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PubMed1986

Exogenous mouse mammary tumor virus proviral DNA isolated from a kidney adenocarcinoma cell line contains alterations in the U3 region of the long terminal repeat.

Mouse mammary tumor virus (MMTV) is a B-type retrovirus which induces predominantly mammary carcinomas after a relatively long latency period. To date, very little is known about the reasons for the strict tissue specificity of MMTV. The BALB/cf/Cd strain of mice, which was infected with milk-borne MMTV (C3H), shows a high incidence of kidney adenocarcinomas, and our data suggest that MMTV might be involved in the formation of these tumors. Newly integrated exogenous MMTV proviruses were found in the genome of transplanted tumor cells as well as in the DNA of a cell line derived from one tumor, but not in normal cells of BALB/cf/Cd mice. The MMTV DNA in these tumor cells was transcribed and viral RNA synthesis was strongly stimulated by glucocorticoid hormones. Viral structural polypeptides, comparable in size and antigenicity to MMTV polypeptides of infected mammary tumor cells were synthesized and processed normally in the cell line and were organized correctly into intracytoplasmic particles. Heteroduplex analysis of the molecularly cloned MMTV proviral DNAs of kidney and mammary tumor origin revealed a high degree of homology in the gag, pol, and env genes. A striking difference, however, was observed in the U3 region of the two LTRs that might relate to the different tissue specificity of the two viruses.

Adenocarcinoma↗

PubMedDate not supplied

The isolation and characterization of a strain of subacute sclerosing panencephalitis measles-related virus.

The isolation of a measles-like virus in CV-1 cell cultures from a biopsy specimen obtained from a case of subacute sclerosing panencephalitis (SSPE) is reported. Evidence of the presence of cell-free virus was obtained from the fifth cell culture passage of the isolated virus. The virus was identified as measles-related by standard immune sera. IF study did not show relevant differences between the isolated virus and the Edmonston strain of measles virus, even though the proportion of cells showing nuclear antigen was higher following the infection with the isolated virus than with the Edmonston strain. Electron microscopy study failed to demonstrate viral structures in the cells from the brain biopsy, while numerous large aggregates of nucleocapsids were found in the cytoplasm of infected cells observed on the third and fourth passage in tissue culture. The neuropathogenicity assay carried out on newborn hamsters, rats and mice using a suspension of disrupted infected cells did not show any clinical pathological effect due to the SSPE strain. These results are partially in contrast with previous findings which indicate relevant differences in the morphology and pathogenicity of SSPE derived strains as compared to reference measles virus.

Antibodies, Viral↗

PubMed1993

Nucleic acid binding proteins in highly purified Creutzfeldt-Jakob disease preparations.

The nature of the infectious agent causing human Creutzfeldt-Jakob disease (CJD), a slowly progressive dementia, is controversial. As in scrapie, no agent-specific proteins or nucleic acids have been identified. However, biological features of exponential replication and agent strain variation, as well as physical size and density data, are most consistent with a viral structure--i.e., a nucleic acid-protein complex. It is often assumed that nuclease treatment, which does not reduce infectious titer, leaves no nucleic acids of > 50 bp. However, nucleic acids of 500-6000 bp can be extracted from highly purified infectious complexes with a mass of approximately 1.5 x 10(7) daltons. It was therefore germane to search for nucleic acid binding proteins that might protect an agent genome. We here use Northwestern blotting to show that there are low levels of nonhistone nucleic acid binding proteins in highly purified infectious 120S gradient fractions. Several nucleic acid binding proteins were clearly host encoded, whereas others were apparent only in CJD, but not in parallel preparations from uninfected brain. Small amounts of residual host Gp34 (prion protein) did not bind any 32P-labeled nucleic acid probes. Most of the minor "CJD-specific" proteins had an acidic pI, a characteristic of many viral core proteins. Such proteins deserve further study, as they probably contribute to unique properties of resistance described for these agents. It remains to be seen if any of these proteins are agent encoded.

Animals↗

PubMed1983

Transformation-defective Rous sarcoma virus mutants with altered p19 of the gag gene and their inhibitory effect on host cell growth.

