyrinth was observed, but no vestibular or auditory dysfunction. These results demonstrate that neonatal middle ear and cochlear cells can be successfully transduced with an AdV vector in vivo, without obvious morphological signs of inflammation or cellular damage. AdV vectors provide a tool for investigation of the role of genes in influencing the development of middle and inner ear structures. Virally mediated expression of protective genes could also be used to rescue hair cells or spiral ganglion cells from congenital degeneration or damage.

Adenoviridae↗

PubMedDate not supplied

Pathological findings in two fin whales (Balaenoptera physalus) with evidence of morbillivirus infection.

Two immature female fin whales stranded on the Belgian and French coastlines, were examined post mortem. The main gross findings were massive parasitic infestation, associated with a large thrombus in one whale, and severe emaciation. Microscopical investigations revealed multinucleated syncytia with large intranuclear inclusion bodies in various tissues, and positive immunolabelling for morbillivirus antigens. Other evidence of morbillivirus infection was provided by the demonstration of specific viral structures in syncytia and in cell cultures, and the detection of neutralizing antibodies to canine distemper virus. To the authors>> knowledge, this is the first firm report of morbillivirus infection in baleen whales.

Animals↗

PubMed1985

Antibody response to individual rubella virus proteins in congenital and other rubella virus infections.

Serum samples from patients with various forms of rubella virus infection were tested for antibodies to each of three viral structural proteins by radioimmunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In most sera antibody to E1 protein was the predominant species. Sera from patients with congenital rubella syndrome, however, contained significantly more E2 antibody relative to E1 antibody than did sera from other rubella patients.

Adolescent↗

PubMedDate not supplied

Prevalence of HTLV-I Tax in a subset of patients with rheumatoid arthritis.

OBJECTIVE: In regions of the world where the human T cell lymphotropic virus type I (HTLV-I) is endemic, it is recognized that infection with this virus is associated with autoimmune diseases such as rheumatoid arthritis (RA). Moreover, mice transgenic for the HTLV-I Tax gene develop a disease akin to RA. The observation that about 8% of healthy American blood donors carry HTLV-I Tax in their lymphocytes (1) prompted studies to determine whether Tax positivity is more prevalent among patients with RA and if so, whether its sequence is homologous with prototypic HTLV-I Tax. This proved to be the case. Of 102 patients with RA tested, one was a carrier of HTLV-I and 25 had the Tax sequences in their mononuclear cells and antibodies to p40 Tax in their sera, while being negative for antibodies to the structural proteins of the virus. METHODS: Blood was collectedfrom 102 RA patients. Lysates of their mononuclear cells were assayed for HTLV-I Tax by PCR/Southern analysis, and in some positive cases Tax sequence analysis was performed. Antibodies to p40 Tax, the gene product of the Tax sequence, were detected by western blot assay using recombinant p40 Tax as antigen. Results Of the 102 patients tested, one proved to be a carrier of the virus, having antibodies and sequences for the viral structural proteins, gag and env in addition to p40 Tax. Twenty-five of the 101 HTLV-I/II seronegative patients carried both HTLV-I Tax sequences in their mononuclear cells and had antibodies to p40 Tax. Sequence analysis confirmed homology with HTLV-I Tax. CONCLUSION: The data show that the prevalence of HTLV-I Tax positivity among patients with RA is -3 times higher than among healthy blood donors. Since Tax is known to be involved in the development of numerous autoimmune diseases, the possibility that it is responsible for the development of RA in a subpopulation of patients with this disease is not remote.

Arthritis, Rheumatoid↗

PubMed1976

Composition of the Zaysan virion.

The sedimentation coefficient of Zaysan virion ribonucleic acid (RNA), estimated by sucrose density gradient centrifugation, was 42 S. Pancreatic ribonuclease digested the viral RNA to acid-soluble fragments. Two viral structural proteins with apparent molecular weight of 50,000 and 30,000 daltons were identified by polyacrylamide gel electrophoresis. A dense, RNA-rich particle, containing the smaller polypeptide, was isolated after mild detergent treatment of Zaysan virus.

