RESUMEN
MaTu is an agent, believed to be derived from a human mammary carcinoma, which displayed several extraordinary properties. These were: RIP and PAGE revealed in MaTu-infected cells only a single protein band of Mr 58 k, the gp 58. This gp 58 was immunoprecipitated by antibodies present in some human sera as well as in some sera of rabbits, sheep, and cattle. MaTu had an extremely restricted host range: it was transmissible only to HeLa cells, but not to human embryo fibroblasts, to three human tumour cell lines (T 47 D, T 24, and HMB 2) or to monkey Vero and rabbit SIRC cells. A retrovirus with a broad host range, used as a helper (X-MLV) enabled the transmission of MaTu to human fibroblasts, but not to Vero or SIRC, which are also permissive for X-MLV. These observations, together with our previous reports, support the view that MaTu might either be a novel type of defective virus, or even a non-viral autonomous genetic element.
Asunto(s)
Virus Defectuosos/patogenicidad , Retroviridae/patogenicidad , Virus de la Estomatitis Vesicular Indiana , Proteínas Virales/análisis , Animales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Virus Defectuosos/análisis , Virus Defectuosos/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Células HeLa , Virus Helper , Humanos , Sueros Inmunes , Virus de la Leucemia Murina , Pruebas de Precipitina , Conejos , Retroviridae/análisis , Retroviridae/aislamiento & purificación , Ovinos , Células Tumorales Cultivadas , Virus de la Estomatitis Vesicular Indiana/análisis , Virus de la Estomatitis Vesicular Indiana/aislamiento & purificación , Virus de la Estomatitis Vesicular Indiana/patogenicidadRESUMEN
Nine measles vaccine preparations, including four different viral strains, provided by eight different manufacturers were analysed by Northern blot for the nature of their nucleocapsid RNAs. Out of nine preparations, six were shown to contain subgenomic RNAs, along with the full length genomic RNA. Presence or absence of the subgenomic RNAs correlated strictly with the viral strains used. The role of the defective interfering particles in measles virus vaccine attenuation, and in its seroconversion efficacy upon vaccination, as well as the potential hazard of the presence of defective interfering particles in live-virus vaccine preparations, is discussed.
Asunto(s)
Vacuna Antisarampión/análisis , Virus del Sarampión/análisis , ARN Viral/análisis , Northern Blotting , Virus Defectuosos/análisis , Virus Defectuosos/genética , Vacuna Antisarampión/normas , Virus del Sarampión/genética , ARN Viral/genética , Estándares de Referencia , Vacunas Atenuadas/análisisRESUMEN
By starting from a thrice-purified wild-type measles virus plaque, the generation of detectable subgenomic RNAs was achieved within a series of five serial infections of Vero cells. The evolution of these subgenomic RNAs was followed for seven serial passages and ended with the preparation of a highly interfering viral stock. On the other hand, the detection of discrete subgenomic RNAs was achieved during the first infection of Vero cells with at least one of three measles virus vaccine preparations tested. These subgenomic RNAs, which interfered very efficiently with the replication of the endogenous standard genomes upon vaccine infection but showed a moderate interfering activity with a standard virus stock derived by plaque purification from the vaccine preparation, resulted from the presence of defective interfering particles in the vaccine preparation. The relevance of this finding for the attenuation, stability, and potential capacity for persistent infection of such a vaccine is discussed.
Asunto(s)
Virus Defectuosos/análisis , Vacuna Antisarampión/análisis , Virus del Sarampión/análisis , Vacunas Atenuadas/análisis , Interferencia Viral , Mapeo Cromosómico , Virus Defectuosos/genética , Amplificación de Genes , Virus del Sarampión/genética , ARN Viral/análisis , ARN Viral/genética , Virión/análisisRESUMEN
The mechanisms (factors) of the measles virus vaccine L-16 strain persistence in HEp-2 cell culture were analysed. Among the known mechanisms, most likely is the reduction of the cell-destroying properties of the persisting virus due to mutations in nucleoprotein gene manifested by changes of the isoelectric point of NP protein and temperature sensitivity of its synthesis.