Mutants (PH2010, PH2011, PH2012) of Rous sarcoma virus which have a growth-inhibitory effect on chicken embryo fibroblasts were isolated from a temperature-sensitive mutant of the Schmidt-Ruppin strain of Rous sarcoma virus (tsNY68). The growth rate of fibroblasts infected with these viruses was about 50 to 60% of that of uninfected fibroblasts. A morphological difference between mutant-infected and uninfected fibroblasts was observed at logarithmic phase but not at stationary phase. Neither the protein p60src nor its associated protein kinase activity was significantly detected by an immunoprecipitation assay in the cells infected with these mutants. Analysis of the unintegrated DNA of the mutant PH2010 showed that a sequence of about 1.4 kilobase pairs at the src gene region is deleted. Further examination of the viral structural proteins in infected cells as well as in virions by immunoprecipitation and peptide mapping revealed that the molecular size of the Pr76 gag protein of the mutant RSV is smaller than that of the mutant tsNY68 because of partial deletion at the p19 gag gene. The peptide maps suggest that the deleted region of the altered p19 of the mutant is near the carboxy terminal of p19. The amount of Prgp92env synthesized in the mutant-infected cells was about fivefold more than that in tsNY68-infected cells.

Animals↗

PubMed1982

Selective vulnerability of neural cells to viral infections.

A number of viruses selectively infect neurons and, in some cases, specific populations of neurons. The susceptible neuron need not be permissively infected to cause acute or chronic disease; therefore, infectious virus may not be recoverable and morphologically identifiable viral structures may not be detectable by ultrastructural structures. Polioviruses and the neurotropic murine retrovirus both cause paralytic disease with major pathological changes in motor neurons of the spinal cord. Both produce disease more readily in later life; in poliovirus because the mature animals are more susceptible to acute infection, and in the neurotropic retrovirus infections because of the long incubation period of the natural infection. In the acute inflammatory poliovirus infections, the motor neurons appear to be selectively infected and lysed by the virus, whereas in the chronic noninflammatory retrovirus infection, the effect may be indirect or may result from nonpermissive infection.

Animals↗

PubMed1981

Amino-terminal sequence analysis of alphavirus polypeptides.

The single late 26S mRNA of Semliki Forest virus (SFV) directs the synthesis of the four viral structural proteins, C, E3, E2, and E1, and the recently described nonstructural protein, 6K. We report here partial NH2-terminal amino acid sequences of the SFV polypeptides E3 and 6K and of p62, the precursor to E3 and E2. In addition, were have determined a partial NH2-terminal sequence of the Sindbis virus homolog of 6K, the 4.2K protein. p62 and E3 of SFV have identical NH2-terminal amino acid sequences. Comparison of the partial NH2-terminal sequences of 6K of SFV and 4.2K of Sindbis virus with the deduced amino acid sequence encoded by the 26S mRNA of each virus reveals that the genes for these peptides are located in each case between those for E2 and E1. The order of the genes on the 26S mRNA of the alphaviruses is therefore 5'-C-E3-E2-6K-E1-3'. We discuss two mechanisms by which the nascent viral glycoproteins may be inserted into the membrane of the endoplasmic reticulum.

Amino Acid Sequence↗

PubMed1988

Carboxyl-terminal mutants of the large tumor antigen of simian virus 40: a role for the early protein late in the lytic cycle.

Simian virus 40 (SV40) mutants dl1066 and dl1140 contain deletions within the region encoding the carboxyl terminus of the large tumor (T) antigen. Although these mutations have little effect on the efficiency of viral DNA replication, they decrease the yield of infectious virus particles by 3-4 orders of magnitude [Pipas, J. (1985) J. Virol. 54, 569-575]. Here we show that the level of late RNA is lower by a factor of 5-15 in CV-1P monkey cells infected with these mutants compared to cells infected with wild-type SV40. Consistent with this decrease in RNA, synthesis of late viral structural proteins VP1 and VP3 decreases by a factor of 5-15. In contrast, the synthesis of SV40 agnoprotein decreases by a factor greater than 100. Intercistronic complementation of these mutants with pm1493 and dl121, two SV40 mutants that are defective in agnoprotein but encode wild-type T antigen, results in an increased synthesis of agnoprotein in the infected cells. These results suggest that the carboxyl-terminal portion of T antigen participates in the posttranscriptional regulation of agnoprotein.

Animals↗

PubMed1976

Alkaline-extracted influenza subunit vaccine.

Treatment of influenza virus concentrates with alkaline solvents releases a major fraction of the viral structural protein content. As determined by polyacrylamide gel electrophoresis, the surface glycoprotein substructures, hemagglutinin and neuraminidase, are the primary solubilized products. Two forms of hemagglutinin antigen are recovered, a 39S active hemagglutinin and a 23S blocking antigen. Dose-response assays in mice demonstrate that hemagglutination-inhibiting and neuraminidase antibodies are induced. Antibody responses are comparable to those resulting from immunization with inactivated whole virus. On the basis of demonstrated purity, high yields of protective antigens, immunogenic potency, and absence of deleterious reagents, alkaline-extracted influenza protein preparations merit consideration as subunit vaccines for human use.

