RESUMEN
None of the enteric caliciviruses except Po/Sapo/GIII/Cowden/80/US replicates in cell culture, which complicates efforts to develop control strategies or to study viral replication. To develop serological assays for bovine noroviruses (BoNVs) and to determine the cross-reactivity of BoNV with human noroviruses, we generated two recombinant baculoviruses, rCV186-OH and rJNCV, to express the capsid genes of Bo/CV186-OH/00/US (Norovirus genogroup III [GIII], genotype 2 [GIII/2]). rCV186-OH expressed the expected 57-kDa capsid protein, but rJNCV expressed a truncated capsid protein of 35 kDa. Sequence analysis of rJNCV identified a single nucleotide deletion in the P domain of the capsid gene, which introduced a stop codon at amino acid 323. The recombinant capsid protein produced by rCV186-OH but not that produced by rJNCV self-assembled into virus-like particles (VLPs) similar to native BoNV. An antibody-capture enzyme-linked immunosorbent assay (ELISA) and antigen-capture ELISA (Ag-ELISA) detected serum antibody and antigen, respectively, from calves infected with Bo/CV186-OH/00/US but not antibodies or antigens to other enteric viruses. In other tests of the GIII/2 BoNV Ag-ELISA, no cross-reactivity was observed with VLPs from one GI and four GII human noroviruses and porcine sapovirus Cowden strain. Because, like human noroviruses, BoNVs do not grow in cell culture, the BoNV VLPs will be useful in the serological assays described for the detection of BoNV antibody and antigen. Consistent with the phylogenetic analysis of the capsid genes of bovine and human noroviruses (M. G. Han, J. R. Smiley, C. Thomas, and L. J. Saif, J. Clin. Microbiol. 42:5214-5224, 2004), the results suggest that GIII/2 BoNV does not share significant antigenic relationships with the five characterized human noroviruses tested.
Asunto(s)
Proteínas de la Cápside/metabolismo , Norovirus/metabolismo , Proteínas Recombinantes/metabolismo , Virión/metabolismo , Ensamble de Virus , Animales , Anticuerpos Antivirales/sangre , Antígenos Virales/análisis , Baculoviridae/genética , Baculoviridae/metabolismo , Infecciones por Caliciviridae/veterinaria , Infecciones por Caliciviridae/virología , Cápside/metabolismo , Proteínas de la Cápside/genética , Bovinos , Enfermedades de los Bovinos/diagnóstico , Enfermedades de los Bovinos/virología , Células Cultivadas , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Gastroenteritis/diagnóstico , Gastroenteritis/veterinaria , Gastroenteritis/virología , Humanos , Norovirus/genética , Proteínas Recombinantes/genética , SpodopteraRESUMEN
Two genetically distinct bovine enteric caliciviruses (BECs) have been identified: the norovirus (NLV) Jena and Newbury Agent-2 (NA-2) BECs, which are genetically related to human noroviruses, and the Nebraska (NB) BECs, which is related to sapoviruses and lagoviruses but may also represent a new calicivirus genus. The prevalence of these two BEC genotypes in cattle is unknown. Although reverse transcription-PCR (RT-PCR) primers for human NLV recognize NLV-BECs, the genetic relationships between NLV from humans and the NLV-BECs commonly circulating in cattle is undefined. In the present study, veal calf fecal samples were assayed for enteric caliciviruses by using six RT-PCR primer sets designed for the detection of human NLVs or BECs. Caliciviruses genetically related to the NLV-BEC Jena and NA-2 strains or to the recently characterized NB BEC strain were identified in three of four and four of four sampled veal herds, respectively. Extended 3'-terminal genome sequences of two NLV-BECs, designated CV95-OH and CV186-OH, encoding the RNA-dependent RNA polymerase (RdRp; open reading frame 1 [ORF-1]), VP1 (ORF-2), and VP2 (ORF-3) genes were determined. Phylogenetic and sequence identity analyses of each genome region demonstrated these viruses to be most closely related to the NLV-BEC Jena and NA-2 strains. In initial testing, the human P289-P290 (P289/290) primer set was found to be the most sensitive for calicivirus detection. However, its failure to identify all positive fecal pools (as determined by other assays) led us to design two new primer sets, CBECU-F/R and NBU-F/R, for the sensitive and specific detection of NLV-BEC (NLV-BEC Jena and NA-2) and BEC-NB-like viruses, respectively. The RT-PCR assays with the new primers were compared against other primer sets, including P289/290. Composite results of the tests completed by using the new assays identified 72% (54 of 75) of veal calf fecal samples as positive, with 21 of 21 sequenced reaction products specific for the target RdRp gene. The same design strategy used for the new BEC assays may also be applicable to the design of similar assays for the detection of human caliciviruses (HuCVs). Our data support the genetic relationship between NLV-BECs and NLV-HuCVs but with the NLV-BECs comprising two clusters within a third NLV genogroup.