Asunto(s)
Virus del Sarampión/fisiología , Células Cultivadas , Efecto Citopatogénico Viral , Virus Defectuosos/análisis , Virus Defectuosos/fisiología , Humanos , Virus del Sarampión/análisis , Mutación , Factores de Tiempo , Interferencia Viral , Proteínas Virales/análisis , Cultivo de VirusRESUMEN
Persistently HIV-infected cell lines were isolated from surviving and proliferating cells after infection of HTLV-I-carrying MT-4 cells with cell-free human immunodeficiency virus (HIV); HTLV-IIIB and LAV. The media of the cloned cell cultures did not cause HIV infection of MT-4, MOLT-4, TALL-1, or HL-60 cells. Most of the constituents of the virus in the media were env proteins and many defective doughnut-shaped particles released from the cells were identified by electron microscopy.
Asunto(s)
Virus Defectuosos/fisiología , Infecciones por Deltaretrovirus/microbiología , Deltaretrovirus/fisiología , VIH/fisiología , Replicación Viral , Línea Celular , Transformación Celular Viral , Células Clonales/microbiología , Células Clonales/ultraestructura , Virus Defectuosos/análisis , Virus Defectuosos/ultraestructura , Infecciones por Deltaretrovirus/patología , VIH/análisis , VIH/ultraestructura , Humanos , Proteínas del Envoltorio Viral/aislamiento & purificaciónAsunto(s)
Hepatitis A/prevención & control , Hepatitis B/prevención & control , Adolescente , Adulto , Portador Sano/prevención & control , Niño , Preescolar , Virus Defectuosos/análisis , Femenino , Hepatitis A/transmisión , Hepatitis B/transmisión , Antígenos de Superficie de la Hepatitis B/análisis , Virus de la Hepatitis B/análisis , Hepatitis C/prevención & control , Homosexualidad , Humanos , Inmunización Pasiva , Lactante , Masculino , Embarazo , Riesgo , Conducta Sexual , Estados Unidos , Vacunas contra Hepatitis Viral/administración & dosificación , Vacunas contra Hepatitis Viral/efectos adversosRESUMEN
The major capsid protein of polyomavirus, VP1, was separated into at least four subspecies by isoelectric focusing. One of these subspecies was selectively extracted from purified virions by mild treatment with sodium dodecyl sulfate, leaving a 140S particle enriched in the other three forms. The two most acidic subspecies were labeled in vivo with [32P]phosphate, and these subspecies are among those identified as being deficient in nontransforming host range (hr-t) mutant virus nonpermissive infection of NIH3T3 cells. Quantitation of VP1 phosphorylation revealed that hr-t mutant virus VP1 is phosphorylated to about 40 to 50% the level of the wild type in NIH3T3 cells, and two-dimensional phosphoamino acid analysis suggested that threonine phosphorylation was affected more than serine phosphorylation. Two results indicate that the VP1 modifications occur before and independent of virus assembly: modified subspecies were detected during wild-type infection within a 2-min pulse-label with [32S]methionine, and VP1 modifications of temperature-sensitive VP1 mutants were the same at both restrictive and permissive temperatures for virus assembly. We conclude that most VP1 modification occurs before viral DNA encapsidation, and that one defect in hr-t mutant virus assembly is in VP1 phosphorylation, primarily affecting threonine.
Asunto(s)
ADN Viral/metabolismo , Virus Defectuosos/análisis , Poliomavirus/análisis , Proteínas Virales/análisis , Virión/análisis , Mutación , Fosforilación , Poliomavirus/metabolismo , Serina/metabolismo , Treonina/metabolismo , Proteínas Virales/metabolismo , Proteínas Estructurales ViralesRESUMEN
Establishment of a persistent infection by echovirus 6 in cloned human WISH cells (PI) was demonstrated. The cloned human WISH cells were maintained for 3.0 years (over 125 passages) and released virus continuously without cellular destruction. Neither temperature-sensitive virus mutants nor interferon appears to play a role in either establishment or maintenance of viral persistence. The majority of the virus produced by cloned human WISH cells is defective (2 X 10(6) particles per PFU) and differs from standard virus in its polypeptide profile and its inability to attach to parental WISH cells.