Alkalies↗

PubMed1989

A comparative study on the cell-free translation of the genomic RNAs of two aphid picorna-like viruses.

The genomic RNAs of aphid lethal paralysis virus (ALPV) and Rhopalosiphum padi virus (RhPV)--two distinct picorna-like viruses found in aphids [Williamson et al. (1988) J Gen Virol 69: 787-795]--were both efficiently translated in rabbit reticulocyte lysates. ALPV RNA was translated into primary products with molecular weights ranging from 92 kDa to 170 kDa. These underwent time-dependent post-translational cleavage to produce smaller polypeptides including some with molecular weights comparable to those of the viral structural proteins. A 92 kDa polypeptide as well as smaller proteins were immunoprecipitated with capsid protein antisera, indicating the presence of at least one large capsid subunit protein precursor. RhPV RNA was translated into products of molecular weights ranging from 45 kDa to 175 kDa. There was no evidence for time-dependent post-translation cleavage of RhPV translation products. However, a 60 kDa polypeptide was precipitated with antiserum to RhPV virions, indicating that at least one capsid protein of RhPV is derived by proteolysis of a precursor protein, like those of ALPV and most other picornaviruses.

Animals↗

PubMed1994

A recombinant immunoblot and ELISA for detection of acute parvovirus B19 infection.

Laboratory diagnosis of parvovirus B19 (B19) infection has been hampered by the limited availability of B19 virus. Recombinant viral proteins are now available for use as antigen in serological assays. We compared detection of anti-B19 IgM by "mu-capture assay" using viral B19 particles to a recombinant (rec.) immunoblot and a rec. enzyme-immunoassay (ELISA) using viral structural proteins as antigens expressed in E. coli. The rec. immunoblot was 94.3% sensitive and 96.4% specific for anti-B19 IgM, and the sensitivity of the rec. ELISA was 94.3% and the specificity, only 72.7%. There was an agreement between the "mu-capture assay" and the rec. immunoblot in 87.8% and the rec. ELISA in only 74.4%. For detection of anti-B19 IgG in patients with acute B19 infection, the rec. immunoblot was 94.3% and the rec. ELISA 85.7% sensitive. The rec. immunoblot is more reliable for detection of acute B19 infection than the rec. ELISA.

Antibodies, Viral↗

PubMed2006

The conserved dileucine- and tyrosine-based motifs in MLV and MPMV envelope glycoproteins are both important to regulate a common Env intracellular trafficking.

BACKGROUND: Retrovirus particles emerge from the assembly of two structural protein components, Gag that is translated as a soluble protein in the cytoplasm of the host cells, and Env, a type I transmembrane protein. Because both components are translated in different intracellular compartments, elucidating the mechanisms of retrovirus assembly thus requires the study of their intracellular trafficking. RESULTS: We used a CD25 (Tac) chimera-based approach to study the trafficking of Moloney murine leukemia virus and Mason-Pfizer monkey virus Env proteins. We found that the cytoplasmic tails (CTs) of both Env conserved two major signals that control a complex intracellular trafficking. A dileucine-based motif controls the sorting of the chimeras from the trans-Golgi network (TGN) toward endosomal compartments. Env proteins then follow a retrograde transport to the TGN due to the action of a tyrosine-based motif. Mutation of either motif induces the mis-localization of the chimeric proteins and both motifs are found to mediate interactions of the viral CTs with clathrin adaptors. CONCLUSION: This data reveals the unexpected complexity of the intracellular trafficking of retrovirus Env proteins that cycle between the TGN and endosomes. Given that Gag proteins hijack endosomal host proteins, our work suggests that the endosomal pathway may be used by retroviruses to ensure proper encountering of viral structural Gag and Env proteins in cells, an essential step of virus assembly.

Amino Acid Motifs↗

PubMed1984

Constitutive production of phagocytosis inducing factor(s) in a monocyte/macrophage lineage cell line (THP-1) by retrovirus-transformed human T cell lines.

