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S A Rice

Publications and source records attributed to S A Rice.

At least 19 recordsLinked to original sources
PubMed1998

The herpes simplex virus immediate-early protein ICP27 shuttles between nucleus and cytoplasm.

ICP27 is an essential herpes simplex virus type 1 (HSV-1) nuclear protein which regulates viral early and late genes during infection. The exact mechanism by which ICP27 modulates viral gene expression is unknown, but considerable evidence suggests that it functions posttranscriptionally. In this study, we have asked whether ICP27, like some other viral and cellular posttranscriptional regulatory proteins, shuttles between the nuclear and cytoplasmic compartments of the cell. Using an interspecies heterokaryon assay, we demonstrate that ICP27, but not the HSV-1 nuclear proteins ICP4 or ICP8, is an efficient shuttling protein. ICP27's shuttling ability does not depend on viral infection or other HSV-1 proteins, as it shuttles even when transiently expressed in uninfected cells. To understand the importance of shuttling for ICP27's regulatory functions, we examined several mutant forms of ICP27 to see whether they exhibited altered shuttling. We identified three ICP27 mutations which partially disrupt shuttling, as well as one mutation, M15, which completely abrogates this activity. The M15 mutation alters residues 465 and 466 near the carboxyl terminus of ICP27 and was previously shown to inactivate ICP27's ability to induce certain viral late mRNAs. These results suggest that ICP27's nuclear shuttling activity is involved in its viral late gene activation function.

Animals↗
PubMed1997

Repetitive sequences found in the chromosome of the myxobacterium Nannocystis exedens are similar to msDNA: a possible retrotransposition event in bacteria.

The first reverse transcriptase (RT) to be found in a prokaryotic cell is encoded by an element called a retron which resides in the chromosome of many different bacteria. In addition, all retrons code for a functionally obscure RNA-DNA satellite molecule called msDNA. msDNA is synthesized from an RNA template by the retron-encoded RT. An unusual retron element is described here from the myxobacterium Nannocystis exedens. This retron does not appear to have a typical RT gene in close proximity (1 kb) to the gene msd (which encodes the DNA strand of msDNA). The gene msr (which encodes the RNA strand of msDNA) appears to be duplicated and flanks both sides of the msd gene. Also discovered throughout the chromosome of this bacterium is a set of repeated sequences related to msDNA. These repeat sequences match only part of the sequences of msDNA and may have become incorporated into the chromosome of this bacterium by reverse transcription.

Base Sequence↗
PubMed1997

Repression of host RNA polymerase II transcription by herpes simplex virus type 1.

Lytic infection of mammalian cells with herpes simplex virus type 1 (HSV-1) results in rapid repression of host gene expression and selective activation of the viral genome. This transformation in gene expression is thought to involve repression of host transcription and diversion of the host RNA polymerase (RNAP II) transcription machinery to the viral genome. However, the extent of virus-induced host transcription repression and the mechanisms responsible for these major shifts in transcription specificities have not been examined. To determine how HSV-1 accomplishes repression of host RNAP II transcription, we assayed transcription patterns on several cellular genes in cells infected with mutant and wild-type HSV-1. Our results suggest that HSV-1 represses RNAP II transcription on most cellular genes. However, each cellular gene we examined responds differently to the transcription repressive effects of virus infection, both quantitatively and with respect to the involvement of viral gene products. Virus-induced shutoff of host RNAP II transcription requires expression of multiple immediate-early genes. In contrast, expression of delayed-early and late genes and viral DNA replication appear to contribute little to repression of host cell RNAP II transcription. Modification of RNAP II to the intermediately phosphorylated (II(I)) form appears unlinked to virus-induced repression of host cell transcription. However, full repression of host transcription is correlated with depletion of the hyperphosphorylated (IIO) form of RNAP II.

Cell Line↗
PubMed1997

Pediatric morning report: an appraisal.

