Biomedical subjects

R C Herman

Publications and source records attributed to R C Herman.

At least 19 recordsLinked to original sources

PubMed1998

The UL4 gene of herpes simplex virus type 1 is dispensable for latency, reactivation and pathogenesis in mice.

The UL4 gene of herpes simplex virus type 1 is predicted to encode a 21.5 kDa protein of 199 amino acids. Although UL4 is dispensable for growth in cell culture, its function is not known. In the present study, the promoter of UL4 was examined and found to contain a cAMP-response element which bound the transcription factor CREB, and was strongly activated by cAMP. A recombinant virus, termed UL4HS, was constructed with a nonsense linker inserted into the UL4 open reading frame, to make a truncated UL4 protein of 60 amino acids. In addition, a marker-rescued virus, UL4HSMR, was constructed. Western immunoblot analysis revealed a 23 kDa band in extracts of wild-type and marker-rescued virus infected cells which was missing for UL4HS. Only modest differences were observed in the abilities of wild-type and UL4-mutant viruses to grow in Vero cells or in contact-inhibited mouse C3H/10T1/2 cells. In addition, there were only modest differences between the ability of UL4HS to replicate in murine corneas and trigeminal ganglia relative to wild-type viruses, and reactivation of UL4HS from latency was unaffected. Taken together, these data demonstrate that UL4 is dispensable for latency and pathogenesis in mice.

Amino Acid Sequence↗

PubMed1997

A novel tetrodotoxin-sensitive, voltage-gated sodium channel expressed in rat and human dorsal root ganglia.

Dorsal root ganglion neurons express a wide repertoire of sodium channels with different properties. Here, we report the cloning from rat, dorsal root ganglia (DRG), cellular expression, and functional analysis of a novel tetrodotoxin-sensitive peripheral sodium channel (PN), PN1. PN1 mRNA is expressed in many different tissues. Within the rat DRG, both the mRNA and PN1-like immunoreactivity are present in small and large neurons. The abundance of sodium channel mRNAs in rat DRG is rBI > PN1 >/= PN3 >>> rBIII by quantitative reverse transcription-polymerase chain reaction analysis. Data from reverse transcription-polymerase chain reaction and sequence analyses of human DRG and other human tissues suggest that rat PN1 is an ortholog of the human neuroendocrine channel. In Xenopus oocytes, PN1 exhibits kinetics that are similar to rBIIa sodium currents and is inhibited by tetrodotoxin with an IC50 of 4.3 +/- 0.92 nM. Unlike rBIIa, the inactivation kinetics of PN1 are not accelerated by the coexpression of the beta-subunits.

Amino Acid Sequence↗

PubMed1996

Structure and function of a novel voltage-gated, tetrodotoxin-resistant sodium channel specific to sensory neurons.

Small neurons of the dorsal root ganglia (DRG) are known to play an important role in nociceptive mechanisms. These neurons express two types of sodium current, which differ in their inactivation kinetics and sensitivity to tetrodotoxin. Here, we report the cloning of the alpha-subunit of a novel, voltage-gated sodium channel (PN3) from rat DRG. Functional expression in Xenopus oocytes showed that PN3 is a voltage-gated sodium channel with a depolarized activation potential, slow inactivation kinetics, and resistance to high concentrations of tetrodotoxin. In situ hybridization to rat DRG indicated that PN3 is expressed primarily in small sensory neurons of the peripheral nervous system.

Amino Acid Sequence↗

PubMed1996

The beta 1 sodium channel subunit modifies the interactions of neurotoxins and local anesthetics with the rat brain IIA alpha sodium channel in isolated membranes but not in intact cells.

Mammalian brain sodium channels consist of an alpha subunit and two smaller beta subunits. The role of the beta 1 subunit in modulating ligand interactions at these channels was examined using a cell line stably expressing human beta1 and rat brain IIA alpha subunits. Coexpression of the beta 1 subunit had no effect on the potencies of sodium channel blockers in inhibiting whole cell [3H]batrachotoxinin A benzoate ([3H]BTX) binding or veratridine-stimulated [14C]guanidinium influx. Coexpression of the beta 1 subunit also had no effect on the potencies of alpha scorpion toxin, brevetoxin, or RU 39568 in stimulating [14C]guanidinium influx. By contrast, coexpression of the beta 1 subunit had dramatic effects on ligand interactions in isolated membranes. In isolated membranes of cells expressing only the alpha subunit, the neurotoxins had no stimulatory effect on [3H]BTX binding and the potencies of local anesthetic-like channel inhibitors were 10-100-fold lower than those at native sodium channels. Whereas in membranes of cells coexpressing the beta 1 subunit, the neurotoxins increased [3H]BTX binding 30-fold and the potencies of the sodium channel inhibitors closely matched those found at native sodium channels. These findings indicate that the beta 1 subunit is not required for the binding of sodium channel activators or inhibitors but rather, that the beta 1 subunit may stabilize the alpha subunit in a functional conformation thereby allowing detection of these interactions in disrupted membranes.

