Biomedical subjects

B Roizman

Publications and source records attributed to B Roizman.

At least 19 recordsLinked to original sources

PubMed2000

Identification of a novel expressed open reading frame situated between genes U(L)20 and U(L)21 of the herpes simplex virus 1 genome.

An open reading frame (ORF) situated between the U(L)20 and U(L)21 genes encodes a protein designated as U(L)20.5. The U(L)20.5 ORF lies 5' and in the same orientation as the U(L)20 ORF. The expression of the U(L)20.5 ORF was verified by RNase protection assays and by in-frame insertion of an amino acid sequence encoding an epitope of an available monoclonal antibody. The tagged U(L)20.5 protein colocalized in small dense nuclear structures with products of the alpha22/U(S)1.5, U(L)3, and U(L)4 genes. Expression of the U(L)20.5 gene was blocked in cells infected and maintained in the presence of phosphonoacetate, indicating that it belongs to the late, or gamma(2), kinetic class. U(L)20.5 is not essential for viral replication inasmuch as a recombinant virus made by insertion of the thymidine kinase gene into the U(L)20.5 ORF replicates in all cell lines tested [J. D. Baines, P. L. Ward, G. Campadelli-Fiume, and B. Roizman (1991) J. Virol. 65, 6414-6424]. The genomic location of the recently discovered genes illustrates the compact nature of the viral genome.

Animals↗

PubMed2000

Small dense nuclear bodies are the site of localization of herpes simplex virus 1 U(L)3 and U(L)4 proteins and of ICP22 only when the latter protein is present.

The herpes simplex virus 1 U(L)3 and U(L)4 open reading frames are expressed late in infection and are not essential for viral replication in cultured cells in vitro. An earlier report showed that the U(L)4 protein colocalizes with the products of the alpha22/U(S)1.5 genes in small nuclear dense bodies. Here we report that the U(L)3 protein also colocalized in these small nuclear dense bodies and the localization of U(L)3 and U(L)4 proteins in these bodies required the presence of alpha22/U(S)1.5 genes. In cells infected with a mutant lacking intact alpha22/U(S)1.5 genes, U(L)3 was diffused throughout the nucleus even though the overall accumulation of the gamma2 U(L)3 protein was decreased. The results suggest that ICP22 acts both as a regulator of U(L)3 accumulation and as the structural component and anchor of these small dense nuclear bodies.

Animals↗

PubMed2000

The herpes simplex virus 1 U(L)34 protein interacts with a cytoplasmic dynein intermediate chain and targets nuclear membrane.

To express the function encoded in its genome, the herpes simplex virus 1 capsid-tegument structure released by deenvelopment during entry into cells must be transported retrograde to the nuclear pore where viral DNA is released into the nucleus. This path is essential in the case of virus entering axons of dorsal root ganglia. The objective of the study was to identify the viral proteins that may be involved in the transport. We report the following findings. (i) The neuronal isoform of the intermediate chain (IC-1a) of the dynein complex pulled down, from lysates of [(35)S]methionine-labeled infected cells, two viral proteins identified as the products of U(L)34 and U(L)31 open reading frames, respectively. U(L)34 protein is a virion protein associated with cellular membranes and phosphorylated by the viral kinase U(S)3. U(L)31 protein is a largely insoluble, evenly dispersed nuclear phosphoprotein required for optimal processing and packaging of viral DNA into preformed capsids. Reciprocal pulldown experiments verified the interaction of IC-1a and U(L)34 protein. In similar experiments, U(L)34 protein was found to interact with U(L)31 protein and the major capsid protein ICP5. (ii) To determine whether U(L)34 protein is transported to the nuclear membrane, a requirement if it is involved in transport, the U(L)34 protein was inserted into a baculovirus vector under the cytomegalovirus major early promoter. Cells infected with the recombinant baculovirus expressed U(L)34 protein in a dose-dependent manner, and the U(L)34 protein localized primarily in the nuclear membrane. An unexpected finding was that U(L)34-expressing cells showed a dissociation of the inner and outer nuclear membranes reminiscent of the morphologic changes seen in cells productively infected with herpes simplex virus 1. U(L)34, like many other viral proteins, may have multiple functions expressed both early and late in infection.

