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N W Fraser

Publications and source records attributed to N W Fraser.

At least 91 records · Page 5Linked to original sources
PubMed1990

A herpes simplex virus type 1 latency-associated transcript mutant reactivates with normal kinetics from latent infection.

The herpes simplex virus type 1 (HSV-1) latency-associated transcripts (LATs) accumulate in neuronal nuclei of latently infected ganglia. Explant reactivation kinetics of LAT deletion mutants in the mouse eye model have suggested a role for the LATs in the reactivation process. This report describes the construction and characterization of an HSV-1 strain HFEM mutant, TB1, disrupted within both copies of the LAT gene. TB1 contains a 440-base-pair segment of bacteriophage lambda DNA in place of a 168-base-pair deletion within the transcribed portion of the LAT gene. The 2.0-kilobase LAT was not produced after infection of tissue culture cells with TB1, but a 0.7- to 0.8-kilobase RNA was expressed. TB1 did establish latent infection after corneal inoculation as efficiently as the parental virus, and its reactivation kinetics from explanted ganglia were similar to those of HFEM. During latent infection with TB1, HSV-1 transcripts were not detectable. Rescuant virus (TB1-R) contained intact LAT genes, synthesized full-length LAT transcripts during productive infection in tissue culture, and reactivated from ganglionic explants of latently infected mice with normal kinetics. Thus, any function these transcripts have in the reactivation process appears to include the region between the putative LAT promoter and the disruption in TB1--a region of approximately 1,600 nucleotides, 800 of which encode the LATs.

Animals↗
PubMed1989

Herpes simplex virus type 1 latency-associated transcripts are evidently not essential for latent infection.

The herpes simplex virus type 1 (HSV-1) transcripts that can be detected during latent infection by Northern blot analysis in human and experimental animal sensory ganglia are encoded by diploid genes. To investigate their role in latent infection we studied HSV-1 variant 1704, which has deleted most of the IRL copy of the coding region of these RNAs and has a 1.2-kb deletion that is immediately upstream of the coding region of the TRL copy. During primary infection, 1704 replicated in trigeminal ganglia with kinetics similar to the parent virus (17+) and established latent infection. However, while explant reactivation of latent HSV-1 from trigeminal ganglia was detected in 100% of 17+ infected mice within 7 days, the reactivation of 1704 was significantly delayed, and 31 days elapsed before eight out of nine mice became virus positive. The recognized HSV-1 latency-associated RNAs were not detected during the latent state of 1704 by Northern blot analysis or in situ hybridization, which implies that the 1.2-kb deletion may contain the promoter or other important regulatory elements. The data indicate that detectable levels of these latency-associated transcripts are not required for viral replication, establishment, or maintenance (greater than 6 weeks) of HSV-1 latency in trigeminal ganglia, but suggest a role in reactivation.

Animals↗
PubMed1989

Detection of HSV-1 proteins prior to the appearance of infectious virus in mouse trigeminal ganglia during reactivation of latent infection.

Following corneal inoculation of mice HSV-1 produces an acute infection and establishes a latent infection in trigeminal ganglia. The latent virus can be reactivated in vitro by explantation of ganglionic tissue. Viral protein expression was studied in trigeminal ganglia during acute infection of mice and explant reactivation of latent infection. HSV-1 proteins were detectable by immunoprecipitation and immunostaining, in mouse ganglia only from 3-5 days post infection. Although during explant reactivation it has been demonstrated that at 24 h post-explant the trigeminal ganglia are all infectious virus negative (Spivack, O'Boyle II and Fraser (1987) J. Virol. 61, 3288-3291), we have found that three HSV-1 proteins, of 175 kDa, 110 kDa and 90 kDa, are present in latently infected trigeminal ganglia as early as 6-21 h post explantation. Initially, only neuronal cells were positive by immunostaining with anti HSV-1 polyclonal serum for HSV-1 antigens, but at later times HSV-1 antigens were seen in non neuronal cells as well. These proteins may play a role in the initial stages of the reactivation process.

Animals↗
PubMed1989

The transcription termination region of the adenovirus 2 major late transcript contains multiple functional elements.

In order to understand the process of transcription termination by eukaryotic RNA polymerase II, the transcription termination region of the advenovirus 2 major late transcription unit was analysed in a transient transfection system. Previously, it had been demonstrated that the entire sequence from map units (m.u.) 97.1 to 100 of the adenovirus 2 genome terminates transcription when inserted into the 5' or 3' untranslated sequences of the chloramphenicol acetyltransferase gene. Using subclones and Bal 31 deletion mutants of the termination region, we have shown that the termination region consists of multiple elements each capable of inhibiting gene expression independently. A DNA sequence analysis reveals the presence of a highly repetitive A-rich sequence motif throughout the entire termination region. The data suggest that the A-rich motif may mediate the transcription termination process.

