RESUMEN

The cytoplasmic domains of many membrane proteins contain sorting signals that mediate their endocytosis from the plasma membrane. VZV gB contains three consensus internalization motifs within its cytoplasmic domain: YMTL (aa 818-821), YSRV (aa 857-860), and LL (aa 841-842). To determine whether VZV gB is internalized from the plasma membrane, and whether these motifs are required for its endocytosis, we compared the internalization of native gB to that of gB containing mutations in each of the predicted internalization motifs. VZV gB present on the surface of transfected cells associated with clathrin and was efficiently internalized to the Golgi apparatus within 60 min at 37 degrees C. VZV gB containing the mutation Y857 failed to be internalized, while gB-Y818A was internalized but did not accumulate in the Golgi. These data indicate that the internalization of VZV gB, and its subsequent localization to the Golgi, is mediated by two tyrosine-based sequence motifs in its cytoplasmic domain.

Asunto(s)

Antígenos Virales/metabolismo , Glicoproteínas/metabolismo , Herpesvirus Humano 3/patogenicidad , Proteínas del Envoltorio Viral/metabolismo , Secuencias de Aminoácidos/genética , Clatrina/metabolismo , Endocitosis , Glicoproteínas/genética , Aparato de Golgi/metabolismo , Herpes Zóster/virología , Herpesvirus Humano 3/metabolismo , Humanos , Mutación , Factores de Tiempo , Células Tumorales Cultivadas , Proteínas del Envoltorio Viral/genética

RESUMEN

Normal herpesvirus assembly and egress depend on the correct intracellular localization of viral glycoproteins. While several post-Golgi transport motifs have been characterized within the cytoplasmic domains of various viral glycoproteins, few specific endoplasmic reticulum (ER)-to-Golgi transport signals have been described. We report the identification of two regions within the 125-amino-acid cytoplasmic domain of Varicella-Zoster virus gB that are required for its ER-to-Golgi transport. Native gB or gB containing deletions and specific point mutations in its cytoplasmic domain was expressed in mammalian cells. ER-to-Golgi transport of gB was assessed by indirect immunofluorescence and by the acquisition of Golgi-dependent posttranslational modifications. These studies revealed that the ER-to-Golgi transport of gB requires a nine-amino-acid region (YMTLVSAAE) within its cytoplasmic domain. Mutations of individual amino acids within this region markedly impaired the transport of gB from the ER to the Golgi, indicating that this domain functions by a sequence-dependent mechanism. Deletion of the C-terminal 17 amino acids of the gB cytoplasmic domain was also shown to impair the transport of gB from the ER to the Golgi. However, internal mutations within this region did not disrupt the transport of gB, indicating that its function during gB transport is not sequence dependent. Native gB is also transported to the nuclear membrane of transfected cells. gB lacking as many as 67 amino acids from the C terminus of its cytoplasmic domain continued to be transported to the nuclear membrane at apparently normal levels, indicating that the cytoplasmic domain of gB is not required for nuclear membrane localization.

Asunto(s)

Citoplasma/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Herpesvirus Humano 3/metabolismo , Señales de Clasificación de Proteína/fisiología , Proteínas del Envoltorio Viral/metabolismo , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Relación Estructura-Actividad , Células Tumorales Cultivadas , Proteínas del Envoltorio Viral/química

RESUMEN

The safety and immunogenicity of a novel hepatitis B virus (HBV) vaccine containing recombinant PreS2 and S antigens combined with MF59 adjuvant (HBV/MF59) was evaluated in healthy adults (N=230) who were randomized to receive 2 or 3 immunizations of either the study vaccine or a licensed control vaccine (Recombivax HB). After a single immunization, 105 of 118 (89%) recipients of HBV/MF59 achieved protective serum levels of anti-HBs antibody (> 10 mIU/ml), compared with 13 of 110 (12%) recipients of licensed vaccine (P < 0.001). The geometric mean titer (GMT) after 2 doses of HBV/MF59 given 2 months apart (13,422 mIU/ml) was more than 5-fold higher than that following 3 doses of licensed vaccine given over 6 months (2,346 mIU/ml; P < 0.001). The GMT following 3 injections of HBV/MF59 (249,917 mIU/ml) was 100-fold higher than licensed vaccine (P < 0.001). Anti-PreS2 antibodies were elicited in over 90% of the subset of HBV/MF59 recipients tested. Both vaccines were well tolerated; transient, mild-to-moderate local inflammation was the major postinjection reaction.

