Biomedical subjects

J R Smiley

Publications and source records attributed to J R Smiley.

At least 37 records · Page 2Linked to original sources

PubMed1996

Identification and characterization of a small modular domain in the herpes simplex virus host shutoff protein sufficient for interaction with VP16.

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.

Amino Acid Sequence↗

PubMed1995

Activation of expression of multiple subfamilies of human Alu elements by adenovirus type 5 and herpes simplex virus type 1.

The nearly one million Alu repetitive elements in the human genome can be grouped into a number of subfamilies. Comparisons between subfamily consensus sequences suggest that Alu evolution is characterized by the sequential amplification and dispersal of a limited number of Alu founder sequences. The S, Sb and Sb1 subfamilies provide an example of such a related series of Alu subfamilies. We have previously demonstrated that adenovirus type 5 and herpes simplex virus type 1 activate RNA polymerase III transcription of endogenous Alu elements in HeLa cells. Here, we report that expression of Alu sequences belonging to the S, Sb and Sb1 subfamilies was activated following infection with these viruses. The data indicate that transpositionally inactive Alu elements can give rise to high levels of pol III transcripts in the presence of appropriate trans-acting factors and demonstrate that the class III promoters of a significant number and variety of Alu sequences are functional in vivo. Multiple subfamilies of Alu sequences were induced in transformed and non-transformed cell types, suggesting that induction of Alu expression may be part of the normal cellular response to viral infection.

Adenoviruses, Human↗

PubMed1995

Mutational analysis of the herpes simplex virus virion host shutoff protein: evidence that vhs functions in the absence of other viral proteins.

Herpes simplex virus (HSV) virions contain one or more factors that trigger rapid shutoff of host protein synthesis and accelerated decay of cellular and viral mRNAs in infected cells. HSV isolates bearing mutations at the virion host shutoff (vhs) locus (gene UL41) are defective for both processes, indicating that the vhs protein is required; however, it is not clear whether the role of vhs in shutoff is direct or indirect and if other virion components are also necessary. We therefore used a transient-cotransfection assay to determine if the vhs protein displays activity in the absence of other viral gene products. We found that a vhs expression vector strongly suppressed expression of a cotransfected lacZ reporter gene and that this effect was eliminated by the vhs1 point mutation that abolishes virion-induced host shutoff during HSV infection. Further evidence for the biological relevance of the transfection assay came from the demonstration that five vhs in-frame linker insertion mutations yielded concordant results when assayed in cotransfected cells and following transfer into the viral genome: three mutations eliminated activity in both assays, while two had no effect. On the basis of these results, we conclude that the vhs protein can trigger host shutoff in the absence of other HSV proteins. The cotransfection assay was used to rapidly assess the activities of a panel of linker insertion mutants spanning the vhs polypeptide. All mutations that mapped to regions conserved among the vhs homologs of alphaherpesvirus inactivated function; in contrast, four of five mutations that mapped to regions that are absent from several vhs homologs had no effect. These results further support the biological relevance of the transfection assay and begin to delineate functional domains of the vhs polypeptide.

Amino Acid Sequence↗

PubMed1994

Activation of RNA polymerase III transcription of human Alu elements by herpes simplex virus.

We found that HSV infection of HeLa cells strongly induces RNA polymerase III transcription of endogenous human Alu elements, resulting in the accumulation of high levels of cytoplasmic RNAs initiated from Alu pol III promoters. Induction required viral protein synthesis and occurred during infection with a viral mutant bearing a null mutation in the immediate-early (IE) gene encoding ICP4, suggesting that one or more IE proteins are sufficient for activation. However, mutations in each of the other four IE genes had no effect on activation of Alu expression. We therefore conclude that HSV most likely encodes at least two proteins that are each sufficient to activate Alu transcription and that at least one of these is an IE protein other than ICP4.

Animals↗

PubMed1994

Herpes simplex virus VP16 forms a complex with the virion host shutoff protein vhs.

Herpes simplex virus (HSV) virions contain at least two regulatory proteins that modulate gene expression in infected cells: the transcriptional activator VP16 and the virion host shutoff protein vhs. VP16 stimulates transcription of the HSV immediate-early genes, and vhs suppresses host protein synthesis and induces accelerated turnover of cellular and viral mRNAs. We report here that vhs binds directly to VP16: vhs and VP16 were coprecipitated from infected cells by an anti-vhs antiserum, and vhs and VP16 protein A fusions each bound intact versions of the other protein in a solid-phase capture assay. In addition, vhs and VP16 interacted in the two-hybrid activator system when coexpressed in Saccharomyces cerevisiae. vhs residues 238 to 344 were sufficient for the interaction, and the VP16 acidic transcriptional activation domain was not required. vhs blocked the ability of VP16 to enter a multiprotein complex on an immediate-early TAATGARATTC consensus sequence, indicating that vhs interacts with one or more regions of VP16 required for promoter recognition. We suggest that this interaction may play a structural role in the assembly of HSV virions and modulate the activity of vhs during infection.