Anesthetics, Local↗

PubMed1994

A herpes simplex virus type 1 ICP22 deletion mutant is altered for virulence and latency in vivo.

The in vivo function of the herpes simplex virus type 1 immediate early gene ICP22 has been investigated in mice and guinea pigs using a deletion mutant (del22Z) of HSV-1(F) that lacks all but 18 nucleotides of the ICP22 coding sequence. This mutant carries the bacterial lacZ gene at the site of the deletion and makes functional beta-galactosidase, but is unable to synthesize any detectable ICP22 messenger RNA or protein in vitro. Del22Z was impaired in its ability to cause death in mice following intracerebral, intraperitoneal, or intravaginal inoculation. The mutant failed to produce lesions or other visible signs of infection after bilateral corneal infection of mice but could be recovered from trigeminal ganglia explanted at day 30 after inoculation. Del22Z replicated poorly after intravaginal inoculation of mice and guinea pigs in comparison to the parental virus, and was not recoverable from the dorsal root ganglia of either species. Nevertheless, del22Z sequences could be detected in the dorsal root ganglia of guinea pigs at day 30 by the polymerase chain reaction. These studies demonstrate that the ICP22 gene product is required for acute infection and virulence in two standard in vivo animal models.

Animals↗

PubMed1993

In vitro characterization of a herpes simplex virus type 1 ICP22 deletion mutant.

We report the construction of a deletion mutant (del22Z) that is unable to synthesize any detectable messenger RNA or protein products from the herpes simplex virus type 1 (HSV-1) immediate early ICP22 gene upon infection. The del22Z deletion mutant lacks all but 18 nucleotides of the ICP22 coding sequence and carries the bacterial lacZ gene at the site of the deletion. No other known open reading frames or flanking sequences were disrupted. Del22Z was able to infect Vero cells productively but was severely restricted in human and rodent cells that were permissive for the parental HSV-1(F). The yield of del22Z was not enhanced significantly, either by increasing the multiplicity of infection or by increasing the duration of the infection. There was a prolonged expression of some early gene products and a delayed appearance of some late gene products in both permissive and restrictive cells. This phenotype of cell-line restricted growth and alteration of the normal gene expression cascade maps specifically to the ICP22 coding region.

Cell Line↗

PubMed1992

Increased synthesis of polycistronic mRNA associated with increased polyadenylation by vesicular stomatitis virus.

Electron microscopy suggested that the mRNA produced in vitro by tsG16(I), a temperature-sensitive mutant of vesicular stomatitis virus, contained an increased proportion of polycistronic mRNAs. Using hybrid selection, we found that the poly(A)+ mRNA synthesized in vitro by tsG16(I) contained approximately two to three times more polycistronic mRNA than did poly(A)+ mRNA synthesized in vitro by the parental wild-type (wt) virus. The increase in polycistronic mRNA occurred at all intergenic junctions examined. In vitro, tsG16(I) has an increased polyadenylation phenotype and a temperature-sensitive transcriptase activity that appear to be due to different mutations. Partial revertants of tsG16(I), which have lost the aberrant polyadenylation phenotype but retain the in vitro thermosensitive transcriptase, produced wt amounts of polycistronic mRNA. This suggested that the increased production of polycistronic mRNA by tsG16(I) may be associated with the increased polyadenylation phenotype of this mutant. These data further support the hypothesis that an increase in size of poly(A) tracts is associated with increased production of polycistronic mRNA.

Base Sequence↗

PubMed1992

Deletion of the herpes simplex virus type 1 ribonucleotide reductase gene alters virulence and latency in vivo.

The role of herpes simplex virus type 1 (HSV-1)-encoded ribonucleotide reductase (RR) has been investigated in mice and guinea pigs using a mutant from which 90% of the large subunit of the enzyme was deleted. The RR mutant was extremely impaired in its ability to induce external vaginal lesions or to cause death in mice following intracerebral, intraperitoneal, or intravaginal inoculation, or in guinea pigs following intraperitoneal or intravaginal inoculation. The RR mutant replicated poorly in the vagina of mice and guinea pigs when compared with the parental virus. Neither infectious nor latent virus was recovered from the trigeminal ganglia of mice or from the dorsal root ganglia of mice and guinea pigs after inoculation with the RR mutant. Using the polymerase chain reaction, RR mutant DNA was, nevertheless, detected in the dorsal root ganglia of guinea pigs. These studies suggest that HSV-1 RR is essential for virulence and may also play a role in the recovery of reactivatable latent virus from ganglia in both mice and guinea pigs.