Asunto(s)
Células Cultivadas/microbiología , Echovirus 6 Humano/crecimiento & desarrollo , Infecciones por Echovirus/microbiología , Enterovirus Humano B/crecimiento & desarrollo , Células Clonales/microbiología , Virus Defectuosos/análisis , Virus Defectuosos/crecimiento & desarrollo , Humanos , Temperatura , Proteínas Virales/análisis , Replicación ViralRESUMEN
DX particles of poliovirus are deletion mutants that do not induce synthesis of capsid proteins or the precursor of capsid proteins (NCVPla) during infection. However, cells infected with DX particles synthesize two proteins, p68 and p25, that are not detected during growth of standard virus, and a protein of 27 000 (p27) which is comparable in molecular weight to VP3. Peptide maps of these proteins were obtained by partial digestion with Staphylococcus aureus V8 protease and elastase. The peptide map of p68 corresponded approximately 70% with the peptide map of NCVPla, and antiserum against virions reacted with p68. These data suggest that p68 is a large fragment of NCVPla. Digestion of purified structural proteins VP1, VP2, and VP3 yielded distinct peptide maps, but p25 was resistant to both V8 protease and elastase and did not react noticeably with anticapsid antibody. Peptide maps obtained for in vivo viral proteins migrating with a molecular weight of 27 000 were complex, indicating the presence of at least two and possibly three proteins. Cells infected with standard gs and gr viruses produced authentic VP3, but cells infected with defective interfering particles did not. However, one gr variant of standard virus contained a mutation in structural protein VP2.
Asunto(s)
Virus Defectuosos/análisis , Poliovirus/análisis , Proteínas Virales/aislamiento & purificación , Cápside/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida/métodos , Endopeptidasas , Células HeLa/análisis , Humanos , Peso Molecular , Fragmentos de Péptidos/análisisRESUMEN
Hepatitis B virus (HBV) DNA was found to be integrated into seven sites in the DNA of the PLC/PRF/5 hepatoma cell line as determined by digestion with the restriction endonuclease HindIII which does not cut through the viral genome. The integration pattern was stable in the cell line, in tumours induced in athymic mice by this line and in cell lines derived from such tumours. Syntheses of hepatitis B surface antigen and alphafoetoprotein were maintained in the induced tumours and derived cell lines. A defective HBV DNA molecule (approx. 2.8 kilobase pairs) appears to be integrated in a head-to-tail tandem arrangement and it is proposed that such defective molecules may be involved in the process of neoplastic transformation by HBV.
Asunto(s)
Carcinoma Hepatocelular/análisis , ADN Viral/análisis , Virus Defectuosos/análisis , Virus de la Hepatitis B/análisis , Neoplasias Hepáticas Experimentales/análisis , Neoplasias Hepáticas/análisis , Animales , Línea Celular , ADN de Neoplasias/análisis , Antígenos de Superficie de la Hepatitis B/análisis , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , alfa-Fetoproteínas/análisisRESUMEN
[3H]uridine-labeled extracellular West Nile virus (WNV) particles produced by cell cultures obtained from genetically resistant C3H/RV and congenic susceptible C3H/HE mice were compared by sucrose density gradient centrifugation as well as by analysis of the particle RNA. Defective interfering (DI) WNV particles were observed among progeny produced during acute infections in both C3H/RV and C3H/HE cells. Although only a partial separation of standard and DI particles was achieved, the DI particles were found to be more dense than the standard virions. Particles containing several species of small RNAs consistently constituted a major proportion of the total population of virus progeny produced by C3H/RV cells, but a minor proportion of the population produced by C3H/HE cells. Decreasing the multiplicity of infection or extensive plaque purification of the WNV inoculum decreased the proportion of small RNAs found in the progeny virus. The ratio of DI particles to standard virus observed in progeny virus was determined by the cell type used to grow the virus. The ratio could be shifted by passaging virus from one cell type to the other. Homologous interference could be demonstrated with WNV produced by C3H/RV cells but not with virus produced by C3H/HE cells. Continued passage of WNV in C3H/HE cells resulted in a cycling of infectivity. However, passage in C3H/RV cells resulted in the complete loss of infectious virus. Four size classes of small viral RNA, with sedimentation coefficients of about 8, 15, 26, and 34S, were observed in the extracellular particles. A preliminary analysis of these RNAs by oligonucleotide fingerprinting indicated that the smaller RNAs were less complex than the 40S RNA and differed from each other. The data are consistent with the conclusion that WNV DI particles interfere more effectively with standard virus replication and are amplified more efficiently in C3H/RV cells than in congenic C3H/HE cells. The relevance of these findings to the further understanding of genetically controlled resistance to flaviviruses is discussed.