Animals↗

PubMed1994

Generation of recombinant herpes simplex virus type 1 to be used for automatic tract tracing.

We generated two insertional beta-galactosidase (lacZ) expressing herpes simplex virus type 1 (HSV-1) with putative mutations in the UL41 or US5 open reading frames. Purified recombinants or wild-type HSV-1 were injected into the left adrenal gland of hamsters. Three days later, virus-infected neurons were visualized with immunohistochemistry in spinal cord sections from all infected hamsters. Neurons were also visualized with X-Gal histochemistry in spinal cord sections from hamsters infected with either of the recombinants but not with the wild-type virus. Insertional mutagenesis of the HSV-1 genome by lacZ did not disrupt the neurotropic properties of the virus. Both recombinant viruses transneuronally labelled the central nervous system (CNS) sympathoadrenal preganglionic neurons and may be useful for the study of synaptic organization of neural circuits. Successful transynaptic transmission of this marker gene into CNS neurons from a peripheral organ suggests that other foreign genes may also be delivered into CNS neurons using HSV-1.

Adrenal Glands↗

PubMed1993

Binding sites for the herpes simplex virus immediate-early protein ICP4 impose an increased dependence on viral DNA replication on simple model promoters located in the viral genome.

We examined the ability of binding sites for the herpes simplex virus immediate-early protein ICP4 to alter the regulation of closely linked promoters by placing strong ICP4 binding sites upstream or downstream of simple TATA promoters in the intact viral genome. We found that binding sites strongly reduced the levels of expression at early times postinfection and that this effect was partially overcome after the onset of viral DNA replication. These data confirm that DNA-bound ICP4 can inhibit the activity of a closely linked promoter and raise the possibility that ICP4 binding sites contribute to temporal regulation during infection.

Animals↗

PubMed1993

Generation of a new adenovirus type 12-inducible fragile site by insertion of an artificial U2 locus in the human genome.

Infection with adenovirus type 12 (Ad12) induces four fragile sites in the human genome (H.F. Stich, G.L. van Hoosier, and J.J. Trentin, Exp. Cell Res. 34:400-403, 1964; H. zur Hausen, J. Virol. 1:1174-1185, 1967). The major site, at 17q21-22, contains the U2 gene cluster, which is specifically disrupted by infection in at least a percentage of the cells (D.M. Durnam, J.C. Menninger, S.H. Chandler, P.P. Smith, and J.K. McDougall, Mol. Cell. Biol. 8:1863-1867, 1988). For direct assessment of whether the U2 locus is the target of the Ad12 effect, an artificial locus, constructed in vitro and consisting of tandem arrays of the U2 6-kbp monomer, was transfected into human cells. We report that integration of this artificial locus on the p arm of chromosome 13 creates a new Ad12-inducible fragile site.

Adenoviruses, Human↗

PubMed1993

Activation of RNA polymerase III transcription of human Alu repetitive elements by adenovirus type 5: requirement for the E1b 58-kilodalton protein and the products of E4 open reading frames 3 and 6.

We found that transcription of endogenous human Alu elements by RNA polymerase III was strongly stimulated following infection of HeLa cells with adenovirus type 5, leading to the accumulation of high levels of Alu transcripts initiated from Alu polymerase III promoters. In contrast to previously reported cases of adenovirus-induced activation of polymerase III transcription, induction required the E1b 58-kDa protein and the products of E4 open reading frames 3 and 6 in addition to the 289-residue E1a protein. In addition, E1a function was not required at high multiplicities of infection, suggesting that E1a plays an indirect role in Alu activation. These results suggest previously unsuspected regulatory properties of the adenovirus E1b and E4 gene products and provide a novel approach to the study of the biology of the most abundant class of dispersed repetitive DNA in the human genome.

Adenovirus E1A Proteins↗

PubMed1993

Construction and preliminary characterization of a nondefective herpes simplex virus recombinant bearing the genome of human papillomavirus type 16.