Arboviruses↗

PubMed2006

Feline lentivirus evolution in cross-species infection reveals extensive G-to-A mutation and selection on key residues in the viral polymerase.

Factors that restrict a virus from establishing productive infection in a new host species are important to understand because cross-species transmission events are often associated with emergent viral diseases. To determine the evolutionary pressures on viruses in new host species, we evaluated the molecular evolution of a feline immunodeficiency virus derived from a wild cougar, Puma concolor, during infection of domestic cats. Analyses were based on the coding portion of genome sequences recovered at intervals over 37 weeks of infection of six cats inoculated by either intravenous or oral-nasal routes. All cats inoculated intravenously, but only one inoculated orally-nasally, became persistently viremic. There were notable accumulations of lethal errors and predominance of G-to-A alterations throughout the genome, which were marked in the viral polymerase gene, pol. Viral structural (env and gag) and accessory (vif and orfA) genes evolved neutrally or were under purifying selection. However, sites under positive selection were identified in reverse transcriptase that involved residues in the nucleotide binding pocket or those contacting the RNA-DNA duplex. The findings of extensive G-to-A alterations in this cross-species infection are consistent with the recently described editing of host cytidine deaminase on lentivirus genomes. Additionally, we demonstrate that the primary site of hypermutation is the viral pol gene and the dominant selective force acting on this feline immunodeficiency virus as it replicates in a new host species is on key residues of the virus polymerase.

Animals↗

PubMed1989

Rev-induced modulation of Nef protein underlies temporal regulation of human immunodeficiency virus replication.

The replication of human immunodeficiency virus type 1 (HIV-1) requires the concerted action of two virus-encoded transactivator proteins, Tat and Rev, and is in turn moderated by the viral transcriptional repressor Nef. We show here that the phenotype of a Rev- HIV-1 provirus was nonreplicating and was distinguished by accumulation of Nef protein and reduced Tat function. Provirus defective in both the rev and nef genes (Rev-Nef-) was also nonreplicating but had normal Tat function. Trans-complementation of the Rev- mutant with Rev caused a decrease of both the steady-state level and the rate of synthesis of Nef. This was accompanied by enhanced synthesis of viral structural proteins. Rev induced even greater levels of virus production from the Rev-Nef- double mutant. In contrast, exogenous Rev did not augment virus production from wild-type provirus. Virus production from Rev- and Rev-Nef- mutants induced by Rev was repressed by exogenous Nef. The repression induced by Nef could not be reversed by exogenous Rev. The ability of Rev to modulate Nef expression solely from the provirus, and thereby relieve the Nef-mediated inhibition of transcription from the viral long terminal repeat, reveals a delicate balance of the functions of these two proteins that might underlie the switch between latency and reactivation.

Base Sequence↗

PubMed1977

Studies on viral DNA protein complexes isolated at different times after infection of monkey kidney cells with simian virus 40.

The major protein components of the DNA complex, isolated from SV40-infected monkey cells, are the major viral structural polypeptide VP1 and cellular histones. At early times (24 h) after infection, VP1 is present in large amounts relative to histones, whereas at late times (48 h), the complex contains mostly histones. The amount of VP1 in the complex can be correlated to the amount of "free" VP1 present in the cells, i.e. VP1 not yet incorporated into virus particles. At early times about 40% of VP1 is "free" VP1; at late times, most of the VP1 is incorporated into virus particles. In contrast, viral DNA is produced in huge excess and only about 13% is incorporated into virions. In agreement with the above result, we find that only about 16% of the DNA in the DNA complex can be chased into virions. There is, apparently, no turnover of newly synthesized VP1 that is associated with the DNA complex at late times after infection.

Animals↗

PubMed1999

Co-expression of a trans-dominant negative mutant of the human immunodeficiency virus type 1 (HIV-1) Rev protein affects the Rev-dependent splicing pattern and expression of HIV-1 RNAs.