Asunto(s)

Adyuvantes Inmunológicos , Vacunas contra Hepatitis B/inmunología , Hepatitis B/prevención & control , Polisorbatos/análisis , Escualeno/análisis , Escualeno/inmunología , Adyuvantes Inmunológicos/efectos adversos , Adolescente , Adulto , Anticuerpos Antivirales/biosíntesis , Femenino , Humanos , Tolerancia Inmunológica , Inmunización Secundaria , Masculino , Polisorbatos/efectos adversos , Escualeno/efectos adversos , Factores de Tiempo

RESUMEN

The varicella-zoster virus (VZV) genes ORF47 and ORF66 are predicted to encode serine/threonine protein kinases, which are homologs of herpes simplex virus 1 (HSV-1) UL13, and US3. When mutants were constructed by inserting stop codons into ORF47 and ORF66, the recombinants ROka47S and ROka66S, as well as intact ROka replicated in tissue culture. In contrast, inoculation of human thymus/liver or skin implants in SCID-hu mice showed that ORF47 protein was required for viral growth in human T cells and skin. Eliminating ORF66 expression inhibited VZV infectivity for T cells partially but did not impair replication in skin compared with ROka. Infectivity for T cells and skin was restored when ROka47S virus was complemented by insertion of ORF47 into a distant, noncoding site. The ORF47 gene product is the first VZV protein identified as necessary for T cell tropism. It also is essential for skin infectivity in vivo, as is glycoprotein C. Expression of ORF66 did not compensate for the absence of the ORF47 protein. The requirement for ORF47 expression in T cells and skin indicates that this gene product, which is dispensable in vitro, has a critical role within differentiated cells that are essential targets for VZV pathogenesis in vivo.

Asunto(s)

Herpesvirus Humano 3/enzimología , Herpesvirus Humano 3/genética , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Piel/virología , Linfocitos T/virología , Animales , Secuencia de Bases , Quimera , Cartilla de ADN/genética , Eliminación de Gen , Expresión Génica , Genes Virales , Herpesvirus Humano 3/patogenicidad , Humanos , Hibridación in Situ , Hígado/patología , Hígado/virología , Masculino , Ratones , Ratones SCID , Sistemas de Lectura Abierta , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Piel/patología , Virulencia/genética , Virulencia/fisiología , Replicación Viral

RESUMEN

Varicella-zoster virus (VZV) open reading frames (ORFs) 47 and 66 encode proteins that are homologous to a family of eukaryotic serine-threonine kinases. Prior studies showed that the VZV ORF47 protein has kinase activity in vitro and is dispensable for replication in cultured cells. To examine the role of the ORF66 protein during infection, we constructed VZV recombinants that are unable to express either the ORF66 protein (ROka 66S) or both the ORF47 and ORF66 proteins (ROka 47S/66S). VZV unable to express ORF66 grew to titers similar to those of the parental VZV (ROka) in vitro; however, VZV lacking both ORF66 and ORF47 grew to titers lower than those of ROka. Nuclear extracts from ROka 66S- or ROka 47S-infected cells showed a 48-kDa phosphoprotein(s); a phosphoprotein with a similar size was not present in nuclear extracts from ROka 47S/66S-infected cells. To determine the role of the ORF66 protein in the phosphorylation of specific VZV-encoded proteins, we immunoprecipitated known VZV phosphoproteins (ORF4, ORF62, ORF63, and ORF68 proteins) from nuclear extracts of phosphate-labeled cells infected with ROka, ROka 66S, or ROka 47S/66S. Each of the VZV phosphoproteins was phosphorylated to a similar extent in the presence or absence of either the ORF66 protein or both the ORF66 and ORF47 proteins. From these studies we conclude (i) neither ORF66 alone nor ORF66 and ORF47 in combination are essential for VZV growth in cultured cells, (ii) ORF66 either is a protein kinase or induces protein kinase activity during infection, and (iii) the VZV phosphoproteins encoded by ORF4, ORF62, ORF63, and ORF68 do not require either ORF66 alone or ORF66 and ORF47 for phosphorylation in vitro.

Asunto(s)

Herpesvirus Humano 3/fisiología , Proteínas Quinasas/metabolismo , Proteínas Virales/metabolismo , Replicación Viral , Secuencia de Bases , Activación Enzimática , Humanos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta

RESUMEN

Varicella-zoster virus (VZV) is an attractive candidate for a live-virus vector for the delivery of foreign antigens. The Oka vaccine strain of VZV is safe and effective in humans, and recombinant Oka VZV (ROka) can be generated by transfecting cells with a set of overlapping cosmid DNAs. By this method, the herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) gene was inserted into an intergenic site in the unique short region of the Oka VZV genome. Expression of gD2 in cells infected with the recombinant Oka strain VZV (ROka-gD2) was confirmed by antibody staining of fixed cells and by immunoblot analysis. Immune electron microscopy demonstrated the presence of gD2 in the envelope of ROka-gD2 virions. The ability of ROka-gD2 to protect guinea pigs against HSV-2 challenge was assessed by inoculating animals with three doses of uninfected human fibroblasts, fibroblasts infected with ROka VZV, or fibroblasts infected with ROka-gD2. Neutralizing antibodies specific for HSV-2 developed in animals immunized with ROka-gD2. Forty days after the third inoculation, animals were challenged intravaginally with HSV-2. Inoculation of guinea pigs with ROka-gD2 significantly reduced the severity of primary HSV-2 infection (P < 0.001). These experiments demonstrate that the Oka strain of VZV can be used as a live virus vector to protect animals from disease with a heterologous virus.