Animals↗

PubMed2000

Bcl-2 blocks a caspase-dependent pathway of apoptosis activated by herpes simplex virus 1 infection in HEp-2 cells.

Earlier reports have shown that herpes simplex virus 1 (HSV-1) mutants induce programmed cell death and that wild-type virus blocks the execution of the cell death program triggered by expression of viral genes, by the Fas and tumor necrosis factor pathways, or by nonspecific stress agents. In particular, an earlier report from this laboratory showed that the mutant virus d120 lacking the genes encoding infected cell protein 4 (ICP4), the major regulatory protein of the virus, induces a caspase-3-independent pathway of apoptosis in human SK-N-SH cells. Here we report that the pathway of apoptosis induced by the d120 mutant in human HEp-2 cells is caspase dependent. Specifically, in HEp-2 cells infected with d120, (i) a broad-range inhibitor of caspase activity, z-vad-FMK, efficiently blocked DNA fragmentation, (ii) cytochrome c was released into the cytoplasm, (iii) caspase-3 was activated inasmuch as poly(ADP-ribose) polymerase was cleaved, and (iv) chromatin condensation and fragmentation of cellular DNA were observed. In parallel studies, HEp-2 cells were transfected with a plasmid encoding human Bcl-2 and a clone (VAX-3) expressing high levels of Bcl-2 was selected. This report shows that Bcl-2 blocked all of the manifestations associated with programmed cell death caused by infection with the d120 mutant. Consistent with their resistance to programmed cell death, VAX-3 cells overproduced infected cell protein 0 (ICP0). An unexpected observation was that ICP0 encoded by the d120 mutant accumulated late in infection in small, quasi-uniform vesicle-like structures in all cell lines tested. Immunofluorescence-based colocalization studies indicated that these structures were not mitochondria or components of the endoplasmic reticulum or the late endosomal compartment. These studies affirm the conclusion that HSV can induce programmed cell death at multiple steps in the course of its replication, that the d120 mutant can induce both caspase-dependent and -independent pathways of programmed cell death, and that virus-induced stimuli of programmed cell death may differ with respect to the pathway that they activate.

Amino Acid Chloromethyl Ketones↗

PubMed2000

The disappearance of cyclins A and B and the increase in activity of the G(2)/M-phase cellular kinase cdc2 in herpes simplex virus 1-infected cells require expression of the alpha22/U(S)1.5 and U(L)13 viral genes.

In uninfected cells the G(2)/M transition is regulated by cyclin kinase complex containing cdc2 and, initially, cyclin A, followed by cyclin B. cdc2 is downregulated through phosphorylation by wee-1 and myt-1 and upregulated by cdc-25C phosphatase. We have examined the accumulation and activities of these proteins in cells infected with wild type and mutants of herpes simplex virus 1. The results were as follows. (i) Cyclin A and B levels were reduced beginning 4 h after infection and were undetectable at 12 to 16 h after infection. (ii) cdc2 protein also decreased in amount but was detectable at all times after infection. In addition, a fraction of cdc2 protein from infected cells exhibited altered electrophoretic mobility in denaturing gels. (iii) The levels of cdk7 or myt-1 proteins remained relatively constant throughout infection, whereas the level of wee-1 was significantly decreased. (iv) cdc-25C formed novel bands characterized by slower electrophoretic mobility that disappeared after treatment with phosphatase. In addition, one phosphatase-sensitive band reacted with MPM-2 antibody that recognizes a phosphoepitope phosphorylated exclusively in M phase. (v) cdc2 accumulating in infected cells exhibited kinase activity. The activity of cdc2 was higher in infected cell lysates than those of corresponding proteins present in lysates of mock-infected cells even though cyclins A and B were not detectable in lysates of infected cells. (vi) The decrease in the levels of cyclins A and B, the increase in activity of cdc2, and the hyperphosphorylation of cdc-25C were mediated by U(L)13 and alpha22/U(S)1.5 gene products. In light of its normal functions, the activated cdc2 kinase may play a role in the changes in the morphology of the infected cell. These results are consistent with the accruing evidence that herpes simplex virus scavenges the cell for useful cell cycle proteins and subverts them for its own use.