We examined and contrasted morning reports at two hospitals, university and community, that have a pediatric residency program. Patient diagnoses assigned at morning report were compared with final diagnoses to assess disease categories discussed and the value of including outpatient follow-up in this educational forum. Data were obtained during morning reports for 6 months by chief residents at university and private community hospitals. Pertinent history, physical examination, and laboratory and radiologic findings were recorded and were assigned a tentative morning report diagnosis based on morning report discussion. Cases were then reviewed at discharge and at 6 months to determine final diagnoses. At the university hospital, 58% of the cases were undiagnosed before presentation at morning report. Of those cases, 23% were assigned a diagnosis at morning report that differed from the final diagnosis. Similarly, at the private community hospital, 28% of cases were undiagnosed before presentation at morning report. Of those cases, 73% were assigned a diagnosis that differed from the final diagnosis. We conclude that the provision of follow-up at morning report is important for maximizing resident education.

Child↗
PubMed1996

The RGG box motif of the herpes simplex virus ICP27 protein mediates an RNA-binding activity and determines in vivo methylation.

ICP27 is an essential herpes simplex virus type 1 nuclear regulatory protein that is required for efficient viral gene expression. Although the mechanism by which ICP27 regulates genes is unknown, a variety of evidence suggests that it functions posttranscriptionally, and recent studies indicate that it is an RNA-binding protein. Previously, we noted that a short arginine- and glycine-rich sequence in ICP27 (residues 138 to 152) is similar to an RGG box motif, a putative RNA-binding determinant found in a number of cellular proteins (W. Mears, V. Lam, and S. Rice, J. Virol. 69:935-947, 1995). In the present study, we have further investigated ICP27's association with RNA and examined the role of the RGG box in RNA binding. We find that ICP27 binds efficiently to RNA homopolymers composed of poly(G) and weakly to poly(U) RNA homopolymers. Poly(G) binding activity maps to the N-terminal 189 residues of ICP27 and requires the RGG box sequence. Using a northwestern blotting assay, we demonstrate that the RGG box alone (residues 140 to 152) can mediate RNA binding when attached to a heterologous protein. As many cellular RGG box proteins are methylated on arginine residues, we also investigated the in vivo methylation status of ICP27. Our results demonstrate that ICP27 is methylated in herpes simplex virus-infected cells. Methylation is dependent on the presence of the RGG box, suggesting that one or more arginine residues in the RGG box sequence are modified. These data demonstrate that ICP27 displays the characteristics of an RGG box-type RNA-binding protein.

Amino Acid Sequence↗
PubMed1996

Attenuation of DNA-dependent protein kinase activity and its catalytic subunit by the herpes simplex virus type 1 transactivator ICP0.

The DNA-dependent protein kinase (DNA-PK) is involved in several fundamental nuclear processes, including DNA double-strand break repair, V(D)J recombination, and transcription by RNA polymerases I and II. In this study, we show that infection of mammalian cells with herpes simplex virus type 1 attenuates DNA-PK activity by specifically depleting the p350/DNA-PKcs catalytic subunit. The half-life of the p350/DNA-PKcs protein decreases from greater than 24 h to less than 4 h following infection. The depletion of DNA-PK activity and p350/DNA-PKcs abundance is dependent on expression of the viral immediate-early protein ICP0. As ICP0 acts as a promoter-independent transactivator of gene expression, these data suggest that ICP0 may function by directly or indirectly targeting the p350/DNA-PKcs subunit of DNA-PK, thereby altering the inhibitory effects of DNA-PK on RNA polymerase II transcription.

Amino Acid Sequence↗
PubMed1995

Bacterial reverse transcriptase and msDNA.

Retrons are a new class of genetic elements found in the chromosome of a large number of different bacteria. These elements code for a reverse transcriptase (RT) that is structurally similar to the polymerases of retroviruses. The retron associated RT is responsible for the production of an unusual extrachromosomal satellite DNA, known as multicopy, single-stranded DNA (msDNA). Synthesis of msDNA is dependent on a novel self-priming mechanism, resulting in the formation of a 2',5'-phosphodiester bond. A comparison of bacterial RTs is presented, noting conserved and unique features of these polymerases. In addition, the origin, means of dissemination, and possible activities of these functionally obscure retroelements are discussed.

Amino Acid Sequence↗
PubMed1995

Phylogenetic comparison of retron elements among the myxobacteria: evidence for vertical inheritance.