Four human T cell lines, MT-2, TCL-Kan, TCL-As 2, and TCL-Haz, established from normal leukocytes by cocultivation with adult T-cell leukemia (ATL) virus (ATLV)-producing cells, produced constitutively phagocytosis inducing factor(s) (PIF) that induced phagocytosis in a human monocytic cell line, THP-1. These cell lines expressed ATLV-associated antigens (ATLA) as well as numerous virus particles, whereas the other twelve leukocyte cell lines tested, including T cell lines, B cell lines, and non-T and non-B cell lines, did not produce detectable amounts of the factor(s) in the culture supernatants. PIF was produced in the absence of serum and was not related to either ATLV-particles or viral structural proteins. Its activity was stable at 56 C for 30 min, but labile at 80 C for 30 min and at pH 2 for 20 hr. MT-2 and TCL-Kan produced large amounts of the factor(s) in the culture supernatants but little interferon-gamma (IFN-gamma) or colony stimulating factor (CSF) activity was detected; furthermore, the activity was not neutralized by rabbit anti-IFN-gamma sera. These observations suggest that some ATLV-transformed T cell lines produce PIF that is different from IFN-gamma and CSF.

Cell Line↗

PubMed1990

Function of Semliki Forest virus E3 peptide in virus assembly: replacement of E3 with an artificial signal peptide abolishes spike heterodimerization and surface expression of E1.

The Semliki Forest virus spike glycoproteins E1 and p62 form a heterodimeric complex in the endoplasmic reticulum (ER) and are transported as such to the cell surface. In the mature virus particle, the heterodimeric association of E1 and E2 (the cleavage product of p62) is maintained, but as a more labile and acid-sensitive oligomer than the E1-p62 complex. The E3 peptide forms the N-terminal part of the p62 precursor and carries the signal for the translocation of p62 into the lumen of the ER. The question of whether E3 is also important in the formation and stabilization of the E1-p62 heterodimer has been addressed here with the aid of an E3 deletion mutant cDNA. In this construct, the entire E3 was replaced with a cleavable, artificial signal sequence which preserved the membrane topology of an authentic E2. The E3 deletion, when expressed via a recombinant vaccinia virus, abolished heterodimerization of the spike proteins. It also resulted in the complete retention of E1 in the ER and almost total inhibition of E2 transport to the plasma membrane. The oligomerization and transport defect of E1 expressed from the E3 deletion mutant could be complemented with a wild-type p62 provided from a separate coding unit in double infections. These results point to a central role of E3 in complex formation and transport of the viral structural components to the site of budding. In conjunction with earlier work (M. Lobigs and H. Garoff, J. Virol. 64:1233-1240, 1990; J. Wahlberg, W. A. M. Boere, and H. Garoff, J. Virol. 63:4991-4997, 1989), the data support a model of spike protein oligomerization control of Semliki Forest virus assembly and disassembly which may be mediated by the presence of E3 in the uncleaved p62 precursor and release of E3 after cleavage.

Animals↗

PubMed2000

Characterization of the coronavirus mouse hepatitis virus strain A59 small membrane protein E.

The small envelope (E) protein has recently been shown to play an essential role in the assembly of coronaviruses. Expression studies revealed that for formation of the viral envelope, actually only the E protein and the membrane (M) protein are required. Since little is known about this generally low-abundance virion component, we have characterized the E protein of mouse hepatitis virus strain A59 (MHV-A59), an 83-residue polypeptide. Using an antiserum to the hydrophilic carboxy terminus of this otherwise hydrophobic protein, we found that the E protein was synthesized in infected cells with similar kinetics as the other viral structural proteins. The protein appeared to be quite stable both during infection and when expressed individually using a vaccinia virus expression system. Consistent with the lack of a predicted cleavage site, the protein was found to become integrated in membranes without involvement of a cleaved signal peptide, nor were any other modifications of the polypeptide observed. Immunofluorescence analysis of cells expressing the E protein demonstrated that the hydrophilic tail is exposed on the cytoplasmic side. Accordingly, this domain of the protein could not be detected on the outside of virions but appeared to be inside, where it was protected from proteolytic degradation. The results lead to a topological model in which the polypeptide is buried within the membrane, spanning the lipid bilayer once, possibly twice, and exposing only its carboxy-terminal domain. Finally, electron microscopic studies demonstrated that expression of the E protein in cells induced the formation of characteristic membrane structures also observed in MHV-A59-infected cells, apparently consisting of masses of tubular, smooth, convoluted membranes. As judged by their colabeling with antibodies to E and to Rab-1, a marker for the intermediate compartment and endoplasmic reticulum, the E protein accumulates in and induces curvature into these pre-Golgi membranes where coronaviruses have been shown earlier to assemble by budding.

Animals↗

PubMed1992

Phosphorylation of the Rex protein of human T-cell leukemia virus type I.