CDC2 Protein Kinase↗

PubMed1999

Accumulation of specific RNAs encoding transcriptional factors and stress response proteins against a background of severe depletion of cellular RNAs in cells infected with herpes simplex virus 1.

Herpes simplex virus 1 encodes several functions to preclude the shutoff of host response to infection, including degradation of mRNA immediately after infection. To determine whether any cellular mRNAs accumulate in infected cells against a background of severe loss of host RNA, we hybridized cDNAs derived from three different cell lines infected with wild type and a mutant virus to a DNA array containing probes for 588 human genes representing different functional groups. The results were that (i) infected cells accumulated at levels above those of mock-infected cells, a small number of transcripts representing transcriptional factors that could regulate gene expression both positively and negatively, and one stress response protein (GADD45), (ii) the amount and nature of the accumulated transcripts showed limited variability depending on the cell and virus, and (iii) at least some of the proteins encoded by the accumulated transcripts could benefit either the virus or the host.

Cell Line↗

PubMed1999

A single amino acid substitution in the cyclin D binding domain of the infected cell protein no. 0 abrogates the neuroinvasiveness of herpes simplex virus without affecting its ability to replicate.

The infected cell protein no. 0 (ICP0) of herpes simplex virus 1 is a promiscuous transactivator shown to enhance the expression of genes introduced into cells by infection or transfection. The protein interacts with several viral and cellular proteins. Earlier studies have shown that ICP0 binds and stabilizes cyclin D3 but does interfere with the phosphorylation of retinoblastoma protein, its major function. Cyclin D3 plays a key role in the transition from G1 to S phase. To define the role of cyclin D3 in productive infection, the ICP0 binding site for cyclin D3 was mapped and mutagenized by substitution of aspartic acid codon 199 with the alanine codon. We report that the substitution precluded the interaction of this protein with cyclin D3 in the yeast two-hybrid system and the stabilization of cyclin D3 in infected cells. A recombinant virus carrying this mutation could not be differentiated from wild-type parent with respect to replication in dividing cells but yielded 10-fold less progeny from infected resting cells and serum-deprived or contact-inhibited human fibroblasts. In mice, the mutant was only slightly less pathogenic than the wild-type parent by intracerebral route but was significantly less neuroinvasive after peripheral inoculation. Replacement of the mutated amino acid with aspartic acid restored wild-type phenotype. Stabilization of cyclin D3 therefore is linked to higher virus yields in nondividing cells and potentially higher virulence in experimental and natural hosts. One function of ICP0 is to scavenge the cell for proteins that could bolster viral replication.

Amino Acid Substitution↗

PubMed1999

Posttranscriptional regulation of US11 in cells infected with a herpes simplex virus 1 recombinant lacking both 222-bp domains containing S-component origins of DNA synthesis.

The US11 gene of herpes simplex virus 1 maps in the unique sequences of the short component of the HSV-1(F) genome approximately 775 bp from the center of the DNA replication origin (OriS) and encodes a virion protein which binds RNA in sequence- and conformation-specific fashion, negatively regulates the accumulation of a prematurely terminated transcript of UL34, associates in the infected cell with the 60S ribosomal subunit, and, late in infection, accumulates in nucleoli. We report the following: (i) Deletion of a 222-bp sequence including OriS (DeltaOriS) negatively affected the accumulation of the US11 protein without decreasing the accumulation of the US11 transcript. (ii) The defect, observed at all times after infection, was multiplicity independent, was unrelated to US11 protein stability, and apparently resulted from a cis-acting element since a coinfecting virus was unable to complement the DeltaOriS virus. (iii) Transcription from the US11 promoter initiated from three sites on the DeltaOriS virus. Transcripts initiated from two of the three initation sites accumulated similarly in cells infected with the DeltaOriS virus or wild-type parent virus. The low-abundance transcript initiating from the third site was apparently unique to the DeltaOriS virus but was not expected to alter the coding capacity of the mRNA. (iv) Infected cells accumulated RNA derived by antisense transcription of the genome domain containing the US11 gene. One transcript accumulated in larger amounts in cells infected with the DeltaOriS virus than in cells infected with parent or repaired virus.