Asunto(s)
Infecciones por Caliciviridae/microbiología , Caliciviridae/aislamiento & purificación , Enfermedades de los Bovinos/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Animales , Caliciviridae/clasificación , Caliciviridae/genética , Infecciones por Caliciviridae/veterinaria , Bovinos , Enfermedades de los Bovinos/epidemiología , Cartilla de ADN , Humanos , Datos de Secuencia Molecular , Norovirus/clasificación , Norovirus/genética , Norovirus/aislamiento & purificación , Prevalencia , ARN Polimerasa Dependiente del ARN/genética , Sensibilidad y Especificidad , Análisis de Secuencia de ADNRESUMEN
Bovine enteric caliciviruses (BEC) are associated with diarrhea in young calves. The BEC strains detected in Europe form a third genogroup within the genus "Norwalk-like viruses" (NLV) of the family Caliciviridae. In this report, we present sequence, clinical, and histological data characterizing a novel enteropathogenic BEC strain, NB, detected in fecal specimens from calves in the United States. The complete RNA genome of the NB virus is 7,453 bases long and is organized into two open reading frames (ORFs). ORF-1 is 2,210 amino acids long and encodes a large nonstructural polyprotein contiguous with the major capsid protein (VP1), similar to the lagoviruses and "Sapporo-like viruses" (SLV). The conserved calicivirus motifs were identified in the nonstructural proteins. ORF-2 is located at the 3' end of the genome and encodes a small basic protein (VP2) of 225 amino acids. The 5' and 3' untranslated regions are 74 and 67 bases long, respectively. Among caliciviruses, NB virus shows amino acid identities of 14.1 to 22.6% over the entire ORF-1 nonstructural-protein sequence with NLV, SLV, vesivirus, and lagovirus strains, while the overall sequence identity of the complete NB VP-1 with other caliciviruses is low, varying between 14.6 and 26.7%. Phylogenetic analysis of the complete VP1 protein, including strains from all four calicivirus genera, showed the closest grouping of NB virus to be with viruses in the genus Lagovirus, which cause liver infections and systemic hemorrhage in rabbits. In gnotobiotic calves, however, NB virus elicited only diarrhea and intestinal lesions that were most severe in the upper small intestine (duodenum and jejunum), similar to the NLV BEC strains. The tissues of major organs, including the lung, liver, kidney, and spleen, had no visible microscopic lesions.