Rex protein, the posttranscriptional regulator of human T-cell leukemia virus type I (HTLV-I), is required for the control of viral structural protein expression and virus replication. Rex is a phosphoprotein found predominantly in the cell nucleolus, whose function is thought to be regulated by its nucleolar localization and phosphorylation. Therefore, we investigated the in vivo phosphorylation of Rex protein in more detail. Phosphorylation of Rex occurred in all HTLV-I-infected cell lines examined in vivo, primarily at serine residues and to a very small extent at threonine residues. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) led to significant but transient enhancement of the incorporation of [32P]orthophosphate into Rex protein. N-terminal truncation of Rex protein abolished TPA-dependent phosphorylation. Chymotryptic digestion of phosphorylated Rex yielded two phosphopeptides. In vivo phosphorylation sites were identified as serine residues 70 and 177 and threonine residue 174. Serine 70 was a TPA-dependent phosphorylation site within a regulatory domain. We have already shown that the protein kinase C inhibitor H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) specifically blocked accumulation of viral unspliced gag-pol mRNA. Therefore, the phosphorylation at serine 70 may be involved in the regulation of Rex function in response to extracellular stimuli.

Amino Acid Sequence↗

PubMed1990

[The results of using the molecular hybridization of nucleic acids in the complex study of nasopharyngeal smears from patients with influenza and other acute respiratory diseases].

A complex study of samples obtained from patients with influenza and other acute respiratory diseases has revealed that the laboratory methods used in this study can be rated in the following order according to their sensitivity: isolation of the virus in chick embryos, analysis of seroconversions in the hemagglutination inhibition test, immunofluorescent determination of viral antigens, determination of viral antigens by enzyme immunoassay (EIA), detection of RNA-containing viral structures by means of molecular hybridization. From the point of view of the possibility of documenting influenza A in patients, the best results are achieved by the combination of molecular hybridization and EIA techniques: 90% and more of all cases. A rational scheme for the examination of samples obtained from patients with a view to epidemiological study, including both traditional and new rapid diagnostic methods, is proposed.

Acute Disease↗

PubMed1984

Host cell glucose metabolism during abortive infection by adenovirus type 12.

Progeny virions were not produced following infection of an established line of African green monkey kidney (Vero) cells with human adenovirus type 12. In addition, viral structural proteins were not synthesized as determined by the immunofluorescent procedure to detect those proteins in infected cells. Cytopathology was observed in the virus-infected Vero cells, but it was less pronounced and differed in some other aspects to the cytopathic effect of adenovirus type 12 during productive infection. Glucose uptake and lactic acid production were monitored as indicators of glucose metabolism over a 48 h period during abortive infection. There was a 50 to 60% increase in glucose uptake and lactic acid production by virus-infected Vero cells compared with parallel cultures of uninfected cells.

Adenoviridae Infections↗

PubMed1999

Dissecting the immune response to moloney murine sarcoma/leukemia virus-induced tumors by means of a DNA vaccination approach.

The intramuscular inoculation of Moloney murine sarcoma/leukemia (M-MSV/M-MuLV) retroviral complex gives rise to sarcomas that undergo spontaneous regression due to the induction of a strong immune reaction mediated primarily by cytotoxic T lymphocytes (CTL). We used a DNA-based vaccination approach to dissect the CTL response against the Gag and Env proteins of M-MSV/M-MuLV in C57BL/6 (B6) mice and to evaluate whether plasmid DNA-immunized mice would be protected against a subsequent challenge with syngeneic tumor cells expressing the viral antigens. Intramuscular DNA vaccination induced CTL against both Gag and Env proteins. A detailed analysis of epitopes recognized by CTL generated in mice inoculated with the whole virus and with the Gag-expressing plasmid confirmed the presence of an immunodominant peptide in the leader sequence of Gag protein (Gag85-93, CCLCLTVFL) that is identical to that described in B6 mice immunized with Friend MuLV-induced leukemia cells. Moreover, CTL generated by immunization with the Env-encoding plasmid recognized a subdominant Env peptide (Env189-196, SSWDFITV), originally described in the B6.CH-2(bm13) mutant strain. B6 mice immunized with the Gag-expressing plasmid were fully protected against a lethal tumor challenge with M-MuLV-transformed MBL-2 leukemia cells, while vaccination with the Env-expressing plasmid resulted in rejection of the tumor in 44% of the mice and in increased survival of an additional 17% of the animals. Taken together, these results indicate the existence of a hierarchy in the capacity of different structural viral proteins to induce a protective immune response against retrovirus-induced tumors.

Animals↗