Autoradiography↗

PubMed1999

Replication-competent, nonneuroinvasive genetically engineered herpes virus is highly effective in the treatment of therapy-resistant experimental human tumors.

A genetically engineered, nonneurotropic herpes simplex virus (R7020) with a proven safety profile in both animals and humans was found effective in the treatment of large xenotransplanted tumors arising from a radiation- and chemotherapy-resistant human epidermoid carcinoma and a hormone-refractory prostate adenocarcinoma. R7020 replicated to high titer and caused rapid regression of the human tumor xenografts. Tumor destruction was accelerated in animals given both R7020 and fractionated ionizing radiation. Tumors arising from cells surviving one treatment with R7020 were fully susceptible to a second dose of virus. We conclude R7020 is an effective antitumor agent for non-central nervous system tumor xenografts with an excellent safety profile.

Adenocarcinoma↗

PubMed1999

The C-terminal domain of the regulatory protein NOVH is sufficient to promote interaction with fibulin 1C: a clue for a role of NOVH in cell-adhesion signaling.

The NOVH protein belongs to the emerging CCN [Connective tissue growth factor (CTGF), Cyr61/Cef10, nephroblastoma overexpressed gene] family of growth regulators sharing a strikingly conserved multimodular organization but exhibiting distinctive functional features. Two members of the family (CYR61 and CTGF) are positive regulators of cell proliferation, whereas NOVH and two other members (ELM1 and RCOP-1) exhibit features of negative regulators of growth. The multimodular structure of these proteins suggests that their biological role(s) may depend on interactions with several factors as well as proteins constitutive of the extracellular matrix. To gain insight into the functionality of these domains, we have used a two-hybrid system to identify proteins interacting with NOVH. We report here that the C-terminal domain confers on the full-length NOVH protein the capacity to bind fibulin 1C, a protein of the extracellular matrix that interacts with several other regulators of cell adhesion. Furthermore, we show that a natural N-truncated isoform of NOVH produced by cells expressing the full-length NOVH protein also binds fibulin 1C with a high affinity, and we hypothesize that the production of truncated isoforms of NOVH (and probably of other CCN proteins) may be a critical aspect in the modulation of their biological activity. These results set the stage for a study of NOVH-fibulin 1C interactions and their potential significance in cell-adhesion signaling in normal and pathological conditions.

Animals↗

PubMed1999

Acyclovir is phosphorylated by the human cytomegalovirus UL97 protein.

Acyclovir (ACV) has shown efficacy in the prophylactic suppression of human cytomegalovirus (HCMV) reactivation in immunocompromised renal transplant patients without the toxicity associated with ganciclovir (GCV). The HCMV UL97 gene product, a protein kinase, is responsible for the phosphorylation of GCV in HCMV-infected cells. This report provides evidence for the phosphorylation of ACV by UL97. Anabolism studies with the HCMV wild-type strain AD169 and with recombinant mutants derived from marker transfer experiments performed by using mutant UL97 DNA from both clinical isolates and a laboratory-derived strain resistant to GCV showed that mutations in the UL97 gene cripple the ability of recombinant virus-infected cells to anabolize both GCV and ACV. These mutant UL97 recombinant viruses were less susceptible to both GCV and ACV than was the wild-type strain. A recombinant herpes simplex virus type 1 strain, in which the thymidine kinase gene is deleted and the UL13 gene is replaced with the HCMV UL97 gene, was able to induce the phosphorylation of ACV in infected cells. Finally, purified UL97 phosphorylated both GCV and ACV to their monophosphates. Our results indicate that UL97 promotes the selective activity of ACV against HCMV.

Acyclovir↗

PubMed1999

Development of a high-throughput quantitative assay for detecting herpes simplex virus DNA in clinical samples.

