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J G Spivack

Publications and source records attributed to J G Spivack.

At least 19 recordsLinked to original sources
PubMed1995

Replication, establishment of latent infection, expression of the latency-associated transcripts and explant reactivation of herpes simplex virus type 1 gamma 34.5 mutants in a mouse eye model.

The herpes simplex virus type 1 (HSV-1) gamma 34.5 gene is located within a region that is transcriptionally active during latent HSV-1 infection. To determine whether the gamma 34.5 gene deletion affects latency-associated transcript (LAT) gene expression or latent HSV-1 infection, a gamma 34.5 gene deletion mutant, 1716, and a stop codon insertion mutant, 1771, were studied in the mouse eye model. Although the gamma 34.5 gene is not essential, 1716 and 1771 replicated poorly in mouse eyes and trigeminal ganglia (TG). When mice were inoculated with 1716, infectious virus was detected in eyes only on the first day post-infection (p.i.), and was not detected at any time point in TG. Following inoculation with 1771, a small amount of virus was detected in the eyes on days 2 and 4 p.i., and in the TG of one animal on day 2 p.i. Reactivation of virus from mice latently infected with 1716 (0/30 TG) and 1771 (1/20 TG) was extremely low compared with the parental strain, 17+, and appropriate rescuants (80 to 100% reactivation), even though latent 1716 DNA was detected by PCR in 50% of TG. These results differ from those obtained following footpad inoculation; in the footpad there was limited 1716 replication and reactivatable latent infection was established in some dorsal root ganglia. The data support the hypothesis that the role of gamma 34.5 may be tissue and/or cell type specific. The synthesis, processing, and stability of the 2.0 kb LAT during 1716 and 1771 replication was not affected by these mutations in the gamma 34.5 gene. However, during latent infection of 1716 in mice the LATs were not detectable in TG by Northern blot, and were present in reduced amounts (approximately 10-fold less) during 1771 latency. The LATs from 1716 were barely detectable in a few neurons by in situ hybridization. Therefore, the gamma 34.5 gene might (i) affect replication in the eye, and reduce the amount of virus available to establish latent infection, be directly involved in (ii) establishment of latency, and/or (iii) the reactivation process.

Animals↗
PubMed1994

The UL55 and UL56 genes of herpes simplex virus type 1 are not required for viral replication, intraperitoneal virulence, or establishment of latency in mice.

A critical determinant for intraperitoneal virulence of Herpes Simplex Virus Type 1 (HSV-1) strain F has been ascribed to the BamHI-B fragment of the genome. This region contains two nonessential genes, UL55 and UL56, of unknown function. To investigate the contribution of these genes to viral pathogenesis, we have generated two recombinant HSV-1 viruses, inUL55 and inUL56, in which the corresponding gene was inactivated in strain F by insertion of an ICP6::lacZ cassette. Strains inUL55 and inUL56, which did not express the UL55 and UL56 RNA, respectively, were virulent in mice inoculated intraperitoneally. Strains inUL55 and inUL56 also established latent infections in the trigeminal ganglia of mice inoculated via the cornea. These results demonstrate that the HSV-1 UL55 and UL56 genes are not critical for intraperitoneal virulence or establishment of latent infection.

Animals↗
PubMed1994

The herpes simplex virus type 1 strain 17+ gamma 34.5 deletion mutant 1716 is avirulent in SCID mice.

Laboratory animal models are important tools for the identification of avirulent herpes simplex virus type 1 (HSV-1) strains which have potential for use in humans as vaccine strains or gene therapy vectors. We have studied an HSV-1 17+ variant, 1716, that has a deletion in the gamma 34.5 gene and which replicates poorly in the footpads of mice and is unable to grow in the mouse central nervous system or dorsal root ganglia (DRG) of the peripheral nervous system following peripheral inoculation. However, 1716 is known to be capable of establishing latent infections in the DRG of mice. Here we show that 1716 is avirulent after ocular infection and has low virulence after intracranial inoculation in SCID mice. Since SCID mice are much more sensitive to HSV-1 infection than immunocompetent mice, our results clearly demonstrate the drastically reduced virulence of the variant 1716 and provide additional support for the hypothesis that this variant would be avirulent in humans.

Animals↗
PubMed1994

Herpes simplex virus type 1 gene expression and reactivation of latent infection in the central nervous system.