Adenoviruses, Human↗
PubMed1989

During latency, herpes simplex virus type 1 DNA is associated with nucleosomes in a chromatin structure.

Latent herpes simplex virus type 1 DNA has a nucleosomal structure similar to that of cellular chromatin, as determined by micrococcal nuclease digestion. All of the major regions, including the transcriptionally active region of the genome, were found to be associated with nucleosomes. Such a chromatin structure is likely to be an important element in the control of herpes simplex virus type 1 gene expression during latency.

Animals↗
PubMed1988

herpes simplex virus infection accelerates the age-related reactivity of mouse trigeminal ganglia neurons with anti-mouse IgG antibody in immunostaining.

The detection of viral proteins is a major goal of research on herpes simplex virus type 1 (HSV-1) latency. We used immunostaining to detect viral proteins in neuronal cells of trigeminal ganglia of Balb/c mice after corneal inoculation with HSV-1 virus. Viral proteins were detected in the neurons during the acute stage of infection, i.e. within one week after inoculation. However, the detection of viral antigens at the latent stage of HSV-1 infection has proven difficult. We have detected age-dependent non-specific reactivity with anti-mouse IgG antibody in the neurons of 10-week-old or older uninfected mice. This reactivity is accelerated in HSV-1 infected mice, being seen at 6 weeks of age (2 weeks post infection). The accelerated reaction and impact of this effect is discussed in relation to detection of viral proteins during latency.

Aging↗
PubMed1988

Latent herpes simplex virus type 1 transcripts in peripheral and central nervous system tissues of mice map to similar regions of the viral genome.

Herpes simplex virus type 1 (HSV-1) DNA and RNA have been detected in peripheral nervous system (PNS) and central nervous system (CNS) tissues of latently infected mice. However, explant methods are successful in reactivating HSV-1 only from latently infected PNS tissues. In this report, latent herpesvirus infections in mouse PNS and CNS tissues were compared by in situ hybridization to determine whether the difference in reactivation was at the level of the virus or the host tissue. It was demonstrated that the HSV-1 transcripts present during latency in the mouse PNS and CNS originated from the same region of the genome, the repeats which bracket the long unique sequence. Therefore, the difference in reactivation with PNS and CNS tissues cannot be accounted for by differences in the extent of the HSV-1 genome transcribed during herpesvirus latency. Latent HSV-1 RNA was detected in the trigeminal ganglia (PNS) and the trigeminal system in the CNS from the mesencephalon to the spinal cord as well as other regions of the CNS not noted previously. Latent HSV-1 RNA was found predominantly in neurons but also in a small number of cells which could not be identified as neuronal cells. It is suggested that host differences in CNS and PNS tissues, rather than differences in latent virus transcription, may be important determinants in the HSV-1 reactivation process in explanted tissues.

Animals↗
PubMed1988

Expression of herpes simplex virus type 1 latency-associated transcripts in the trigeminal ganglia of mice during acute infection and reactivation of latent infection.

Herpes simplex virus type 1 (HSV-1) establishes a latent infection in the trigeminal ganglia of mice infected via the eye. In these ganglia three viral transcripts, of 2.0, 1.5, and 1.45 kilobases (kb), which are at least partially colinear, have been identified by Northern (RNA) blot analysis. These RNAs partially overlap ICPO, but are transcribed in the opposite direction (J. G. Spivack and N. W. Fraser, J. Virol. 61:3841-3847, 1987). The accumulation of these latency-associated transcripts, as well as other viral RNAs, was studied during an acute infection and the reactivation of a latent HSV-1 infection in mice. The 2.0-kb latency-associated transcript was detected in trigeminal ganglia of mice as early as 4 days postinfection, and the 1.45- and 1.5-kb RNA doublet was detected at 14 days postinfection. The levels of these latency-associated transcripts increased steadily over a 60-day period. In contrast, other HSV-1 transcripts were detected at 2 to 3 days postinfection, reached a peak on day 4, and rapidly declined below detectable levels by day 7. The data indicate that the temporal expression of the latency-associated genes during acute infection in the trigeminal ganglia of mice is different from the temporal expression of genes involved in HSV-1 replication. During the reactivation of latent HSV-1 from explanted trigeminal ganglia, the latency-associated RNAs decreased about twofold, but were present at significant levels even after HSV-1 DNA increased and infectious virus was recovered. The decrease of the latency-associated transcripts occurred when reactivation was blocked by phosphonoacetic acid or novobiocin, which suggests that this decrease may be an early event in the entry of latent HSV-1 into the viral replication cycle.

Animals↗
PubMed1988

Expression of herpes simplex virus type 1 (HSV-1) latency-associated transcripts and transcripts affected by the deletion in avirulent mutant HFEM: evidence for a new class of HSV-1 genes.