We have developed a high-throughput, semiautomated, quantitative fluorescence-based PCR assay to detect and type herpes simplex virus (HSV) DNA in clinical samples. The detection assay, which uses primers to the type-common region of HSV glycoprotein B (gB), was linear from <10 to 10(8) copies of HSV DNA/20 microl of sample. Among duplicate samples in reproducibility runs, the assay showed less than 5% variability. We compared the fluorescence-based PCR assay with culture and gel-based liquid hybridization system with 335 genital tract specimens from HSV type 2 (HSV-2)-seropositive persons attending a research clinic and 380 consecutive cerebrospinal fluid (CSF) samples submitted to a diagnostic virology laboratory. Among the 162 culture-positive genital tract specimens, TaqMan PCR was positive for 157 (97%) specimens, whereas the quantitative-competitive PCR was positive for 144 (89%) specimens. Comparisons of the mean titer of HSV DNA detected by the two assays revealed that the mean titer detected by the gel-based system was slightly higher (median, 1 log). These differences in titers were in part related to the fivefold difference in the amount of HSV DNA used in the amplicon standards with the two assays. Among the 380 CSF samples, 42 were positive by both assays, 13 were positive only by the assay with the agarose gel, and 3 were positive only by the assay with the fluorescent probe. To define the subtype of HSV DNA detected in the screening assay, we also designed one set of primers which amplifies the gG regions of both types of HSV and probes which are specific to either HSV-1 (gG1) or HSV-2 (gG2). These probes were labeled with different fluorescent dyes (6-carboxyfluorescein for gG2 and 6-hexachlorofluorescein for gG1) to enable detection in a single PCR. In mixing experiments the probes discriminated the correct subtype in mixtures with up to a 7-log-higher concentration of the opposite subtype. The PCR typing results showed 100% concordance with the results obtained by assays with monoclonal antibodies against HSV-1 or HSV-2. Thus, while the real-time PCR is slightly less sensitive than the gel-based liquid hybridization system, the high throughput, the lack of contamination during processing, the better reproducibility, and the better ability to type the isolates rapidly make the real-time PCR a valuable tool for clinical investigation and diagnosis of HSV infection.

DNA, Viral↗

PubMed1999

The U(L)3 protein of herpes simplex virus 1 is translated predominantly from the second in-frame methionine codon and is subject to at least two posttranslational modifications.

The U(L)3 open reading frame (ORF) has the coding capacity of 235 codons. The proteins reacting with the anti-U(L)3 antibody form in denaturing polyacrylamide gel bands with apparent M(r)s of 34,000, 35,000, 38,000, 40,000, 41,000, and 42,000 and designated 1 to 6, respectively. Studies on their origins revealed the following. (i) The U(L)3 proteins forming all six bands were present in lysates of cells infected with wild-type virus and treated with tunicamycin or monensin or in cells infected with the mutant lacking the gene encoding the U(S)3 protein kinase. (ii) The proteins contained in the slower-migrating bands were absent from cells infected with the mutant lacking the U(L)13 protein kinase. Bands 1 and 3, however were phosphorylated in cells infected with this mutant. (iii) Band 2 protein was absent from cells transfected with a plasmid carrying a substitution of the predicted first methionine codon of the U(L)3 ORF and superinfected with the U(L)3(-) mutant. (iv) Band 1 and 3 proteins were absent from lysates of cells transfected with a plasmid carrying a substitution of the second (M12) methionine codon of the U(L)3 ORF and superinfected with the U(L)3(-) mutant. (v) Cells superinfected with mutants lacking both methionine codons did not accumulate any of the proteins contained in the six bands. (vi) In vitro transcription-translation studies indicated that the translation of band 1 protein was initiated from the second (M12) methionine codon and that band 3 protein represented a U(L)13-independent, posttranslationally processed form of these proteins. The results indicate that the U(L)3 protein of herpes simplex virus 1 is translated predominantly from the second in-frame methionine codon and is subject to at least two posttranslational modifications.

Amino Acid Sequence↗

PubMed1999

Herpes simplex virus 1 blocks caspase-3-independent and caspase-dependent pathways to cell death.