Restricted gene expression takes place during latent infection of herpes simplex virus type 1 (HSV-1) in the human peripheral nervous system and has been linked with viral reactivation. The state of HSV-1 gene expression in the central nervous system (CNS) during latency is unclear and we, therefore, examined gene expression in the brainstem of experimental mice and normal humans. Only part of the transcription pattern present during latent infection in peripheral sensory ganglia (PSG) was identified in the human brainstem by in situ hybridization and Northern blot analysis for HSV-1-specific transcripts. Instead of three HSV-1 latency-associated transcripts (LATs) present in PSG and demonstrated by Northern blot analysis, only one was identified in mouse brainstem and none was detected in human brainstem. These findings might be attributed to the relatively low amounts of HSV-1-specific latency-associated RNAs in brainstem tissue. Combined with our inability to reactivate HSV-1 from explanted mouse brainstem, these findings suggest that tissue levels of latency-associated gene expression play a role in HSV-1 reactivation and have relevance to the very low incidence of HSV-1-induced CNS disease compared with peripheral mucocutaneous disease.

Animals↗
PubMed1994

Two open reading frames (ORF1 and ORF2) within the 2.0-kilobase latency-associated transcript of herpes simplex virus type 1 are not essential for reactivation from latency.

The herpes simplex virus type 1 (HSV-1) latency-associated transcripts (LATs) are dispensable for establishment and maintenance of latent infection. However, the LATs have been implicated in reactivation of the virus from its latent state. Since the reported LAT deletion and/or insertion variants that are reactivation impaired contain deletions in the putative LAT promoter, it is not known which LAT sequences are involved in reactivation. To examine the role of the 2.0-kb LAT in the process of reactivation and the functional importance of the putative open reading frames (ORF1 and ORF2) contained within the 2.0-kb LAT, we have constructed an HSV-1 variant that contains a precise deletion and insertion within the LAT-specific DNA sequences using site-directed mutagenesis. The HSV-1 variant FS1001K contains an 1,186-bp deletion starting precisely from the 5' end of the 2.0-kb LAT and, for identification, a XbaI restriction endonuclease site insertion. The FS1001K genome contains no other deletions and/or insertions as analyzed by a variety of restriction endonucleases. The deletion in FS1001K removes the entire 556-bp intron within the 2.0-kb LAT, the first 229 nucleotides of ORF1, and the first 159 nucleotides of ORF2 without having an affect on the RL2 (ICP0) gene. Explant cocultivation reactivation assays indicated that this deletion had a minimal effect on reactivation of the variant FS1001K compared with the parental wild-type virus using a mouse eye model. As expected, Northern (RNA) blot analyses have shown that the variant virus (FS1001K) does not produce the 2.0-kb LAT or the 1.45- to 1.5-kb LAT either in vitro or in vivo; however, FS1001K produces an intact RL2 transcript in tissue culture. These data suggest that the 2.0-kb LAT putative ORF1 and ORF2 (or the first 1,186 bp of the 2.0-kb LAT) are dispensable for explant reactivation of latent HSV-1.

Animals↗
PubMed1993

Modification of discrete nuclear domains induced by herpes simplex virus type 1 immediate early gene 1 product (ICP0).

The outcome of herpes simplex virus type 1 (HSV-1) infection depends upon the interplay of both host and viral factors. During lytic infection, HSV-1 causes a loss of immunofluorescent staining of discrete nuclear domains (ND10). This elimination of the host's ND10 staining occurs under conditions that allow only HSV-1 immediate early viral gene expression. Western blot analysis indicates that the loss of ND10 staining is due to ND10 redistribution, rather than protein degradation or turnover. When deletion mutants of all of the HSV-1 immediate early genes were tested, only infection with an immediate early gene 1 product (ICP0) deletion mutant, d11403, was unable to eliminate ND10 antigen staining. Also, ICP0 transiently colocalized with ND10 antigens, after which ND10 antigens became undetectable. At late times during infection with d11403, the host ND10 antigens were retained in virus-induced structures which were never observed during wild-type HSV-1 infection. These results suggested that ICP0 may be directly involved in the modification of the host nuclear domain. Infection with an adenovirus recombinant that expressed ICP0 demonstrated that in the absence of other HSV-1 proteins ICP0 was sufficient for the change in nuclear distribution of host antigens located at ND10. We postulate that the trans-activation function of ICP0 during viral replication may be mediated by replacing, modifying or reorganizing nuclear host factors.

Animals↗
PubMed1991

The HSV-1 latency associated transcript (LAT) variants 1704 and 1705 are glycoprotein C negative.