Asunto(s)
Infecciones por Caliciviridae/veterinaria , Caliciviridae/genética , Enfermedades de los Bovinos/virología , Animales , Secuencia de Bases , Caliciviridae/clasificación , Caliciviridae/aislamiento & purificación , Infecciones por Caliciviridae/virología , Cápside/genética , Proteínas de la Cápside , Bovinos , ADN Viral , Diarrea/virología , Genoma Viral , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , Homología de Secuencia de Ácido Nucleico , Proteínas no Estructurales Virales/genéticaAsunto(s)
Simplexvirus/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Northern Blotting , Electroforesis en Gel de Agar , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Ribonucleasas , Homología de Secuencia de Aminoácido , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Infectious bursal disease virus strains U2, 586, L1, and Q2 were isolated from pooled bursal samples collected from commercially reared broilers. These viruses were propagated in specific-pathogen-free (SPF) embryonated chicken eggs for 24 or 25 passages. Nucleotide sequences of a 743-bp reverse transcription (RT)/polymerase chain reaction (PCR) product containing the VP2 hypervariable region were compared before and after passage of the viruses in embryonated chicken eggs. To determine the genetic stability of the viruses, each isolate was compared with its egg-passed ancestor; virus isolates were not compared with each other. When the restriction enzymes BstNI and MboI were used, no differences were observed in the restriction fragment length polymorphism profiles of the RT/ PCR products after embryo passage. After embryo passage, six nucleotide changes were identified in the viruses. Among the four viruses examined, these nucleotide changes resulted in a total of five amino acid changes. The amino acid changes were S-222-L in virus 586, K-249-N in viruses U2, L1, and Q2, and G-281-V in virus Q2. Three of the five amino acid changes occurred at residue 249. The convergent nature of this residue shift in three of four of the chick embryo-passed viruses suggests the occurrence of a functional, as opposed to random, mutation. The original isolates caused typical signs of infectious bursal disease in 3-wk-old SPF chicks. Their embryo-passed ancestors also produced typical signs of infectious bursal disease in 3-wk-old SPF chicks, suggesting the amino acid mutations observed did not affect virulence of the viruses.
Asunto(s)
Regiones Determinantes de Complementariedad/genética , Virus de la Enfermedad Infecciosa de la Bolsa/genética , Proteínas Estructurales Virales/genética , Animales , Bolsa de Fabricio/virología , Embrión de Pollo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Virus de la Enfermedad Infecciosa de la Bolsa/patogenicidad , Polimorfismo de Longitud del Fragmento de Restricción , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Organismos Libres de Patógenos EspecíficosRESUMEN
The virion host shutoff protein (vhs) of herpes simplex virus (HSV) triggers global shutoff of host protein synthesis and accelerated mRNA turnover during virus infection and induces endoribonucleolytic cleavage of exogenous RNA substrates when it is produced in a rabbit reticulocyte (RRL) in vitro translation system. Although vhs induces RNA turnover in the absence of other HSV gene products, it is not yet known whether cellular factors are required for its activity. As one approach to addressing this question, we expressed vhs in the budding yeast Saccharomyces cerevisiae. Expression of vhs inhibited colony formation, and the severity of this effect varied with the carbon source. The biological relevance of this effect was assessed by examining the activity of five mutant forms of vhs bearing previously characterized in-frame linker insertions. The results indicated a complete concordance between the growth inhibition phenotype in yeast and mammalian host cell shutoff. Despite these results, expression of vhs did not trigger global mRNA turnover in vivo, and cell extracts of yeast expressing vhs displayed little if any vhs-dependent endoribonuclease activity. However, activity was readily detected when such extracts were mixed with RRL. These data suggest that the vhs-dependent endoribonuclease requires one or more mammalian macromolecular factors for efficient activity.
Asunto(s)
Endorribonucleasas/metabolismo , Simplexvirus/fisiología , Proteínas Virales/fisiología , Animales , Fenotipo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Conejos , Ribonucleasas , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Virus infection induces an antiviral response that is predominantly associated with the synthesis and secretion of soluble interferon. Here, we report that herpes simplex virus type 1 virions induce an interferon-independent antiviral state in human embryonic lung cells that prevents plaquing of a variety of viruses. Microarray analysis of 19,000 human expressed sequence tags revealed induction of a limited set of host genes, the majority of which are also induced by interferon. Genes implicated in controlling the intracellular spread of virus and eliminating virally infected cells were among those induced. Induction of the cellular response occurred in the absence of de novo cellular protein synthesis and required viral penetration. In addition, this response was only seen when viral gene expression was inhibited, suggesting that a newly synthesized viral protein(s) may function as an inhibitor of this response.