Animals↗

PubMed1992

The herpes simplex virus type 1 ICP6 gene is regulated by a 'leaky' early promoter.

Expression from the promoter for the large subunit (ICP6) of the ribonucleotide reductase encoded by herpes simplex virus type 1 (HSV-1) has been examined. Using the lacZ reporter gene fused in-frame with ICP6 regulatory sequences to assay expression quantitatively, we showed that the ICP6 promoter responded very weakly to the alpha-transinducing factor (TIF) in the absence of all other viral gene products, but much more strongly to immediate early proteins. Similar patterns of regulation were observed when the reporter gene construct was located at two different positions within the the viral genome or in a stably transfected Vero cell line. Infection of the stably transfected cells with various HSV-1 mutants identified ICP0 as the major transactivator of the ICP6 promoter.

Animals↗

PubMed1990

Genetic evidence for multiple functions of the matrix protein of vesicular stomatitis virus.

TsO82, a spontaneous temperature-sensitive (ts) mutant of vesicular stomatitis virus (VSV) isolated in chick embryo fibroblasts (CEFs), complements the five prototype ts mutants of the virus. The data presented here indicate that the defect in tsO82 is localized in the M gene. The mutation changed a methionine to an arginine at position 51 of the M protein. Only true revertants could be isolated, and their frequency was low, perhaps due to the type of substitution required to return to the wild-type phenotype. TsO82 does not exhibit hypertranscription, in contrast to the data reported for all of the other ts mutants affected in the M protein. Moreover, tsO82 is conditionally ts, since it grows normally in BHK-21 cells at all temperatures. It exhibits no c.p.e. at the non-permissive temperature in CEFs. Our data argue for multiple functions of the M protein of VSV, the domain affected by the tsO82 mutation possibly being implicated both in the shut-off of cellular RNA synthesis, and for the recognition of a cellular factor required for efficient viral RNA synthesis.

Animals↗

PubMed1989

Alternatives for the initiation of translation.

New evidence of exceptions to the scanning mechanism for the initiation of translation has been recently obtained. These data suggest that ribosomes can bind and initiate internally on certain mRNAs without having to scan from the 5' end.

Base Sequence↗

PubMed1989

Synthesis of respiratory syncytial virus RNA in cell-free extracts.

A cell-free system has been used to synthesize respiratory syncytial virus (RSV) RNA in vitro. Polyadenylated species representing all size classes of RSV mRNAs were labelled. Some of the labelled RNA was seen in a CsCl gradient at the density characteristic of negative-strand viral nucleocapsids. Experiments using a thiotriphos-phorylated nucleotide indicate that RNA chains are initiated de novo in the cell-free system.

Cell-Free System↗

PubMed1987

Characterization of the internal initiation of translation on the vesicular stomatitis virus phosphoprotein mRNA.

Internal initiation of translation on the vesicular stomatitis virus (VSV) phosphoprotein (P) mRNA leads to the synthesis of a second protein [Herman, R. C. (1986) J. Virol. 58, 797-804]. Characterization of this phenomenon shows that initiation at the 5'-proximal and internal AUG codons has different optima for mono- and divalent cations in the reticulocyte lysate. Whereas 5' initiation is stimulated by increasing concentration of K+ over the endogenous level, internal initiation is inhibited. Internal initiation is much less sensitive to the effects of the cap analogue 7mGpppG in both the reticulocyte lysate and the wheat-germ extract under conditions that reduce 5'-proximal initiation to only about 4-5% of the control level. These results imply that 5'-proximal and internal initiations are distinct biochemical processes.

Animals↗

PubMed1986

Internal initiation of translation on the vesicular stomatitis virus phosphoprotein mRNA yields a second protein.