Asunto(s)
Virus Defectuosos/fisiología , Infecciones por Togaviridae/microbiología , Interferencia Viral , Fiebre del Nilo Occidental/microbiología , Virus del Nilo Occidental/fisiología , Animales , Células Cultivadas , Centrifugación por Gradiente de Densidad , Virus Defectuosos/análisis , Inmunidad Innata , Ratones , Ratones Endogámicos C3H , ARN Viral/análisis , ARN Viral/biosíntesis , Ensayo de Placa Viral , Replicación Viral , Fiebre del Nilo Occidental/genética , Virus del Nilo Occidental/análisisRESUMEN
We have used an antiserum directed against a synthetic v-mos peptide (anti-C3 serum) to screen ts110 murine sarcoma virus (MuSV)-infected cells for the presence of v-mos-encoded proteins. Anti-C3 serum specifically recognized an 85,000-dalton protein doublet (P85) from [35S]methionine-labeled ts110 MuSV-infected producer cells grown at 32 degrees C, the permissive temperature for transformation. The P85 doublet was also recognized by an antiserum directed against the viral gag protein p15. P85 was present but at 2- to 10-fold-lower levels in ts110 MuSV-infected producer cells grown at 39 degrees C, the restrictive temperature for transformation. The P85gag-mos fusion product was the only v-mos protein reproducibly detected in this ts110 MuSV-transformed cell line. Immunoprecipitation of 32P-labeled cells with anti-C3 serum revealed that the upper band of the P85 doublet is phosphorylated, containing mostly phosphoserine and some phosphothreonine. Cells acutely infected with ts110 MuSV contained slightly higher levels of P85 than did the ts110 MuSV-infected producer cell line. Anti-C3 serum specifically recognized a 33,000-dalton protein (p33) in the acutely infected cells labeled with [35S]methionine. p33 was present in trace amounts and may represent a previously unidentified ts110 MuSV-encoded v-mos protein.
Asunto(s)
Virus Defectuosos/análisis , Virus de la Leucemia Murina de Moloney/análisis , Virus del Sarcoma Murino/análisis , Proteínas Virales/análisis , Animales , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Electroforesis en Gel de Poliacrilamida , Productos del Gen gag , Ratones , Fosfoserina/análisis , Fosfotreonina/análisis , Ratas , Proteínas del Núcleo Viral , Proteínas Virales/inmunologíaRESUMEN
Two murine leukemia viruses were isolated from JLS-V9 cells which had been infected with Rauscher plasma virus. One virus was XC positive and failed to grow on mink or cat cells and thus was an ecotropic virus. The other virus formed cytopathic foci on mink cells, was XC negative, and fell into the mink cell focus-forming (MCF) viral interference group and was thus an MCF virus. The glycoproteins of the two viruses could be distinguished immunologically, by peptide mapping, and by size in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The MCF virus produced gp69, and the ecotropic virus produced gp71, explaining the origin of the heterogeneous glycoprotein (gp69 and gp71) of Rauscher leukemia virus. Amino-terminal sequences of gp69 and gp71 were determined. The MCF sequence was distinct from the ecotropic sequence, but retained partial homology to it. The data show that the glycoproteins are encoded by related yet distinct genes. The protein structural data support the proposal that MCF virus gp70 molecules have nonecotropic sequences at the amino terminus, with ecotropic sequences occurring at the 3' end of the gene. The Rauscher MCF virus glycoprotein lacks a glycosylation site found at position 12 of the ecotropic sequence.
Asunto(s)
Gammaretrovirus/análisis , Virus Rauscher/análisis , Proteínas Virales/análisis , Animales , Gatos , Células Cultivadas , Virus Defectuosos/análisis , Fibroblastos/análisis , Gammaretrovirus/genética , Gammaretrovirus/inmunología , Glicoproteínas/análisis , Ratones , Ratones Endogámicos AKR/microbiología , Visón , Virus Rauscher/genética , Virus Rauscher/inmunología , Proteínas del Envoltorio Viral , Proteínas Virales/genéticaRESUMEN
Fujinami sarcoma virus (FSV) encodes a transforming protein of 130,000 daltons (P130) which is associated with a tyrosine-specific protein kinase activity. To elucidate mechanisms involved in cell transformation by FSV, we have studied the intracellular location of P130 in rat cells nonproductively infected with FSV. Immunofluorescent staining of several FSV-transformed rat cell lines with a tumor regressor antiserum specific against the fps sequences of P130 showed that the major staining was localized in the cytoplasm. Staining was also seen in cell ruffles and in some cases at areas of cell contact. The cytoplasmic location of P130 staining in cells infected with temperature-sensitive mutants of FSV was unchanged when they were grown at permissive or nonpermissive temperature. Cell fractionation of FSV-transformed cells under various conditions showed that the ionic strength used during cell fractionation had a striking effect on the distribution of P130. At 10 mM NaCl, 70% of P130 sedimented in the large granule fraction, whereas at 500 mM NaCl 70 to 90% of P130 was recovered in the cytosol fraction. Furthermore, a combination of ionic and nonionic detergents that effectively solubilized subcellular membranes was insufficient to solubilize P130 unless the salt concentration was raised. We conclude that the majority of P130 and its associated protein kinase activity are localized in the cytoplasm and that P130 is not an integral membrane protein.