The latency associated transcripts (LAT) of herpes simplex virus type 1 (HSV-1) are encoded by diploid genes that are expressed during latent infections. Two LAT variant viruses, 1704 and 1705, were compared with the parental strain 17+. Variant 1705 has a deletion affecting one copy of the LAT genes, expresses LATs during latent infection and reactivates normally. Variant 1704 has deletions affecting both LAT gene copies, does not express LATs during latent infection, and its reactivation is impaired (Steiner et al., 1989). Comparison of infected cell proteins by immunoprecipitation and Western blot analysis revealed one significant difference between HSV-1 strain 17+, 1704 and 1705; glycoprotein C (gC) was not synthesized by 1704 or 1705. Since both 1704 and 1705 are gC minus, the reactivation defect of 1704 is most likely related to the absence of the LATs, and not to the absence of gC.

Animals↗
PubMed1991

Investigation of herpes simplex virus type 1 (HSV-1) gene expression and DNA synthesis during the establishment of latent infection by an HSV-1 mutant, in1814, that does not replicate in mouse trigeminal ganglia.

In previous studies, the herpes simplex virus type 1 (HSV-1) mutant, in1814, which lacks the trans-inducing function of Vmw65, did not replicate in the trigeminal ganglia of mice following corneal inoculation but did establish a reactivatable latent infection in the ganglia 12 to 24 h after ocular infection. Since in1814 did not replicate in vivo, the molecular events during the establishment phase of latent HSV-1 infection could be characterized without the complications of concurrent productive viral infection. In comparison to parental HSV-1 strain 17+, the expression of viral immediate early (IE), early and late genes and the levels of viral DNA in the trigeminal ganglia of mice following in1814 infection were greatly reduced. However, accumulation of latency-associated transcripts, a prominent feature of latent HSV-1 infection, occurred in a wild-type fashion. Furthermore, low levels of viral gene expression and an increase in the level of viral DNA in the in1814-infected ganglia were not detected until 1 to 2 days after the establishment of HSV-1 latency. Thus, IE gene expression and replication of viral DNA in the trigeminal ganglia are not prerequisites for the establishment of HSV-1 latency. These results suggest that the pathways leading to productive and latent infections in neurons may diverge at an early stage of the host-HSV-1 interaction and that the level of viral IE gene expression has a key role in determining the outcome of infection.

Animals↗
PubMed1991

Identification of a novel latency-specific splice donor signal within the herpes simplex virus type 1 2.0-kilobase latency-associated transcript (LAT): translation inhibition of LAT open reading frames by the intron within the 2.0-kilobase LAT.

Herpes simplex virus type 1 establishes latent infection in trigeminal ganglia of mice infected via the eye. A family of three colinear viral transcripts (LATs), 2.0, 1.5, and 1.45 kb, is present in latently infected ganglia. To characterize these LATs, lambda gt10 cDNA libraries were constructed with RNAs isolated from the trigeminal ganglia of latently infected mice. A series of recombinant bacteriophage were isolated containing cDNA inserts covering 1.7 kb of the 2.0-kb LAT. Splice junctions of the smaller LATs and the 3' end of the 2.0-kb LAT were identified by sequence analysis of RNA polymerase chain reaction products. No splice acceptor site, which does not support the hypotheses that the 2.0-kb LAT is an intron. However, the data are consistent with the possibility of a short leader sequence or multiple LAT transcription start sites. To generate the smaller 1.5- and 1.45-kb LATs, there is a 559-nucleotide intron spliced from the 2.0-kb LAT in strain F and a 556-nucleotide intron in strain 17+. The nucleotide sequences at the 5' and 3' ends of these introns are characteristic of spliced transcripts from eukaryotic protein-encoding genes, with one significant difference; i.e., the 5' end of the LAT intron is GC instead of the consensus sequence GT. This splice donor sequence is conserved in herpes simplex virus type 1 strains F, 17+, and KOS. Processing of the 2.0-kb LAT to form the spliced LATs preserves two open reading frames (ORFs) at the 3' end of the LATs; no new ORFs are created. Splicing of the LATs positions a 276-nucleotide leader sequence close to these ORFs and removes an intron that inhibits their translation in vitro. The novel 5' structure of the intron within the 2.0-kb LAT may be part of a control mechanism for transcription processing that results in splicing of the LATs only in sensory neurons during latent infection and reactivation but not during the viral replication cycle.