Asunto(s)
Regulación de la Expresión Génica , Herpesvirus Humano 1/fisiología , Proteínas Virales , Animales , Línea Celular , Etiquetas de Secuencia Expresada , Proteína Vmw65 de Virus del Herpes Simple/genética , Proteína Vmw65 de Virus del Herpes Simple/metabolismo , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/patogenicidad , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Interferones/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Células Tumorales Cultivadas , Ensayo de Placa Viral , Virión/inmunologíaRESUMEN
The virion host shutoff (vhs) protein of herpes simplex virus (HSV) triggers global shutoff of host protein synthesis and accelerated turnover of host and viral mRNAs during HSV infection. As well, it induces endoribonucleolytic cleavage of RNA substrates when produced in a rabbit reticulocyte lysate (RRL) in vitro translation system. The vhs1 point mutation (Thr 214-->Ile) eliminates vhs function during virus infection and in transiently transfected mammalian cells and was therefore previously considered to abolish vhs activity. Here we demonstrate that the vhs1 mutant protein induces readily detectable endoribonuclease activity on RNA substrates bearing the internal ribosome entry site of encephalomyocarditis virus in the RRL assay system. These data document that the vhs1 mutation does not eliminate catalytic activity and raise the possibility that the vhs-dependent endoribonuclease employs more than one mode of substrate recognition.
Asunto(s)
Herpesvirus Humano 1/metabolismo , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Animales , Endorribonucleasas/metabolismo , Herpesvirus Humano 1/patogenicidad , Mutación Puntual , ARN Viral/metabolismo , Conejos , Reticulocitos , Ribonucleasas , ViriónRESUMEN
Herpes simplex virus (HSV) ICP27 is an essential and multifunctional regulator of viral gene expression that modulates RNA splicing, polyadenylation, and nuclear export. We have previously reported that ICP27 causes the cytoplasmic accumulation of unspliced alpha-globin pre-mRNA. Here we examined the effects of a series of ICP27 mutations that alter important functional regions of the protein on the processing and nuclear transport of alpha-globin and HSV ICP0 RNA. The results demonstrate that ICP27 mutants that are impaired for growth in noncomplementing cells, including mutants in the N- and C-terminal regions, are defective in the accumulation of alpha-globin pre-mRNA. Unexpectedly, several mutants that are competent to repress the expression of reporter genes in transient transfection assays failed to accumulate unspliced RNA, implying that different mechanisms are responsible for transrepression and pre-mRNA accumulation. Several mutants caused a marked increase in the length and heterogeneity of the alpha-globin mRNA poly(A) tail, suggesting that ICP27 may directly or indirectly affect the regulation of poly(A) polymerase. ICP27 was also required for the accumulation of multiple ICP0 intron-bearing transcripts, but this effect displayed a mutational sensitivity profile different from that of accumulation of unspliced alpha-globin RNA. Moreover, unlike spliced and unspliced alpha-globin RNAs, which were efficiently exported to the cytoplasm, spliced and intron-containing ICP0 transcripts were predominantly nuclear in localization, and ICP27 was not required for nuclear retention of the spliced message. We propose that these transcript- and ICP27 allele-specific differences may be explained by the presence of a strong cis-acting ICP27 response element in the alpha-globin transcript.
Asunto(s)
Globinas/genética , Herpesvirus Humano 1/genética , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/fisiología , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Globinas/metabolismo , Células HeLa/virología , Herpesvirus Humano 1/metabolismo , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Intrones/genética , Mutación , Poli A , Empalme del ARN , ARN Mensajero/genética , ARN Viral/genética , ARN Viral/metabolismo , Fracciones Subcelulares , Transcripción Genética , Ubiquitina-Proteína Ligasas , Replicación ViralRESUMEN
During infection with herpes simplex virus type 1 (HSV-1), VP16 serves multiple functions, including transcriptional activation of viral immediate early genes and downregulation of the virion host shutoff protein vhs. Furthermore, VP16 has been shown to be involved in some aspect of virus assembly and/or maturation. Experiments with a VP16 null virus, 8MA, suggested that VP16 plays a direct role in virion assembly, since removal of VP16 from the HSV-1 genome results in reduced levels of encapsidated DNA and a failure to produce extracellular enveloped particles. However, VP16 null mutants display a severe translational arrest due to unrestrained vhs activity, thus complicating interpretation of these data. We examine here the role of VP16 in virion assembly and egress in the context of a vhs null background, using the virus 8MA/DeltaSma (VP16(-) vhs(-)). Comparison of 8MA and 8MA/DeltaSma with respect to viral DNA accumulation and encapsidation and accumulation of the major capsid protein, VP5, revealed that the 8MA lethal phenotype is only partially due to uncontrolled vhs activity, indicating that VP16 is required in HSV-1 virion formation. Electron microscopy confirmed these results and further showed that VP16 is required for HSV-1 egress beyond the perinuclear space. In addition, we describe the isolation and characterization of an 8MA derivative capable of propagation on Vero cells, due to second site mutations in the vhs and UL53 (gK) genes. Taken together, these results show that VP16 is required for viral egress downstream of the initial envelopment step and further underscore the importance of VP16 in controlling vhs activity within an infected cell.