Asunto(s)
Virus del Sarcoma Aviar/análisis , Citoplasma/análisis , Virus Defectuosos/análisis , Proteínas Quinasas/análisis , Animales , Virus del Sarcoma Aviar/genética , Células Cultivadas , Virus Defectuosos/genética , Detergentes , Embrión de Mamíferos , Fibroblastos , Técnica del Anticuerpo Fluorescente , Proteínas Tirosina Quinasas , Ratas , Cloruro de Sodio , TemperaturaRESUMEN
It has previously been shown that infectious bursal disease virus is a naked icosahedral particle with a diameter of about 60 nm and a genome consisting of two segments of double-stranded RNA (Müller et al., J. Virol. 31:584-589, 1979). One of the two major structural polypeptides (molecular weight, 40,000) of this virus could not be found in lysates of infected cells; it is derived from a precursor polypeptide demonstrable inside the cells in relatively large quantities and seems to be processed during virus assembly or later. The precursor molecule is regularly present in the infectious virus particle (buoyant density, 1.33 g/ml) in minor proportions, but it represents an outstanding structural element of incomplete noninfectious particles ("top components"; buoyant density, 1.29 g/ml) which contain viral RNA. This type of incomplete particles is mainly produced by chicken embryo fibroblasts in contrast to lymphoid cells from the bursa of Fabricius. Precursor-product relationships also seem to exist in the biosynthesis of the other viral polypeptides. In contrast to some other viruses with a segmented double-stranded RNA genome, none of the structural proteins of infectious bursal disease virus is appreciably glycosylated.
Asunto(s)
Virus Defectuosos/metabolismo , Virus de la Enfermedad Infecciosa de la Bolsa/metabolismo , Reoviridae/metabolismo , Proteínas Virales/biosíntesis , Animales , Células Cultivadas , Embrión de Pollo , Pollos , Virus Defectuosos/análisis , Virus de la Enfermedad Infecciosa de la Bolsa/análisis , Virus de la Enfermedad Infecciosa de la Bolsa/crecimiento & desarrollo , Péptidos/análisis , Precursores de Proteínas/biosíntesis , Proteínas Virales/análisis , Proteínas Estructurales ViralesRESUMEN
The arrangement of endogenous ecotropic retroviruses in selected high- and low-ecotropic-virus-producing mouse strains was examined by Southern blot hybridization analysis, using an ecotropic retrovirus-specific DNA probe. High-ecotropic-virus-producing mouse strains of the AKR family displayed heterogeneity with respect to the number of copies and the sites of insertion of endogenous ecotropic specific DNA. This diversity was seen even among individuals of the same AKR subline. Contrastingly, individuals within the same low-ecotropic-retrovirus-producing mouse strain showed no evidence of variability in their endogenous ecotropic proviral sequences. These results favored the hypothesis that germ line proviral reinsertion was responsible for the proviral sequence heterogeneity observed in high-ecotropic-virus-producing mouse strains.
Asunto(s)
Virus de la Leucemia Murina AKR/análisis , Virus de la Leucemia Murina/análisis , Ratones Endogámicos AKR/microbiología , Animales , Enzimas de Restricción del ADN , ADN Viral/análisis , Virus Defectuosos/análisis , Ratones , Ratones Endogámicos BALB C/microbiología , Ratones Endogámicos C57BL/microbiología , Ratones Endogámicos DBA/microbiología , Hibridación de Ácido NucleicoRESUMEN
HEp-2 and L-41 culture chronically infected with mumps virus at late passage levels produced 10(2)--10(3) PFU/ml small-plaque slow-replicating virus and defective interfering particles (DIP) in significant amounts. The DIP were characterized by the polypeptide composition similar to that of the virus, the presence of subgenome sise of RNA, and marked interfering activity with regard to the original mumps virus.