Twenty-eight myxobacterial strains, representing members from all three subgroups, were screened for the presence of retron elements, which are novel prokaryotic retroelements encoding reverse transcriptase. The presence of retrons was determined by assaying strains for a small satellite DNA produced by reverse transcription called multicopy, single-stranded DNA (msDNA). An msDNA-producing retron appeared to be absent from only one of the strains surveyed. DNA hybridization experiments revealed that retron elements similar to retron Mx162, first identified in Myxococcus xanthus, were found only among members of the Myxococcus subgroup; that is, each of the seven different genera which constitute this subgroup contained a Mx162 homolog. Another retron element also appeared to have a clustered distribution, being found exclusively within the Nannocystis subgroup of the myxobacteria. A retron element of the Mx162 type was cloned from Melittangium lichenicola, and its DNA sequence was compared with those of similar elements in M. xanthus and Stigmatella aurantiaca. Together, the degree of sequence diversity, the codon bias of the reverse transcriptase genes, and the clustered distribution of these retrons suggest a possible evolutionary scenario in which a common ancestor of the Myxococcus subgroup may have acquired this retroelement.

Amino Acid Sequence↗
PubMed1995

Identification of nuclear and nucleolar localization signals in the herpes simplex virus regulatory protein ICP27.

Previous work has shown that the herpes simplex virus type 1 (HSV-1) regulatory protein ICP27 localizes to the cell nucleus and that certain mutant ICP27 polypeptides localize preferentially in nucleoli. To map the signals in ICP27 which mediate its nuclear localization, we identified the portions of ICP27 which can direct a cytoplasmic protein, pyruvate kinase (PK), to nuclei. Our results demonstrate that ICP27 contains multiple nuclear localization signals (NLSs) that function with differing efficiencies. First, ICP27 possesses a strong NLS, mapping to residues 110 to 137, which bears similarity to the bipartite NLSs found in Xenopus laevis nucleoplasmin and other proteins. Second, ICP27 possesses one or more weak NLSs which map to a carboxyl-terminal portion of the protein between residues 140 and 512. Our PK-targeting experiments also demonstrate that ICP27 contains a relatively short sequence, mapping to residues 110 to 152, that can function as a nucleolar localization signal (NuLS). This signal includes ICP27's strong NLS as well as 15 contiguous residues which consist entirely of arginine and glycine. This latter sequence is very similar to an RGG box, a putative RNA-binding motif found in a number of cellular proteins which are involved in nuclear RNA processing. To confirm the results of the PK-targeting experiments, we mutated the ICP27 gene by deleting sequences encoding either the strong NLS or the RGG box. Deletion of the strong NLS (residues 109 to 138) resulted in an ICP27 molecule that was only partially defective for nuclear localization, while deletion of the RGG box (residues 139 to 153) resulted in a molecule that was nuclear localized but excluded from nucleoli. Recombinant HSV-1s bearing either of these deletions were unable to replicate efficiently in Vero cells, suggesting that ICP27's strong NLS and RGG box carry out important in vivo functions.

Amino Acid Sequence↗
PubMed1995

Herpes simplex virus immediate-early protein ICP22 is required for viral modification of host RNA polymerase II and establishment of the normal viral transcription program.

Infection of cells with herpes simplex virus type 1 (HSV-1) results in a rapid alteration of phosphorylation on the large subunit of cellular RNA polymerase II (RNAP II), most likely on its C-terminal domain (S. A. Rice, M. C. Long, V. Lam, C. A. Spencer, J. Virol. 68:988-1001, 1994). This phosphorylation modification generates a novel form of the large subunit which we have designed IIi. In this study, we examine roles that HSV-1 gene products play in this process. An HSV-1 mutant defective in the immediate-early transcriptional activator protein ICP4 is able to efficiently induce IIi. Viruses having mutations in the genes for the ICP0, ICP6, or ICP27 proteins are also competent for IIi formation. In contrast, 22/n199, an HSV-1 mutant which contains a nonsense mutation in the gene encoding the immediate-early protein ICP22, is significantly deficient in IIi induction. This effect is seen in Vero cells, where 22/n199 grows relatively efficiently, and in human embryonic lung (HEL) cells, where 22/n199 growth in more restricted. RNAP II is recruited into viral replication compartments in 22/n199-infected cells, indicating that altered phosphorylation of RNAP II is not a prerequisite for nuclear relocalization of RNAP II. In addition, we show by nuclear run-on transcription analysis that viral gene transcription is deficient in HEL cells infected with 22/n199. Viral late gene transcription does not occur efficiently, and antisense transcription throughout the genome is diminished compared with that of the wild-type HSV-1 infection. These transcriptional effects cannot be explained by differences in viral DNA replication, since 22/n199 replicates its DNA efficiently in HEL cells. Our results demonstrated that ICP22 is necessary for virus-induced aberrant phosphorylation of RNAP II and for normal patterns of viral gene transcription in certain cell lines.