Asunto(s)
Proteína Vmw65 de Virus del Herpes Simple/fisiología , Herpesvirus Humano 1/genética , Ensamble de Virus , Animales , Chlorocebus aethiops , Regulación hacia Abajo , Eliminación de Gen , Proteína Vmw65 de Virus del Herpes Simple/genética , Herpesvirus Humano 1/fisiología , Herpesvirus Humano 1/ultraestructura , Immunoblotting , Microscopía Electrónica , Mutagénesis Sitio-Dirigida , Fenotipo , Ribonucleasas , Células Vero , Ensayo de Placa Viral , Proteínas Virales/genéticaRESUMEN
Transcripts of most intron-bearing cellular genes must be processed by the splicing machinery in order to efficiently accumulate and gain access to the cytoplasm. However, we found that herpes simplex virus induces cytoplasmic accumulation of both spliced and unspliced polyadenylated alpha-globin RNAs in infected HeLa cells. Accumulation of the unspliced RNA required the immediate-early protein ICP27, and ICP27 was sufficient (in combination with ICP4) to produce this effect in a transient-transfection assay. However, expression of ICP27 did not markedly alter the levels of fully spliced alpha-globin transcripts in infected cells. These data demonstrate that the previously documented effects of ICP27 on the cellular splicing apparatus do not greatly inhibit splicing of alpha-globin RNA and argue that ICP27 induces a splicing-independent pathway for alpha-globin RNA accumulation and nuclear export.
Asunto(s)
Globinas/genética , Proteínas Inmediatas-Precoces/metabolismo , Poli A/metabolismo , Empalme del ARN , Citoplasma/metabolismo , Células HeLa , Humanos , Proteínas Inmediatas-Precoces/genética , Precursores del ARN/metabolismoRESUMEN
Interferon (IFN) is an important immune system molecule capable of inducing an antiviral state within cells. Herpes simplex virus type 1 (HSV-1) replication is somewhat reduced in tissue culture in the presence of IFN, presumably due to decreased viral transcription. Here, we show mutations that inactivate immediate-early (IE) gene product ICP0 render HSV-1 exquisitely sensitive to IFN inhibition, resulting in greatly decreased levels of viral mRNA transcripts and the resulting polypeptides and a severe reduction in plaque formation ability. Mutations in other HSV-1 genes, including the genes coding for virion transactivator VP16 and the virion host shutoff protein vhs, IE gene ICP22, and the protein kinase UL13 gene, do not increase the IFN sensitivity of HSV-1. Interestingly, ICP0 mutants demonstrate the same level of sensitivity to IFN as wild-type virus on U2OS cells, an osteosarcoma cell line that is known to complement mutations in ICP0 and VP16. Thus, in some cell types, functional ICP0 is required for HSV-1 to efficiently bypass the inhibitory effects of IFN in order to ensure its replication. The significance of this link between ICP0 and IFN resistance is discussed.