Studies of the molecular biology of human papillomavirus type 16 have been limited by the lack of a tissue culture system that is fully permissive for virus replication; as a result, high-titre stocks of infectious virus are not readily available. Therefore, studies of viral gene expression have relied on analysis of transformed or tumour cell lines harbouring latent or integrated viral genomes, or on the behaviour of transfected reporter gene constructs. To provide a method of efficiently delivering papillomavirus information into the nuclei of mammalian cells, we constructed a herpes simplex virus type 1 recombinant bearing the entire human papillomavirus type 16 genome. The resulting recombinant was capable of lytic replication and induced the accumulation of papillomavirus mRNAs initiated from the p97 early promoter during infection of Vero cells. This and other herpes simplex - papillomavirus recombinants should facilitate molecular analysis of the life cycle of human papillomavirus type 16.

Base Sequence↗

PubMed1992

The herpes simplex virus type 1 immediate-early polypeptide ICP4 is required for expression of globin genes located in the viral genome.

We infected Vero cells with ICP4-deficient herpes simplex virus recombinants bearing the rabbit beta-globin and human alpha 2-globin genes under the control of their own promoters and found that globin gene expression occurred only when ICP4 was provided in trans. These results demonstrate that ICP4 is required for the activity of globin promoters located in the viral genome and support the hypothesis that these cellular promoters are functionally equivalent to HSV early regulatory regions.

Animals↗

PubMed1992

Identification and characterization of the virion-induced host shutoff product of herpes simplex virus gene UL41.

The virion-induced host shutoff product of the herpes simplex virus UL41 gene is required for shutoff of host translation and degradation of cellular mRNAs. We employed a rabbit antipeptide antiserum to identify a 58K UL41-related phosphoprotein in infected cells. We also provide evidence that this protein is a component of the virus particle, consistent with its role in virion-induced shutoff.

Amino Acid Sequence↗

PubMed1992

The herpes simplex virus type 1 (HSV-1) a sequence serves as a cleavage/packaging signal but does not drive recombinational genome isomerization when it is inserted into the HSV-2 genome.

We inserted the terminal repeat (a sequence) of herpes simplex virus type 1 (HSV-1) strain KOS into the tk gene of HSV-2 strain HG52 in order to assess the ability of the HSV-1 a sequence to provoke genome isomerization events in an HSV-2 background. We found that the HSV-1 a sequence was cleaved by the HSV-2 cleavage/packaging machinery to give rise to novel genomic termini. However, the HSV-1 a sequence did not detectably recombine with the HSV-2 a sequence. These results demonstrate that the viral DNA cleavage/packaging system contributes to a subset of genome isomerization events and indicate that the additional recombinational inversion events that occur during infection require sequence homology between the recombination partners.

Base Sequence↗

PubMed1992

The ICP4 binding sites in the herpes simplex virus type 1 glycoprotein D (gD) promoter are not essential for efficient gD transcription during virus infection.

Activation of the early and late genes of herpes simplex virus type 1 during infection in tissue culture requires functional immediate-early regulatory protein ICP4. ICP4 is a specific DNA-binding protein which recognizes a variety of DNA sequences, many of which contain the consensus ATCGTC. In general, mutations which impair the ability of ICP4 to bind to DNA also eliminate its ability to activate viral early and late promoters both in transfection assays and in the infected cell. However, the role of ICP4 binding sites in the viral genome is unclear; many early and late promoters do not contain consensus binding sites in their vicinity. The glycoprotein D (gD) gene contains two well-characterized ICP4 binding sites upstream of its promoter and a third downstream of the transcription start site. Multimerization of one of these sites has been shown to increase the response of the gD promoter to ICP4 in transfection assays, while their removal reduces stimulation of the gD promoter by ICP4 in vitro. To assess the role of these binding sites during virus infection, we have constructed a recombinant viral genome which has mutations affecting all three. Comparison of the amounts of gD RNA synthesized by the recombinant and wild-type viruses indicated that the mutations had little or no effect on the activity of the gD promoter. Therefore, either the sites have no essential role in gD promoter regulation in the presence of all of the herpes simplex virus type 1 IE polypeptides during a normal infection or they can be functionally substituted by other ICP4 binding sites elsewhere in the genome.

Animals↗

PubMed1991

Regulation of herpes simplex virus true late gene expression: sequences downstream from the US11 TATA box inhibit expression from an unreplicated template.