Animals↗
PubMed1994

Amino acid substitution mutations in the herpes simplex virus ICP27 protein define an essential gene regulation function.

ICP27 is an essential herpes simplex virus type 1 (HSV-1) alpha protein that is required for the transition from the beta to the gamma phase of infection. To identify functional regions of ICP27, we constructed 16 plasmids that contain nucleotide substitution mutations in the ICP27 gene. The mutations created XhoI restriction sites, altered one or two codons, and were spaced at semiregular intervals throughout the coding region. Three mutations completely inactivated an essential function of ICP27, as demonstrated by the inability of the transfected plasmids to complement the growth of an HSV-1 ICP27 deletion mutant. These mutations, M11, M15, and M16, mapped in the carboxyl-terminal one-third of ICP27 at residues 340 and 341, 465 and 466, and 488, respectively. In cotransfection assays, all three defective-plasmid mutants retained the transrepression function of ICP27 but were defective at transactivation. To define the lytic functions that are mediated by the transactivation activity of ICP27, we engineered HSV-1 recombinants containing the M11, M15, or M16 mutation. All three viral mutants failed to grow in Vero cells and possessed similar phenotypes. The viral mutants replicated their DNA similarly to the wild-type virus but showed several defects in viral gene expression. These were a failure to down-regulate alpha and beta genes at late times after infection and an inability to induce certain gamma-2 genes. Our results demonstrate that the transactivation function of ICP27 (as it is defined in cotransfection assays) mediates an essential gene regulation function during the HSV-1 infection. This activity is not required for ICP27-dependent enhancement of viral DNA replication. Our work supports and extends previous studies which suggest that ICP27 carries out two distinct regulatory activities during the HSV-1 infection.

Amino Acids↗
PubMed1994

RNA polymerase II is aberrantly phosphorylated and localized to viral replication compartments following herpes simplex virus infection.

During lytic infection, herpes simplex virus subverts the host cell RNA polymerase II transcription machinery to efficiently express its own genome while repressing the expression of most cellular genes. The mechanism by which RNA polymerase II is directed to the viral delayed-early and late genes is still unresolved. We report here that RNA polymerase II is preferentially localized to viral replication compartments early after infection with herpes simplex virus type 1. Concurrent with recruitment of RNA polymerase II into viral compartments is a rapid and aberrant phosphorylation of the large subunit carboxy-terminal domain (CTD). Aberrant phosphorylation of the CTD requires early viral gene expression but is not dependent on viral DNA replication or on the formation of viral replication compartments. Localization of RNA polymerase II and modifications to the CTD may be instrumental in favoring transcription of viral genes and repressing specific transcription of cellular genes.

Animals↗
PubMed1994

UL69 of human cytomegalovirus, an open reading frame with homology to ICP27 of herpes simplex virus, encodes a transactivator of gene expression.