Asunto(s)

Herpes Genital/fisiopatología , Herpesvirus Humano 2/inmunología , Herpesvirus Humano 3 , Vacunas Sintéticas , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales , Animales , Southern Blotting , ADN Viral/análisis , Femenino , Cobayas , Herpes Genital/inmunología , Herpes Genital/prevención & control , Herpesvirus Humano 3/genética , Humanos , Inmunización , Microscopía Inmunoelectrónica , Proteínas del Envoltorio Viral/análisis , Proteínas del Envoltorio Viral/biosíntesis

RESUMEN

To investigate the role of varicella-zoster virus (VZV) open reading frame 47 (ORF47) protein kinase during infection, a VZV mutant was generated in which two contiguous stop codons were introduced into ORF47, thus eliminating expression of the ORF47 kinase. ORF47 kinase was not essential for the growth of VZV in cultured cells, and the growth rate of the VZV mutant lacking ORF47 protein was indistinguishable from that of parental VZV. Nuclear extracts from cells infected with parental VZV contained several phosphorylated proteins which were not detected in extracts from cells infected with the ORF47 mutant. The herpes simplex virus type 1 (HSV-1) UL13 protein (the homolog of VZV ORF47 protein) is responsible for the posttranslational processing associated with phosphorylation of HSV-1 ICP22 (the homolog of VZV ORF63 protein). Immunoprecipitation of 32P-labeled proteins from cells infected with parental virus and those infected with ORF47 mutant virus yielded similar amounts of the VZV phosphoproteins encoded by ORF4, ORF62, ORF63, and ORF68 (VZV gE), and the electrophoretic migration of these proteins was not affected by the lack of ORF47 kinase. Therefore, while the VZV ORF47 protein is capable of phosphorylating several cellular or viral proteins, it is not required for phosphorylation of the ORF63 protein in virus-infected cells.

Asunto(s)

Herpesvirus Humano 3/fisiología , Proteínas Inmediatas-Precoces/metabolismo , Sistemas de Lectura Abierta , Proteínas Quinasas/metabolismo , Proteínas del Envoltorio Viral , Proteínas Virales , Replicación Viral , Línea Celular , Núcleo Celular , Herpesvirus Humano 3/enzimología , Herpesvirus Humano 3/genética , Humanos , Melanoma , Mutagénesis , Nucleoproteínas/aislamiento & purificación , Nucleoproteínas/metabolismo , Fosfoproteínas/aislamiento & purificación , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Recombinantes/metabolismo , Simplexvirus/fisiología , Transfección , Células Tumorales Cultivadas , Proteínas Reguladoras y Accesorias Virales

RESUMEN

Cells infected with varicella-zoster virus (VZV) express a viral ribonucleotide reductase which is distinct from that present in uninfected cells. VZV open reading frames 18 and 19 (ORF18 and ORF19) are homologous to the herpes simplex virus type 1 genes encoding the small and large subunits of ribonucleotide reductase, respectively. We generated recombinant VZV by transfecting cultured cells with four overlapping cosmid DNAs. To construct a virus lacking ribonucleotide reductase, we deleted 97% of VZV ORF19 from one of the cosmids. Transfection of this cosmid with the other parental cosmids yielded a VZV mutant with a 2.3-kbp deletion confirmed by Southern blot analysis. Virus-specific ribonucleotide reductase activity was not detected in cells infected with VZV lacking ORF19. Infection of melanoma cells with ORF19-deleted VZV resulted in plaques smaller than those produced by infection with the parental VZV. The mutant virus also exhibited a growth rate slightly slower than that of the parental virus. Chemical inhibition of the VZV ribonucleotide reductase has been shown to potentiate the anti-VZV activity of acyclovir. Similarly, the concentration of acyclovir required to inhibit plaque formation by 50% was threefold lower for the VZV ribonucleotide reductase deletion mutants than for parental virus. We conclude that the VZV ribonucleotide reductase large subunit is not essential for virus infection in vitro; however, deletion of the gene impairs the growth of VZV in cell culture and renders the virus more susceptible to inhibition by acyclovir.

Asunto(s)

Eliminación de Gen , Herpesvirus Humano 3/crecimiento & desarrollo , Ribonucleótido Reductasas/genética , Aciclovir/farmacología , Secuencia de Bases , Southern Blotting , Cósmidos , ADN Recombinante/genética , ADN Viral/genética , Relación Dosis-Respuesta a Droga , Herpesvirus Humano 3/efectos de los fármacos , Herpesvirus Humano 3/enzimología , Herpesvirus Humano 3/genética , Humanos , Immunoblotting , Melanoma Experimental , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Ribonucleótido Reductasas/efectos de los fármacos , Células Tumorales Cultivadas , Ensayo de Placa Viral , Replicación Viral/genética