During latent herpes simplex virus type 1 (HSV-1) infection in the trigeminal ganglia of mice, three virus-specific transcripts, 2.0, 1.5, and 1.45 kilobases (kb), are detectable by Northern (RNA) blot analysis, but only the 2.0-kb transcript can be detected in HSV-1-infected tissue culture cells (J.G. Spivack and N. W. Fraser, J. Virol. 61:3842-3847, 1987). Since these latency-associated genes map to a diploid region of the genome, transcription from the deletion mutant HFEM, which contains only one complete copy of these genes, was investigated to determine the effect of gene dosage. The 4.1-kb HFEM deletion is located between the alpha genes ICP0 and ICP27. ICP0 mRNA and the 2.0-kb latency-associated transcript were present at normal levels during HFEM infection, but ICP27 mRNA and 0.9- and 1.1-kb transcripts that map near the deletion were not readily detectable. The levels of expression of one or more of these genes might be an important determinant of HSV-1 virulence in animal hosts. ICP27 mRNA accumulated when protein synthesis was inhibited before HFEM infection, implying that the deletion may affect ICP27 regulatory rather than coding elements. Expression of the 2.0-kb latency-associated transcript was characterized in infected CV-1 cells with metabolic inhibitors and strand-specific probes. On the basis of metabolic inhibitor studies, the gene encoding the 2.0-kb latency-associated transcript is not an alpha gene. During HSV-1 replication in infected tissue culture cells, the beta and gamma genes require the prior expression of alpha gene products. However, the latency-associated RNAs are expressed in the absence of detectable levels of alpha transcripts in latently infected mice. Thus, this latency-associated gene family appear to be regulated quite differently than alpha, beta, or gamma genes. For these reasons, and because the latency-associated genes may perform latent rather than replicative functions, we propose that they should be considered members of a new HSV-1 gene class, the lambda genes.

Cell Line↗
PubMed1988

Latent herpes simplex virus type 1 transcription in human trigeminal ganglia.

We studied latent herpes simplex virus type 1 gene expression in human trigeminal ganglia. Two transcripts were mapped to a 3.0-kilobase region within the long repeat region and appeared to be located in neuronal nuclei. These viral RNAs were not abundant during lytic replication and may represent an alternative pattern of herpes simplex virus type 1 gene expression involved in the pathogenesis of latent infection.

Adult↗
PubMed1987

Physical state of the latent herpes simplex virus genome in a mouse model system: evidence suggesting an episomal state.

Herpes simplex virus (HSV) can establish latent infections in tissues of the nervous system. We have examined the HSV-1 genome in both acutely and latently infected mice by CsCl buoyant density gradient centrifugation. Viral sequences, in gradient fractions, were detected by a spot blot technique using nick-translated HSV-1 cloned DNA as probe, and mouse chromosomal DNA was located by measuring optical density at 260 nm. Most HSV-1-specific DNA from both acutely and latently infected mouse brains was found to band at the buoyant density of virion DNA. However, some HSV-1-specific hybridization banded at the density of the mouse chromosomal DNA. Further rounds of CsCl density gradient centrifugation of this chromosomal DNA from acutely infected brains released most of the HSV-1-specific hybridizing material, suggesting that the association was due to trapping. In the case of the recycled chromosomal DNA from a latent infection, all the HSV-1-specific hybridization remained chromosomal DNA associated. However, the amount of hybridization was not significantly greater in quantity than the cross-hybridization between HSV-1 and chromosomal DNA from uninfected mice. AW-Ramos, an EBV-transformed cell line containing one integrated copy of the viral genome, was centrifuged under similar conditions and showed little if any shearing; the EBV DNA banded at the density of the host cell chromosomal DNA. We conclude that the majority of the latent HSV-1 DNA exists in an extrachromosomal state in the mouse model.

Animals↗
PubMed1987

RNA from an immediate early region of the type 1 herpes simplex virus genome is present in the trigeminal ganglia of latently infected mice.

Transcription of the type 1 herpes simplex virus (HSV-1) genome in trigeminal ganglia of latently infected mice was studied using in situ hybridization. Probes representative of each temporal gene class were used to determine the regions of the genome that encode the transcripts present in latently infected cells. Probes encoding HSV-1 sequences of the five immediate early genes and representative early (thymidine kinase), early-late (major capsid protein), and late (glycoprotein C) genes were used in these experiments. Of the probes tested, only those encoding the immediate early gene product infected-cell polypeptide (ICP) 0 hybridized to RNA in latently infected tissues. Probes containing the other immediate early genes (ICP4, ICP22, ICP27, and ICP47) and the representative early, early-late, and late genes did not hybridize. Two probes covering approximately equal to 30% of the HSV-1 genome and encoding over 20 early and late transcripts also did not hybridize to RNA in latently infected tissues. These results, with probes spanning greater than 60% of the HSV-1 genome, suggest that transcription of the HSV-1 genome is restricted to one region in latently infected mouse trigeminal ganglia.