Animals↗
PubMed1991

In vivo and in vitro reactivation impairment of a herpes simplex virus type 1 latency-associated transcript variant in a rabbit eye model.

Many recent studies of latent herpes simplex virus type 1 (HSV-1) infections within the nervous system have focused on the diploid genes encoding the latency-associated transcripts (LATs). The impaired explant reactivation of LAT variants from mouse trigeminal ganglia has implicated the LATs in the efficiency or speed of the reactivation process (D. A. Leib, C. L. Bogard, M. Kosz-Vnenchak, K. A. Hicks, D. M. Coen, D. M. Knipe, and P. A. Schaffer, J. Virol. 63:2893-2900, 1989; I. Steiner, J. G. Spivack, R. P. Lirette, S. M. Brown, A. R. MacLean, J. H. Subak-Sharpe, and N. W. Fraser, EMBO J. 8:505-511, 1989). However, it is not known how closely explant reactivation mimics the reactivation process in vivo. In the current study, a LAT variant (1704), parental strain (17+), and rescuant (1704R) were compared in vivo for reactivation of latent infection by iontophoresis in the rabbit eye model and in vitro by explant cocultivation of trigeminal ganglia from rabbits. Following iontophoresis, 17+ and 1704R reactivated in vivo from 76 and 64% of rabbits, respectively, while 1704 reactivated only from 4% (1 of 25) of the animals. In explant reactivation experiments, 17+ and 1704R reactivated from 98 and 67% of rabbit trigeminal ganglia, while 1704 reactivated from only 28% of trigeminal ganglia. The mean time required for the appearance of reactivated 1704 in explant culture, 17 days, was significantly longer than for 17+ and 1704R, 8 to 9 days. Thus, the explant reactivation kinetics in rabbit trigeminal ganglia reflect the behavior of LAT variant 1704 in vivo in the rabbit eye model. These data support the role of the LATs in the reactivation process and support the hypothesis that explant reactivation is a suitable system for analyzing the biological behavior of HSV-1 variants with defined genetic alterations in the LAT gene.

Animals↗
PubMed1991

A review of the molecular mechanism of HSV-1 latency.

The neurotropic herpes viruses, as typified by herpes simplex virus type 1, are noted for their ability to form latent infections. The latent infection differs from the acute infection both in gene expression and the physical state of the viral genome. Latency can be divided into several stages--establishment, maintenance of reactivation--each of which are active areas of research. This review describes the molecular biology of HSV-1 latency and presents the current level of understanding of the molecular mechanism of HSV-1 latency.

Animals↗
PubMed1990

A herpes simplex virus type 1 mutant containing a nontransinducing Vmw65 protein establishes latent infection in vivo in the absence of viral replication and reactivates efficiently from explanted trigeminal ganglia.

Vmw65, a herpes simplex virus type 1 (HSV-1) tegument protein, in association with cellular proteins, transactivates viral immediate early genes. In order to examine the role of Vmw65 during acute and latent infection in vivo, a mutant virus (in1814), containing a 12-base-pair insertion in the Vmw65 gene, which lacks the transactivating function of Vmw65 (C. I. Ace, T. A. McKee, J. M. Ryan, J. M. Cameron, and C. M. Preston, J. Virol. 63:2260-2269, 1989) was examined in mice. Following corneal inoculation, the parental virus (17+) and the revertant (1814R) replicated effectively in eyes and trigeminal ganglia with 30 to 60% mortality. At either equal PFU or equal particle numbers, in1814 did not replicate in trigeminal ganglia and none of the infected mice died. Although in1814 did not replicate following corneal inoculation, it established latent infection in trigeminal ganglia. HSV-1 in1814 reactivated at explant as efficiently and rapidly as did 17+ and 1814R. Even low amounts of inoculated in1814 (10(2) PFU) were sufficient to establish latent infection in some animals. Since infectious in1814 was not detected at any time in mouse trigeminal ganglia, in1814 provided a unique opportunity to determine how soon after primary infection latency begins. Latent in1814 infection was detected shortly after virus reached the sensory ganglia, between 24 to 48 h postinfection. Thus, though Vmw65 may be required for lytic infection in vivo, it is dispensable for the establishment of and reactivation from latent infection. These data support the hypotheses that the latent and lytic pathways of HSV-1 are distinct and that latency is established soon after infection without a requirement for viral replication. However, the levels of Vmw65 reaching neuronal nuclei may be a critical determinant of whether HSV-1 forms a lytic or latent infection.

Animals↗
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↗
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↗