Earlier reports have shown that herpes simplex virus 1 (HSV-1) mutants induce programmed cell death and that wild-type HSV blocks the execution of the cell death program triggered by viral gene products, by the effectors of the immune system such as the Fas and tumor necrosis factor pathways, or by nonspecific stress agents such as either osmotic shock induced by sorbitol or thermal shock. A report from this laboratory showed that caspase inhibitors do not block DNA fragmentation induced by infection with the HSV-1 d120 mutant. To identify the events in programmed cell death induced and blocked by HSV-1, we examined cells infected with wild-type virus or the d120 mutant or cells infected and exposed to sorbitol. We report that: (i) the HSV-1 d120 mutant induced apoptosis by a caspase-3-independent pathway inasmuch as caspase 3 was not activated and DNA fragmentation was not blocked by caspase inhibitors even though the virus caused cytochrome c release and depolarization of the inner mitochondrial membrane. (ii) Cells infected with wild-type HSV-1 exhibited none of the manifestations associated with programmed cell death assayed in these studies. (iii) Uninfected cells exposed to osmotic shock succumbed to caspase-dependent apoptosis inasmuch as cytochrome c was released, the inner mitochondrial potential was lost, caspase-3 was activated, and chromosomal DNA was fragmented. (iv) Although caspase-3 was activated in cells infected with wild-type HSV-1 and exposed to sorbitol, cytochrome c outflow, depolarization of the inner mitochondrial membrane, and DNA fragmentation were blocked. We conclude that although d120 induces apoptosis by a caspase-3-independent pathway, the wild-type virus blocks apoptosis induced by this pathway and also blocks the caspase-dependent pathway induced by osmotic shock. The block in the caspase-dependent pathway may occur downstream of caspase-3 activation.

Apoptosis↗

PubMed1999

A novel cellular protein, p60, interacting with both herpes simplex virus 1 regulatory proteins ICP22 and ICP0 is modified in a cell-type-specific manner and Is recruited to the nucleus after infection.

Herpes simplex virus 1 encodes two multifunctional regulatory proteins, infected-cell proteins 22 and 0 (ICP22 and ICP0). ICP0 is a promiscuous transactivator, whereas ICP22 is required in vivo and for efficient replication and expression of a subset of late (gamma2) genes in rodent or rabbit cell lines and in primary human cell strains (restrictive cells) but not in HEp-2 or Vero (permissive) cells. We report the identification in the yeast two-hybrid system of a cellular protein designated p60 that interacts with ICP22. This protein (apparent Mr of 60,000) has not been previously described and has no known motifs. Analyses of p60 revealed the following. (i) p60 bound fast-migrating, underprocessed wild-type ICP22 and ICP22 lacking the carboxyl-terminal 24 amino acids but not ICP22 lacking the carboxyl-terminal 40 amino acids, whereas the previously identified cellular protein p78 (R. Bruni and B. Roizman, J. Virol. 72:8525-8531, 1998) bound all forms of ICP22. The interaction of p60 with only one isoform of ICP22 supports that hypothesis that each isoform of herpes simplex virus proteins performs a specific function that may be different from that of other isoforms. (ii) p60 also bound ICP0; the binding of ICP0 was independent of that of ICP22. (iii) p60 localized in uninfected rabbit skin cells in both nuclei and cytoplasm. In rabbit skin cells infected with wild-type virus, p60 was posttranslationally processed to a higher apparent Mr but was not redistributed. Posttranslational processing required the presence of the genes encoding ICP22 and UL13 protein kinase. (iv) In uninfected HEp-2 cells, p60 localized primarily in nuclei. Soon after infection with wild-type virus, the p60 localized in discrete small nuclear structures with ICP0. Late in infection, both ICP0 and p60 tended to disperse but p60 did not change in apparent Mr. The localization of p60 was independent of ICP22, but p60 tended to be more localized in small nuclear structures and less dispersed in cells infected with mutants lacking the genes encoding the UL13 or US3 protein kinases. The results suggest that posttranslational modification of p60 is mediated either by ICP0 (permissive cells) or by ICP22 and UL13 protein kinase (restrictive rabbit skin cells) and that the restrictive phenotype of rabbit skin cells may be related to the failure to process p60 by mutants lacking the genes encoding UL13 or ICP22.

Amino Acid Sequence↗

PubMed1999

Functional anatomy of herpes simplex virus 1 overlapping genes encoding infected-cell protein 22 and US1.5 protein.

