RESUMEN
HIV-1 retrovirus-like particles can be produced in VERO cells that have been transfected with an expression construct encoding HIV-1 structural proteins. The particles are entirely non-infectious although structurally they resemble infectious virus particles. This makes them a promising candidate for use as an HIV-1 vaccine. In order to ensure their safety and enhance their immunogenicity, the retrovirus-like particles were modified in a number of ways. A large deletion in the HIV-1 pol gene has eliminated reverse transcriptase and integrase activities. Deletion of RNA packaging signals in the RNA untranslated leader sequence and in Gag reduced packaged RNA to 5% of that in HIV-1 virus. Replacement of the existing HIV-1LAI envelope protein with that of HIV-1MN has ensured that immune responses to the particles are relevant to those against the majority of HIV-1 clade B isolates. In addition to these changes in particle composition, yields of the modified particles were increased using a superior method of inducing the expression construct promoter, and an effective scheme for particle purification was developed. Immunization of non-human primates demonstrated that the particles were capable of generating anti-HIV-1 neutralizing antibodies. The technological refinements reported here will permit retrovirus-like particles to be tested safely in humans, and the change in envelope proteins should allow a more realistic evaluation of the immunogenicity of these particles. Experience gained in engineering these refinements will greatly facilitate other modifications that may be required to achieve maximum efficacy as a vaccine against HIV-1.
Asunto(s)
Vacunas contra el SIDA/inmunología , VIH-1/inmunología , Vacunas Sintéticas/inmunología , Vacunas contra el SIDA/genética , Secuencia de Aminoácidos , Animales , Chlorocebus aethiops , Seguridad de Productos para el Consumidor , Expresión Génica , Productos del Gen gag/genética , Productos del Gen gag/inmunología , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/inmunología , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , VIH-1/genética , VIH-1/patogenicidad , VIH-1/fisiología , Humanos , Macaca mulatta , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Vacunas Sintéticas/genética , Células Vero , Virión/fisiología , Ensamble de VirusRESUMEN
We have established two cell lines that constitutively synthesize a temperature-sensitive form of ICP4, the herpes simplex virus immediate-early protein that activates early and late transcription. ICP4 in both cell lines was confirmed to be functionally temperature sensitive when tested by complementation of an ICP4 deletion mutant virus for expression of viral early and late genes. When grown at the permissive temperature the two cell lines contained approximately 5 and 25%, respectively, of the ICP4 present in control HSV-infected cells. If the cells were grown at the nonpermissive temperature, ICP4 levels were reduced by approximately fourfold; a twofold reduction was observed in control cells synthesizing the wild-type protein. The lower levels of ICP4 at the nonpermissive temperature were the result of two effects: a decrease in mRNA which was similar in cells producing the mutant or wild-type form of ICP4 and a more rapid turnover of the protein which was greater for the mutant than for the wild-type form. Our observations of lower levels of ICP4 in producer cells differ from published reports of overproduction of immediate-early proteins at the nonpermissive temperature in human or hamster cells infected with ICP4 temperature-sensitive mutant viruses. This discrepancy may be related to cell species differences since we observed only a modest twofold overproduction of immediate-early proteins at the nonpermissive temperature in infections of mouse cell lines with an ICP4 temperature-sensitive mutant virus.
Asunto(s)
Proteínas Inmediatas-Precoces , Simplexvirus/metabolismo , Factores de Transcripción/biosíntesis , Proteínas Virales/biosíntesis , Animales , Línea Celular , Regulación de la Expresión Génica , Ratones , Simplexvirus/genética , Temperatura , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Proteínas Virales/genética , Proteínas Virales/fisiologíaRESUMEN
It has been previously reported that immediate early proteins of pseudorabies and cytomegalo viruses can substitute for the products of the human adenovirus type 5 (Ad5) E1A gene in the activation of early Ad5 transcription. In the present report the effect of one of the herpes simplex virus type 1 (HSV-1) immediate early genes, ICP4, on Ad5 early gene expression has been examined using mouse cell lines that constitutively express ICP4. These lines as well as nonproducers were infected with wild-type (wt) Ad5 or with various Ad5 E1A mutants and the levels of expression of the Ad5 E2A 72K DNA binding protein were measured by immunoprecipitation with a monoclonal antibody specific for 72K. With dl 312, which lacks E1A, some 72K expression was seen in nonproducer lines but levels were considerably higher in the producer lines. A similar result was also obtained using dl 312-infected nonproducer cells that were superinfected with HSV-1 virions. These data suggest that HSV-1 ICP4 can substitute for E1A in the activation of expression of early Ad5 proteins. With wt Ad5, 72K was also expressed at high levels in nonproducer mouse cells, however, in the ICP4 producer cell lines, a marked inhibition of 72K expression was observed and this inhibition correlated with the amount of ICP4 present. Using the E1A mutants pm 975 and hr 1, this inhibition was found to be specific for the products of the 1.1-kb E1A mRNA. These data suggest that ICP4 and E1A proteins either directly inhibit each other, or more likely, operate independently and competitively on factors required for viral gene activation.
Asunto(s)
Adenovirus Humanos/genética , Proteínas Oncogénicas Virales/genética , Simplexvirus/genética , Proteínas Precoces de Adenovirus , Animales , Anticuerpos Monoclonales , Secuencia de Bases , Línea Celular , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Genes , Genes Virales , Humanos , Células KB/citología , Ratones , Peso Molecular , Proteínas Oncogénicas Virales/aislamiento & purificación , Timidina Quinasa/genéticaRESUMEN
To study the role of herpes simplex virus type 1 immediate-early proteins in the transcriptional activation of herpes simplex virus genes, we isolated stably transformed cells expressing herpes simplex virus type 1 ICP4, an immediate-early protein known from previous studies to be necessary for delayed-early and late transcription. These cells efficiently expressed six delayed-early herpes simplex virus genes introduced by viral superinfection, in the absence of de novo viral protein synthesis. In contrast, the delayed-early gene encoding alkaline exonuclease and the late gene encoding the capsid protein VP5 were expressed at much lower levels. Expression of a second late gene, that for glycoprotein C, was undetectable under the same experimental conditions. These results suggest that many, but not all, delayed-early genes are efficiently activated by ICP4; in addition, they demonstrate that although the late gene for VP5 is detectably activated by ICP4, its full expression requires additional factors.
Asunto(s)
Genes Virales , Simplexvirus/genética , Proteínas Virales/genética , Animales , Secuencia de Bases , Activación Enzimática , Exonucleasas/genética , Timidina Quinasa/análisis , Proteínas Virales/análisis , Proteínas Virales/farmacología , Proteínas Estructurales ViralesRESUMEN
The double-stranded RNA segments of infectious pancreatic necrosis virus were extracted from virions by a method which avoids proteinase. In contrast to proteinase-treated RNA, such segments (i) exhibited a lower electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels and agarose gels, (ii) had a slightly lower buoyant density, and (iii) demonstrated a marked tendency toward aggregation as observed by electron microscopy. A small amount of protein tightly bound to the RNA could account for the above properties, and a 110,000-dalton protein was liberated from purified virion RNA by sequential digestion with RNase III and RNase A. The amount of radioactivity associated with RNA from virions labeled in vivo with [35S]methionine suggested that an average of 1.4 molecules was bound per RNA segment. Interactions between RNA segments seen in electron micrographs appeared to occur only among the ends of the segments, suggesting these were the exclusive sites of protein attachment.