In vitro translation of a mixture of the vesicular stomatitis virus (VSV) polyadenylated mRNAs yielded a previously undetected protein with a molecular weight of approximately 7,000 (7K protein). Hybrid-arrested translation demonstrated that both the 7K protein and the VSV phosphoprotein (P protein) were encoded by the P protein message. Immunoprecipitation of the 7K protein with monoclonal antiserum directed against the P protein indicated that the two products were encoded in the same open reading frame. A protein of approximately the same size was immunoprecipitated from cytoplasmic extracts of VSV-infected cells by both the polyclonal and monoclonal antisera, and it is likely that it was a previously unrecognized viral gene product. Translational mapping of the P protein mRNA in vitro indicated that the 7K protein was encoded in the 3' one-third of the sequence. The synthesis of the 7K protein in vitro was unaffected by hybrid arrest conditions which blocked the 5' two-thirds of the mRNA and inhibited synthesis of the P protein. These results imply that the ribosomes bind and initiate translation internally on the P protein mRNA at a site located hundreds of nucleotides downstream from the capped 5' end.

Amino Acid Sequence↗

PubMed1985

Nonpermissive infection of lymphoblastoid cells by vesicular stomatitis virus. II. Effect on viral morphogenesis.

The human B-lymphoblastoid cell line Raji is nonpermissive for infection by vesicular stomatitis virus (VSV). The VSV particles released from Raji cells display a more heterogeneous distribution in equilibrium sucrose density gradients than particles released from BHK cells. The particles released from Raji cells contain approximately one-half to one-third as much viral matrix protein, relative to the nucleocapsid protein, as is normal. They also contain a higher proportion of the unglycosylated form of the G protein. The particles released from Raji cells are unstable and many disintegrate in the growth medium. Most of them deform when subjected to ultracentrifugation prior to fixation. The ratio of plaque-forming units to physical particles is much lower for the virions released from Raji cells.

B-Lymphocytes↗

PubMed1984

Nonpermissive infection of lymphoblastoid cells by vesicular stomatitis virus. I. Synthesis and function of the viral transcripts.

The human B-lymphoblastoid cell line Raji is nonpermissive for infection by vesicular stomatitis virus (VSV) (Nowakowski et al. (1973) J. Virol. 12, 1272-1278). Viral-specific transcription begins immediately after infection, but Raji cells synthesize only about one-twentieth as much viral RNA as is synthesized by a permissive host. The viral primary transcripts appear to be unstable in Raji cells when prevented from engaging in protein synthesis by the addition of cycloheximide. The messages are undermethylated in the 5'-terminal cap structure and have a relatively short 3'-polyadenylate tail. Nevertheless, the subcellular distribution of the messages indicates that many of these RNAs are present in large polyribosomes. Analyses of the effects of a temperature-sensitive mutation in the viral matrix protein indicate that mRNA synthesis in Raji cells is limited only by the amount of available nucleocapsid templates and not by a specific defect in transcription.

B-Lymphocytes↗

PubMed1984

Nucleotide sequence of an aberrant glycoprotein mRNA synthesized by the internal deletion mutant of vesicular stomatitis virus.

The transcriptionally active internal deletion mutant (DI-LT) of vesicular stomatitis virus synthesizes an abnormal mRNA (G*) containing a transcript of the remnant polymerase gene covalently linked to the 3' end of the glycoprotein message (R.C. Herman and R.A. Lazzarini, J. Virol. 40:78-86, 1981). A complementary DNA copy of the 3' end of the G* transcript was molecularly cloned and then chemically sequenced. The results showed that the deletion removed the last 54 nucleotides of the normal glycoprotein gene, the intergenic dinucleotide, and all but the last 258 nucleotides of the polymerase gene. The sequence of DI-LT at the deletion site was compared to that of the transcriptionally inactive DI-LT2 particle.

Animals↗

PubMed1983

Conditional synthesis of an aberrant glycoprotein mRNA by the internal deletion mutant of vesicular stomatitis virus.

The internal deletion mutant (DI-LT) derived from the heat-resistant strain of vesicular stomatitis virus synthesizes an aberrant polyadenylated mRNA (G*) containing a transcript of the partially deleted polymerase gene covalently linked to the 3' end of the glycoprotein message (R. C. Herman and R. A. Lazzarini, J. Virol. 40:78-86, 1981). The heat-resistant polymerase appears to play a role in the synthesis of the abnormal G* RNA. The synthesis of G* correlated directly with the presence of the heat-resistant L protein on the defective interfering particle template. Chimeric defective interfering particles produced by passaging DI-LT with a helper virus that encodes the wild-type vesicular stomatitis virus polymerase did not synthesize G*. The subsequent passage of the chimeric DI-LT with a heat-resistant helper virus restored the ability to synthesize the G* transcript. These results imply that the regulatory signals normally present at the vesicular stomatitis virus G/L intercistronic boundary may be preserved in DI-LT. These sequences are only conditionally functional because they are recognized correctly by the wild-type but not by the heat-resistant polymerase.

Defective Viruses↗