Earlier studies have shown that (i) the coding domain of the alpha22 gene encodes two proteins, the 420-amino-acid infected-cell protein 22 (ICP22) and a protein, US1.5, which is initiated from methionine 147 of ICP22 and which is colinear with the remaining portion of that protein; (ii) posttranslational processing of ICP22 mediated largely by the viral protein kinase UL13 yields several isoforms differing in electrophoretic mobility; and (iii) mutants lacking the carboxyl-terminal half of the ICP22 and therefore DeltaUS1.5 are avirulent and fail to express normal levels of subsets of both alpha (e.g., ICP0) or gamma2 (e.g., US11 and UL38) proteins. We have generated and analyzed two sets of recombinant viruses. The first lacked portions of or all of the sequences expressed solely by ICP22. The second set lacked 10 to 40 3'-terminal codons of ICP22 and US1. 5. The results were as follows. (i) In cells infected with mutants lacking amino-terminal sequences, translation initiation begins at methionine 147. The resulting protein cannot be differentiated in mobility from authentic US1.5, and its posttranslational processing is mediated by the UL13 protein kinase. (ii) Expression of US11 and UL38 genes by mutants carrying only the US1.5 gene is similar to that of wild-type parent virus. (iii) Mutants which express only US1. 5 protein are avirulent in mice. (iv) The coding sequences Met147 to Met171 are essential for posttranslational processing of the US1.5 protein. (v) ICP22 made by mutants lacking 15 or fewer of the 3'-terminal codons are posttranslationally processed whereas those lacking 18 or more codons are not processed. (vi) Wild-type and mutant ICP22 proteins localized in both nucleus and cytoplasm irrespective of posttranslational processing. We conclude that ICP22 encodes two sets of functions, one in the amino terminus unique to ICP22 and one shared by ICP22 and US1.5. These functions are required for viral replication in experimental animals. US1.5 protein must be posttranslationally modified by the UL13 protein kinase to enable expression of a subset of late genes exemplified by UL38 and US11. Posttranslational processing is determined by two sets of sequences, at the amino terminus and at the carboxyl terminus of US1.5, respectively, a finding consistent with the hypothesis that both domains interact with protein partners for specific functions.

Amino Acid Sequence↗

PubMed1999

Cellular elongation factor 1delta is modified in cells infected with representative alpha-, beta-, or gammaherpesviruses.

Earlier reports (Y. Kawaguchi, R. Bruni, and B. Roizman, J. Virol. 71:1019-1024, 1997; Y. Kawaguchi, C. Van Sant, and B. Roizman, J. Virol. 72:1731-1736, 1998) showed that herpes simplex virus 1 (HSV-1) infection causes the hyperphosphorylation of translation elongation factor 1delta (EF-1delta) and that the modification of EF-1delta is the consequence of direct phosphorylation by a viral protein kinase encoded by the UL13 gene of HSV-1. The UL13 gene is conserved in members of all herpesvirus subfamilies. Here we report the following. (i) In various mammalian cells, accumulation of the hyperphosphorylated form of EF-1delta is observed after infection with alpha-, beta-, and gammaherpesviruses, including HSV-2, feline herpesvirus 1, pseudorabiesvirus, bovine herpesvirus 1, human cytomegalovirus (HCMV), and equine herpesvirus 2. (ii) In human lung fibroblast cells infected with recombinant HSV-1 lacking the UL13 gene, the hypophosphorylated form of EF-1delta is a minor species, whereas the amount of the hyperphosphorylated form of EF-1delta significantly increases in cells infected with the recombinant HSV-1 in which UL13 had been replaced by HCMV UL97, a homologue of UL13. These results indicate that the posttranslational modification of EF-1delta is conserved herpesvirus function and the UL13 homologues may be responsible for the universal modification of the translation factor.

Alphaherpesvirinae↗

PubMed1999

Colocalization of the herpes simplex virus 1 UL4 protein with infected cell protein 22 in small, dense nuclear structures formed prior to onset of DNA synthesis.

Herpes simplex virus 1 infected cell protein 22 (ICP22) localizes in small, dense nuclear bodies of primate cells early in infection and in the more diffuse replicative complexes after the onset of DNA synthesis. UL4, a gamma2 protein, localizes in cytoplasm and in the small nuclear structures containing ICP22 but not in replicative complexes. In rabbit skin cells, both ICP22 and UL4 localize in the dense nuclear bodies late in infection. The results suggest that the small nuclear structures perform a function involving both proteins late in infection.

Animals↗