Asunto(s)
Antivirales/farmacología , Herpesvirus Humano 1/efectos de los fármacos , Proteínas Inmediatas-Precoces/fisiología , Interferón-alfa/farmacología , Animales , Chlorocebus aethiops , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/patogenicidad , Humanos , Proteínas Inmediatas-Precoces/genética , Mutagénesis , Ubiquitina-Proteína Ligasas , Células Vero , Ensayo de Placa ViralRESUMEN
The herpes simplex virus (HSV) proteins VP16 and ICP0 play key roles in stimulating the onset of the viral lytic cycle. We sought to explore the regulatory links between these proteins by studying the phenotypes of viral mutants in which the activation functions of both were simultaneously inactivated. This analysis unexpectedly revealed that truncation of the C-terminal transcriptional activation domain of VP16 (allele V422) in an ICP0-deficient background almost completely eliminated immediate-early gene expression and virus replication in Vero and HEL cells. The doubly mutant viral genome persisted in a quiescent state for at least 10 days in HEL cells infected at high multiplicity and could be reactivated by superinfection with wild-type HSV. In contrast, the in1814 VP16 mutation produced a markedly less severe phenotype in the same ICP0-deficient background. These data demonstrate that expression of the immediate-early genes requires ICP0 when the C-terminal activation domain of VP16 is deleted and raise the possibility that the in1814 form of VP16 retains a residual ability to stimulate gene expression during virus infection.
Asunto(s)
Regulación Viral de la Expresión Génica , Proteína Vmw65 de Virus del Herpes Simple/metabolismo , Herpesvirus Humano 1/genética , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Activación Transcripcional , Proteínas Virales , Animales , Sitios de Unión , Chlorocebus aethiops , Proteína Vmw65 de Virus del Herpes Simple/genética , Humanos , Mutagénesis , Células Tumorales Cultivadas , Ubiquitina-Proteína Ligasas , Células Vero , Ensayo de Placa Viral , Proteínas Reguladoras y Accesorias ViralesRESUMEN
The herpes simplex virus (HSV) virion host shutoff (vhs) protein (UL41 gene product) is a component of the HSV virion tegument that triggers shutoff of host protein synthesis and accelerated mRNA degradation during the early stages of HSV infection. vhs displays weak amino acid sequence similarity to the fen-1 family of nucleases and suffices to induce accelerated RNA turnover through endoribonucleolytic cleavage events when it is expressed as the only HSV protein in a rabbit reticulocyte in vitro translation system. Although vhs selectively targets mRNAs in vivo, the basis for this selectivity remains obscure, since in vitro activity is not influenced by the presence of a 5' cap or 3' poly(A) tail. Here we show that vhs activity is greatly altered by placing an internal ribosome entry site (IRES) from encephalomyocarditis virus or poliovirus in the RNA substrate. Transcripts bearing the IRES were preferentially cleaved by the vhs-dependent endoribonuclease at multiple sites clustered in a narrow zone located immediately downstream of the element in a reaction that did not require ribosomes. Targeting was observed when the IRES was located at the 5' end or placed at internal sites in the substrate, indicating that it is independent of position or sequence context. These data indicate that the vhs-dependent nuclease can be selectively targeted by specific cis-acting elements in the RNA substrate, possibly through secondary structure or a component of the translational machinery.
Asunto(s)
Virus de la Encefalomiocarditis/genética , Poliovirus/genética , ARN Viral/metabolismo , Ribosomas/genética , Proteínas Virales/metabolismo , Animales , Secuencia de Bases , Northern Blotting , Electroforesis en Gel de Agar , Virus de la Encefalomiocarditis/metabolismo , Datos de Secuencia Molecular , Iniciación de la Cadena Peptídica Traduccional , Plásmidos/genética , Poliovirus/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , Conejos , Ribonucleasas , Ribosomas/metabolismo , Simplexvirus/genética , Simplexvirus/metabolismo , Transcripción Genética , Proteínas Virales/genéticaRESUMEN
The herpes simplex virus virion host shutoff (vhs) protein (UL41 gene product) is a component of the HSV virion tegument that triggers shutoff of host protein synthesis and accelerated mRNA degradation during the early stages of HSV infection. Previous studies have demonstrated that extracts from HSV-infected cells and partially purified HSV virions display vhs-dependent RNase activity and that vhs is sufficient to trigger accelerated RNA degradation when expressed as the only HSV protein in an in vitro translation system derived from rabbit reticulocytes. We have used the rabbit reticulocyte translation system to characterize the mode of vhs-induced RNA decay in more detail. We report here that vhs-dependent RNA decay proceeds through endoribonucleolytic cleavage, is not affected by the presence of a 5' cap or a 3' poly(A) tail in the RNA substrate, requires Mg(2+), and occurs in the absence of ribosomes. Intriguingly, sites of preferential initial cleavage were clustered over the 5' quadrant of one RNA substrate that was characterized in detail. The vhs homologue of pseudorabies virus also induced accelerated RNA decay in this in vitro system.