Animals↗
PubMed1987

Latent herpes simplex virus type 1 DNA is not extensively methylated in vivo.

The methylation pattern of herpes simplex virus type 1 (HSV-1) DNA, present in the central nervous system of latently infected mice, was examined by digestion of the DNA with methylation-sensitive restriction endonucleases and Southern blot hybridization. Using the enzymes SmaI, XmaI, SalI and SacII, the data indicate no extensive methylation of latent HSV-1 DNA in vivo. Thus, extensive methylation of the viral genome is not a necessary condition for, or a consequence of maintaining, the latent state in vivo.

Animals↗
PubMed1987

Novobiocin and coumermycin A1 inhibit viral replication and the reactivation of herpes simplex virus type 1 from the trigeminal ganglia of latently infected mice.

Herpes simplex virus type 1 was reactivated from the trigeminal ganglia of latently infected mice in a quantitative and time-dependent manner. Novobiocin and coumermycin A1 reversibly inhibited the reactivation of herpes simplex virus type 1. They did not inhibit viral replication in permissive cells (CV-1) but did inhibit replication in cells of neuronal origin (C1300) and acutely infected trigeminal ganglia.

Aminocoumarins↗
PubMed1987

Detection of herpes simplex virus type 1 transcripts during latent infection in mice.

A latent infection can be established in the trigeminal ganglia of mice after corneal inoculation of herpes simplex virus type 1 (HSV-1). With a virion DNA probe, three transcripts (2.0, 1.5, and 1.45 kilobases [kb]) were detected by Northern blot (RNA blot) analysis of RNAs isolated from the ganglia of latently infected mice. All three transcripts hybridized to a nick-translated HSV-1 DNA probe from BamHI restriction fragment B (strain F). These RNAs were mapped with subfragments of BamHI-B and with strand-specific probes. They are at least partially colinear with each other, map to a 3.0-kb PstI-MluI subfragment of BamHI-B, and are transcribed from left to right. The latent HSV-1 RNAs partially overlap the 3' end of ICP0 mRNA but are transcribed in the opposite direction. The latent RNAs were not as extensively poly(A)+ as actin mRNA. The HSV-1 transcripts detected in latently infected trigeminal ganglia did not correspond with any that have been previously identified in permissively infected cells in tissue culture. However, the 2.0-kb HSV-1 RNA present during latency was detectable at reduced levels in the trigeminal ganglia of acutely infected mice and in infected tissue culture cells. The data indicate that the pattern of viral gene expression during HSV-1 latency in the trigeminal ganglia of mice does not represent restriction of the genes actively transcribed during the lytic replication cycle in tissue culture.

Animals↗
PubMed1987

DNA sequences downstream of the adenovirus type 2 fiber polyadenylation site contain transcription termination signals.

The major late transcription unit of adenovirus type 2 (Ad2) terminates in a region near the end of the linear DNA genome at map units 98.2 to 100. Specific 3' ends mapping in the transcription termination region were detected in nuclear but not cytoplasmic RNA isolated at 16 to 18 h postinfection. Using S1 nuclease protection analysis, a major nuclear RNA species with a 3' terminus at map unit 98.9 was detected. With the use of recombinant expression vectors and run-on transcription in isolated nuclei, we demonstrated that the Ad2 sequences from map units 97.1 to 100, inserted into either the 5' or 3' untranslated sequences of the chloramphenicol acetyltransferase gene (cat), terminated transcription in transfected cells. Termination occurred only when the 97.1- to 100-map-unit sequence was in the same direction of transcription as the major late transcript and was not observed with Ad2 sequences upstream of map unit 97.1.

Acetyltransferases↗
PubMed1986

Chromosomal organization of the herpes simplex virus genome during acute infection of the mouse central nervous system.

After corneal inoculation, herpes simplex virus type 1 replicates in the mouse eye, trigeminal ganglia, and brainstem, producing first an acute and then a latent infection. Previous work from this laboratory focused on the structure of the viral DNA in this system. We have now examined the structure of the viral genome at the chromosome level by using micrococcal nuclease digestion. Studies with disaggregated cell preparations made from the brainstems of acutely infected mice show that the majority of the viral DNA is in a nonnucleosomal form; however, a nucleosomelike fraction was also consistently detected. A similar result was obtained for viral DNA in herpes simplex virus type 1-infected C1300 (clone NA) neuroblastoma cells (a neuronal cell line).

Acute Disease↗