The UL69 open reading frame of human cytomegalovirus (HCMV) is homologous to the immediate-early protein ICP27 of herpes simplex virus, an essential viral regulatory protein involved in the transition from early to late gene expression. Genes with homology to ICP27 have been detected in all subclasses of herpesviruses so far. While the respective proteins in alpha- and gammaherpesviruses have been defined as trans-regulatory molecules, nothing is known about these genes in betaherpesviruses. This study was therefore undertaken in order to investigate expression from the UL69 gene locus of HCMV. Northern (RNA) blot experiments revealed a complex pattern of transcripts that changed during the time course of the HCMV replicative cycle: two transcripts of 2.7 and 3.5 kb that were regulated differentially could be detected as early as 7 h after infection. However, these transcripts could not be detected in the presence of cycloheximide. Additional, larger transcripts were present exclusively at late times after infection. To analyze protein expression from the UL69 gene region, the UL69 open reading frame was expressed as a histidine-tagged protein in Escherichia coli. A specific antiserum was generated and used to detect the UL69 protein in HCMV-infected cells which revealed its localization within the intranuclear inclusions that are characteristic for HCMV infection. In cotransfection experiments, an HCMV true late promoter could not be activated by UL69, whereas an early promoter and several heterologous promoters were stimulated about 10-fold. Complementation studies showed that the UL69 protein cannot substitute for ICP27 in the context of the HSV infection, suggesting functional differences between these two proteins. In summary, these experiments define a novel regulatory protein encoded by HCMV that is expressed as an early-late gene and appears to exert a broad stimulatory effect on gene expression.

Amino Acid Sequence↗
PubMed1994

A poxvirus protein with a RING finger motif binds zinc and localizes in virus factories.

Shope fibroma virus (SFV) is a Leporipoxvirus closely related to the highly virulent myxoma virus. The DNA sequence of the BamHI N fragment of the SFV DNA genome was determined, and the single complete open reading frame (N1R) was characterized. The protein encoded by the N1R gene was found to contain a C3HC4 RING finger motif at the C terminus. This C3HC4 motif is the hallmark of a growing family of proteins, many of which are involved in regulation of gene expression, DNA repair, or DNA recombination. Complete homologs of the SFV N1R gene were also detected in variola virus, myxoma virus, and vaccinia virus strain IHD-W. In contrast, the gene is completely absent from vaccinia virus strain Copenhagen, and in vaccinia virus strain WR, the open reading frame is truncated prior to the zinc binding domain because of an 11-bp deletion, thus producing a frameshift and premature stop codon. Recombinant N1R protein from SFV was expressed in Escherichia coli and shown to bind zinc in a specific manner. Using fluorescence microscopy to visualize a peptide epitope tag (derived from ICP27 of herpes simplex virus) fused to the N terminus of the poxvirus proteins, we observed that the N1R protein of SFV and its homologs in myxoma virus and vaccinia virus IHD-W were localized primarily to the virus factories in the cytoplasm of infected cells and, to a lesser degree, the host cell nucleus. The truncated protein of vaccinia virus strain WR failed to localize in this manner but instead was observed throughout the cytoplasm.

Amino Acid Sequence↗
PubMed1993

The clinical spectrum of patients with aneurysms of the ascending aorta.

Aneurysms of the ascending aorta are often unsuspected, yet they can quickly lead to death from aortic rupture or dissection. To examine the clinical spectrum of patients with aneurysms of the ascending aorta, we searched the University of California, San Francisco (USCF) Echocardiography Data Base for all patients with aneurysms of the ascending aorta (> or = 5.0 cm in diameter) seen over a 7-year period. The echocardiograms and clinical courses of these patients were then reviewed. We identified 15 patients with aneurysms of the ascending aorta: five had aneurysms > 7.0 cm in diameter, three had aneurysms 6.0 to 6.9 cm, and seven had aneurysms 5.0 to 5.9 cm in diameter. Among the five patients < 50 years of age, four had Marfan's syndrome, and among the 10 patients > or = 50 years of age, eight had evidence of atherosclerotic vascular disease. At presentation, 13 patients had nonspecific symptoms, and two were asymptomatic. Echocardiography demonstrated that 12 patients had at least mild aortic insufficiency and that five had aortic dissections. One of the seven patients who underwent surgical resection died of an intraoperative cardiac arrest, and two of the eight patients treated medically died within 1 week of presentation. We conclude that the clinical spectrum of patients with aneurysms of the ascending aorta is wide. Because these aneurysms are often unsuspected, physicians should have a low threshold for imaging the ascending aorta in patients with Marfan's syndrome or atherosclerotic vascular disease, particularly when aortic insufficiency is present.

Adult↗