Asunto(s)
Endorribonucleasas/metabolismo , Proteínas Virales/metabolismo , Animales , Secuencia de Bases , Perros , Endorribonucleasas/genética , Células HeLa , Herpesvirus Suido 1 , Humanos , Magnesio , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Caperuzas de ARN/metabolismo , ARN Mensajero/metabolismo , Conejos , Ribonucleasas , Ribosomas , Partícula de Reconocimiento de Señal/metabolismo , Especificidad por Sustrato , Proteínas Virales/genéticaRESUMEN
Infectious bursal disease virus (IBDV), family Birnaviradae, is the etiologic agent of a commercially important, globally distributed, contagious, immunosuppressive disease of young chickens. A restriction enzyme-compatible ssRNA internal control was developed for an IBDV reverse transcription/polymerase chain reaction-restriction fragment length polymorphism (RT/PCR-RFLP) diagnostic assay. An 841-bp bacteriophage-lambda DNA fragment was directionally ligated to 3' and 5' oligonucleotide linkers containing the IBDV RT/PCR target primer sequences. A pGEM-3Zf (+) transcription vector containing the internal control construct was used in an in vitro transcription reaction to produce ssRNA. After RT and PCR amplification, the transcripts produced an 882-bp cDNA product, larger than, co-amplifiable with, and free of the restriction sites used to prepare RFLP patterns of the 743-bp IBDV cDNA target product. The limit of detection of the transcripts in the RT/PCR test is 3.2 femtograms. With the internal control, a test inhibition rate of 7.7% (20/261) was determined for the IBDV RT/PCR assay. By identifying inhibited tests, the assay was improved through a reduction in the number of false-negative results.
Asunto(s)
Infecciones por Birnaviridae/diagnóstico , Virus de la Enfermedad Infecciosa de la Bolsa/genética , Enfermedades de las Aves de Corral/diagnóstico , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Animales , Infecciones por Birnaviridae/genética , Pollos , Reacciones Falso Negativas , Virus de la Enfermedad Infecciosa de la Bolsa/patogenicidad , Polimorfismo de Longitud del Fragmento de Restricción , Enfermedades de las Aves de Corral/genética , ARN/análisis , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
We examined the phenotype of a herpes simplex virus (HSV) type 1 mutant (V422) in which the C-terminal acidic activation domain of the virion transactivator VP16 is truncated at residue 422. The efficiency of plaque formation by V422 on Vero cells was boosted by approximately 100-fold by including hexamethylene bis-acetimide (HMBA) in the growth medium, as previously observed with the in1814 VP16 linker insertion mutant isolated by Preston and colleagues. V422 displayed severely reduced levels of the immediate-early transcripts encoding ICP0 and ICP4 during infection in the presence of cycloheximide, and this defect was partially overcome by the addition of HMBA. The defect in plaque formation exhibited by V422 and in 1814 was efficiently complemented in U2OS osteosarcoma cells, which had previously been shown to complement ICP0 null mutations. Taken in combination, these data confirm the key role of VP16 in triggering the onset of the HSV lytic cycle.