Trans-dominant negative mutants of the human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev inhibit the function of wild-type Rev in a dose-dependent manner. This was previously shown to be caused by nuclear retention of the wild-type protein. In the present work, further analysis of the trans-dominant negative effect was performed using cotransfection experiments with different constructs encoding HIV-1 Rev and viral structural proteins together with a plasmid encoding a trans-dominant negative Rev mutant. Thus, one species of pre-mRNA was transcribed from the reporter plasmids. This pre-mRNA was then either spliced or exported by Rev as unspliced RNA for translation of the HIV structural proteins. An immunofluorescence assay and Western blot analysis were used for analysis of protein expression. In situ hybridization was applied for labelling of unspliced mRNA in transfected cells, and RNase protection analysis was used to determine the relative amount of unspliced versus spliced mRNAs. The experiments confirmed that the transdominant negative mutant inhibited nuclear export of unspliced mRNA. It was, in addition, demonstrated for the first time that the trans-dominant negative mutant also affected a Rev-dependent regulatory step connected with viral pre-mRNA splicing. As a consequence, proteins expressed from unspliced and singly spliced HIV mRNAs decreased while there was an increase in protein products encoded by spliced and alternatively spliced mRNAs.

Alternative Splicing↗

PubMed1996

Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8).

The genome of the Kaposi sarcoma-associated herpesvirus (KSHV or HHV8) was mapped with cosmid and phage genomic libraries from the BC-1 cell line. Its nucleotide sequence was determined except for a 3-kb region at the right end of the genome that was refractory to cloning. The BC-1 KSHV genome consists of a 140.5-kb-long unique coding region flanked by multiple G + C-rich 801-bp terminal repeat sequences. A genomic duplication that apparently arose in the parental tumor is present in this cell culture-derived strain. At least 81 ORFs, including 66 with homology to herpesvirus saimiri ORFs, and 5 internal repeat regions are present in the long unique region. The virus encodes homologs to complement-binding proteins, three cytokines (two macrophage inflammatory proteins and interleukin 6), dihydrofolate reductase, bcl-2, interferon regulatory factors, interleukin 8 receptor, neural cell adhesion molecule-like adhesin, and a D-type cyclin, as well as viral structural and metabolic proteins. Terminal repeat analysis of virus DNA from a KS lesion suggests a monoclonal expansion of KSHV in the KS tumor.

Base Sequence↗

PubMed1977

Feline oncornavirus-associated cell membrane antigen: expression in transformed nonproducer mink cells.

The feline oncornavirus-associated cell membrane antigen (FOCMA) is a target for naturally occurring immunity that protects the cat against development of fibrosarcoma and leukemia. Feline sarcoma virus-transformed "nonproducer" mink cells express high levels of FOCMA, but not the major viral structural proteins. Transformation of the same cells by murine sarcoma virus, or infection with feline leukemia virus, which is nontransforming for epithelial or fibroblastic cells, did not induce FOCMA. Thus, FOCMA expression in mind lung cells is specifically associated with transformation by feline sarcoma virus.

Animals↗

PubMed1987

Complete nucleotide sequence of the genome of coxsackievirus B1.

The complete nucleotide sequence of the genome of the coxsackievirus B1, a human enterovirus that belongs to the Picornaviridae, was determined by using molecular cloning and rapid sequence analysis techniques. Sequence analysis of the cloned cDNAs revealed that the virion RNA was 7389 nucleotides long and polyadenylylated at the 3' terminus. Similar to other picornavirus genomes, a single large open reading frame was identified. The translated sequence starts at nucleotide position 742 and ends at 7287 of the genome. Thus, the viral polyprotein should consist of 2182 amino acids. When the predicted amino acid sequence of the viral polyprotein was compared with those of other human enteroviruses such as polioviruses, a striking sequence homology was observed, especially in viral proteins 1B, 2C, and 3D. This allowed us to predict precise map locations of the viral structural and nonstructural proteins on the genome, although two proteolytic processing sites, between 1D and 2A and between 2B and 2C, were obscure. The result presented here implied important information with respect to the genetical variation of human enteroviruses.

Amino Acid Sequence↗