The true late genes of herpes simplex virus type 1 (HSV-1) are expressed only after the onset of viral DNA replication. Previous studies demonstrated that late promoters lack elements upstream of the TATA box and suggested that only a subset of TATA elements can function in the context of true late promoters. We determined which structural features of true late promoters are responsible for the stringent requirement for viral DNA replication by inserting a series of simple model constructs into the HSV-1 genome in place of one of the two promoters of the UL24 gene. An oligonucleotide consisting of 19 nucleotides spanning the TATA box of the HSV-1 true late US11 gene drove barely detectable levels of expression; by contrast, the corresponding regions of the Adenovirus type 2 major late promoter and the HSV-1 true late glycoprotein C promoter were much more active. Transcripts driven from all of these minimal TATA box promoters accumulated without viral DNA replication. The activity of the US11 TATA box was stimulated by adding upstream Sp1-binding sites or placing the US11 or rabbit beta-globin cap/leader region (-11 to +39) downstream. The Sp1-TATA and TATA-beta-globin cap/leader constructs remained replication independent, while the TATA-US11 cap/leader promoter displayed true late regulation. These results demonstrate that sequences located within the US11 cap/leader region impose a strict requirement for viral DNA replication on a minimal TATA box promoter.

Adenoviruses, Human↗

PubMed1990

Sequence requirements for DNA rearrangements induced by the terminal repeat of herpes simplex virus type 1 KOS DNA.

We investigated the sequence requirements for the site-specific DNA cleavages and recombinational genome isomerization events driven by the terminal repeat or a sequence of herpes simplex virus type 1 KOS DNA by inserting a series of mutated a sequences into the thymidine kinase locus in the intact viral genome. Our results indicate that sequences located at both extremities of the a sequence contribute to these events. Deletions entering from the Ub side of the a sequence progressively reduced the frequency of DNA rearrangements, and further deletion of the internal DR2 repeat array had an additional inhibitory effect. This deletion series allowed us to map the pac1 site-specific DNA cleavage signal specifying the S-terminal cleavage to a sequence that is conserved among herpesvirus genomes. Constructs lacking this signal were unable to directly specify the S-terminal cleavage event but retained a reduced ability to give rise to S termini following recombination with intact a sequences. Deletions entering from the Uc side demonstrated that the copy of direct repeat 1 located adjacent to the Uc region plays an important role in the DNA rearrangements induced by the a sequence: mutants lacking this sequence displayed a reduced frequency of novel terminal and recombinational inversion fragments, and further deletions of the Uc region had a relatively minor additional effect. By using a construct in which site-specific cleavage was directed to heterologous DNA sequences, we found that the recombination events leading to genome segment inversion did not occur at the sites of DNA cleavage used by the cleavage-packaging machinery. This observation, coupled with the finding that completely nonoverlapping portions of the a sequence retained detectable recombinational activity, suggests that inter-a recombination does not occur by cleavage-ligation at a single specific site in herpes simplex virus type 1 strain KOS. The mutational sensitivity of the extremities of the a sequence leads us to hypothesize that the site-specific DNA breaks induced by the cleavage-packaging system stimulate the initiation of recombination.

Animals↗

PubMed1990

Differential regulation of endogenous and transduced beta-globin genes during infection of erythroid cells with a herpes simplex virus type 1 recombinant.

We infected murine erythroleukemia cells with a nondefective herpes simplex virus (HSV) type 1 recombinant bearing the rabbit beta-globin gene under the control of its own promoter, in order to compare the regulation of a cellular gene residing in the viral genome to that of its active endogenous counterpart. We found that the viral globin gene was activated by HSV immediate-early polypeptides, whereas expression of the endogenous beta-globin gene was strongly suppressed: transcription was greatly inhibited, and beta-globin mRNA was rapidly degraded. Degradation of globin mRNA was induced by a component of the infecting virion and required a functional UL41 gene product. These results demonstrate that HSV products can have opposing effects on the expression of homologous genes located in the cellular and viral genomes and suggest that the preferential expression of HSV genes that occurs during infection is not achieved solely through sequence-specific differentiation between viral and cellular promoters or mRNAs.

Animals↗

PubMed1989

Regulation of cellular genes transduced by herpes simplex virus.

Previous studies demonstrated that the rabbit beta-globin gene is transcribed from its own promoter and regulated as a herpes simplex virus (HSV) early gene following insertion into the early HSV thymidine kinase gene in the intact viral genome (J. R. Smiley, C. Smibert, and R. D. Everrett, J. Virol. 61:2368-2377, 1987). We report here that the beta-globin promoter remained under early control after insertion into the late HSV gene encoding glycoprotein C. On the basis of these findings, we concluded that the beta-globin promoter is functionally equivalent to an HSV early-control region. We found that a transduced human alpha-globin gene was also regulated as an early HSV gene, while two linked Alu elements mimicked the behavior of HSV late genes. These results demonstrate that certain aspects of HSV temporal regulation can be duplicated by cellular elements and provide strong support for the hypothesis that the regulation of HSV gene expression can occur through mechanisms that do not rely on recognition of virus-specific temporal control signals.

Animals↗