Asunto(s)
Proteína Vmw65 de Virus del Herpes Simple/fisiología , Activación Transcripcional , Animales , Chlorocebus aethiops , Proteína Vmw65 de Virus del Herpes Simple/química , Proteínas Inmediatas-Precoces/genética , Mutación , Fenotipo , Regiones Promotoras Genéticas , Ubiquitina-Proteína Ligasas , Células VeroRESUMEN
Globin genes are normally expressed only in erythroid cell lineages. However, we found that the endogenous alpha-globin gene is activated following infection of human fibroblasts and HeLa cells with herpes simplex virus (HSV), leading to accumulation of correctly initiated transcripts driven by the alpha-globin promoter. The alpha1- and alpha2-globin genes were both induced, but expression of beta- or zeta-globin genes could not be detected. Experiments using HSV mutants showed that null mutations in the genes encoding the viral immediate-early proteins ICP4 and ICP22 reduced induction approximately 10-fold, while loss of ICP0 function had a smaller inhibitory effect. Transient transfection experiments showed that ICP0 and ICP4 are each sufficient to trigger detectable expression of the endogenous gene, while ICP22 had no detectable effect in this assay. ICP4 also strongly enhanced expression of transfected copies of the alpha2-globin gene. In contrast, the adenovirus E1a protein did not activate the endogenous gene and inhibited expression of the plasmid-borne alpha2-globin gene. Previous studies have led to the hypothesis that chromosomal alpha-globin genes are subject to chromatin-dependent repression mechanism that prevents expression in nonerythroid cells. Our data suggest that HSV ICP0 and ICP4 either break or bypass this cellular gene silencing mechanism.
Asunto(s)
Regulación de la Expresión Génica , Globinas/genética , Herpesvirus Humano 1/genética , Proteínas Inmediatas-Precoces/genética , Proteínas Virales , Proteínas E1A de Adenovirus/genética , Animales , Línea Celular , Chlorocebus aethiops , Expresión Génica , Células HeLa , Humanos , Mutagénesis , ARN/metabolismo , ARN Mensajero , Factores de Tiempo , Transfección , Ubiquitina-Proteína Ligasas , Células Vero , Proteínas Reguladoras y Accesorias ViralesRESUMEN
Herpes simplex virus (HSV) virions contain two regulatory proteins that facilitate the onset of the lytic cycle: VP16 activates transcription of the viral immediate-early genes, and vhs triggers shutoff of host protein synthesis and accelerated turnover of cellular and viral mRNAs. VP16 and vhs form a complex in infected cells, raising the possibility of a regulatory link between them. Here we show that viral protein synthesis and mRNA levels undergo a severe decline at intermediate times after infection with a VP16 null mutant, culminating in virtually complete translational arrest. This phenotype was rescued by a transcriptionally incompetent derivative of VP16 that retains vhs binding activity, and was eliminated by inactivating the vhs gene. These results indicate that VP16 dampens vhs activity, allowing HSV mRNAs to persist in infected cells. Further evidence supporting this hypothesis came from the demonstration that a stably transfected cell line expressing VP16 was resistant to host shutoff induced by superinfecting HSV virions. Thus, in addition to its well known function as a transcriptional activator, VP16 stimulates viral gene expression at a post-transcriptional level, by sparing viral mRNAs from degradation by one of the virus-induced host shutoff mechanisms.
Asunto(s)
Regulación Viral de la Expresión Génica , Proteína Vmw65 de Virus del Herpes Simple/fisiología , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Simplexvirus/fisiología , Proteínas Virales/metabolismo , Replicación Viral/fisiología , Animales , Secuencia de Bases , Chlorocebus aethiops , Genes Inmediatos-Precoces , Genes Virales , Sustancias Macromoleculares , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Viral/genética , Ribonucleasas , Simplexvirus/genética , Transcripción Genética , Células Vero , Proteínas Virales/biosíntesis , Proteínas Virales/genéticaRESUMEN
The herpes simplex virus transactivator VP16 and the virion host shutoff protein vhs are viral structural components that direct the activation of immediate-early gene expression and the arrest of host protein synthesis, respectively, during an infection. Recent studies show that VP16 and vhs physically interact with each other in vitro and in infected cells, suggesting that their respective regulatory functions are coupled. In this report, we used the yeast two-hybrid system and affinity chromatography with purified VP16 fusion proteins to precisely map a region in vhs that directs interaction with VP16. Deletion analysis of vhs demonstrated that a 21-amino-acid-long domain spanning residues 310 to 330 (PAAGGTEMRVSWTEILTQQIA) was sufficient for directing complex formation with VP16 in vivo and in vitro when fused to a heterologous protein. Site-directed mutagenesis of this region identified tryptophan 321 as a crucial determinant for interaction with VP16 in vitro and in vivo and additional residues that are important for stable complex formation in vitro. These findings indicate that vhs residues 310 to 330 constitute an independent and modular binding interface that is recognized by VP16.