Biomedical subjects

Nigel W Fraser

Publications and source records attributed to Nigel W Fraser.

8 recordsLinked to original sources

PubMed2003

Establishment and maintenance of HSV latent infection is mediated through correct splicing of the LAT primary transcript.

To study the effect of the splicing of HSV-1 latency-associated transcript (LAT) on viral latency, we constructed two mutant viruses (FHlambda+ and FHlambda-) in which the 168-bp HpaI-HpaI fragment within the 2-kb LAT intron was replaced by a 447-bp bacteriophage lambda sequence. The lambda DNA was inserted in opposite orientations in FHlambda+ and FHlambda-. The mutation in FHlambda+ disrupted the splicing of LAT primary transcript and altered both LAT exon and intron, whereas the mutation in FHlambda- virus preserved the wild-type splice sites and the wild-type exon. Quantitative PCR analysis revealed that during latency there was a reduction in the number of viral genomes in mouse trigeminal ganglia infected with FHlambda+ but not in those infected with FHlambda-. The decrease in the latent genome numbers was not due to a defect in viral replication during the acute stage of infection. Furthermore, trigeminal ganglia from mice latently infected with FHlambda+ displayed a slower reactivation kinetics compared to those infected with the parental strain. To elucidate the mechanism, we examined the antiapoptotic properties of these LAT constructs. A plasmid containing the pHlambda+ construct was found to be less protective for cells against apoptosis than plasmid containing the wild-type or pHlambda- constuct. These results suggest that the splicing of LAT primary transcript, and thus the correctly spliced exon product, play an important role in promoting the establishment and/or maintenance of viral latency.

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PubMed2003

Requirement of an integrated immune response for successful neuroattenuated HSV-1 therapy in an intracranial metastatic melanoma model.

Neuroattenuated herpes simplex virus ICP34.5 mutants slow progression of preformed tumors and lead to complete regression of some tumors. Although this was previously thought to be due to viral lysis of infected tumor cells, it is now understood that there is an immune component to tumor destruction. We have previously shown that no difference in survival is seen in lymphocyte-depleted mice after viral or mock therapy of syngeneic intracranial melanomas. We have also demonstrated the presence of a wide spectrum of immune cells following viral therapy, including larger percentages of CD4+ T cells and macrophages. In this paper, the contribution of the immune system to tumor destruction has been further delineated. Viral therapy of intracranial melanoma induces a tumor-specific cytotoxic and proliferative T cell response. However, there is no increase following viral therapy in either serum tumor antibody levels or viral-neutralizing antibodies. Thus specific T cell responses appear to mediate viral-elicited prolongation in survival. These data suggest that designing new viruses capable of augmenting T cell responses may induce stronger tumor destruction upon viral therapy.

Animals↗

PubMed2003

HSV-1 therapy of primary tumors reduces the number of metastases in an immune-competent model of metastatic breast cancer.

The HSV-1 1716 mutant virus and similar oncolytic herpesviruses deficient in the gamma 34.5 neurovirulence gene are able to reduce the growth of tumors in mice. Here we demonstrate that HSV-1 1716 therapy moderately reduced the growth of tumors of the highly malignant, spontaneously metastasizing 4T1 mouse mammary carcinoma model. This moderate effect on 4T1 tumor growth was likely due to poor replication kinetics of HSV-1 1716 in 4T1 cells. Interestingly, HSV-1 therapy of the primary tumor increased the survival time of mice. Coincident with this increase was a reduction in metastases as determined by quantification of the number of metastatic cells in the lungs. HSV-1 therapy of the primary tumor was also able to reduce the establishment of a second challenge of 4T1 tumors. Moreover, infiltrates of both CD4(+) and CD8(+) T cells were detected in HSV-1 1716-treated tumors. An important role for the T cell infiltrates was confirmed when HSV-1 therapy did not reduce the growth of 4T1 tumors in SCID mice. Collectively, these results demonstrate that an HSV-dependent anti-tumor immune response is required for the reduction in primary 4T1 tumor growth and for the reduction in the establishment of metastases in this tumor model.

Animals↗

PubMed2003

Herpes simplex virus latency-associated transcript gene function.

A major area of interest in the study of herpes simplex virus type 1 (HSV-1) involves the persistence of the virus within a latent state in neuronal cells of infected humans. The latency-associated transcripts (LATs) are believed to play a key role during HSV-1 latency. This review will discuss the most recent findings on the involvement of the LAT region with apoptotic pathways and how this relates to other potential functions of the LATs.

Animals↗

PubMed2002

Stability and circularization of herpes simplex virus type 1 genomes in quiescently infected PC12 cultures.

Herpes simplex virus type 1 (HSV-1) DNA has been shown to exist as a linear, double-stranded molecule in the virion and as a non-linear (endless), episomal, nucleosomal form in latently infected trigeminal ganglia. The kinetics of the formation and appearance of endless viral genomes and the stability of linear genomes in neuronal cells are not well understood. Nerve growth factor (NGF)-differentiated PC12 cells can sustain long-term, quiescent infections with HSV-1. In this report, the structure and stability of HSV-1 viral DNA in NGF-differentiated PC12 cells was studied as a function of time following infection using both wild-type and replication-defective virus. Unexpectedly, unencapsidated linear genomes were stable in the nucleus of NGF-differentiated PC12 cells for up to 2-3 weeks following infection, beyond the period at which there is no detectable viral gene expression. However, following infection with wild-type HSV, the majority of quiescent viral genomes were in an endless form after 3-4 weeks. These data suggest that the stability and fate of HSV-1 DNA in non-mitotic neuronal-like cells is different from that in productively infected cells and thus there is a significant cellular role in this process. The relevance to the virus life-cycle in neurones in vivo is discussed.

Animals↗

PubMed2002

Herpes simplex virus type 1 infection prevents detachment of nerve growth factor-differentiated PC12 cells in culture.

In tissue culture, rat pheochromocytoma (PC12) cells differentiated with nerve growth factor (NGF) cease division, extend neuritic processes and acquire many properties characteristic of neuronal cells. In previous work, we have shown that NGF-differentiated PC12 cells can survive infection with herpes simplex virus type 1 (HSV-1) and maintain the viral genome in a quiescent but reactivatable state. In this study, we report that uninfected NGF-differentiated PC12 cells uniformly and predictably detach from the culture flask substratum after approximately 7 weeks. Although uninfected cells were uniformly lost from the culture by 7 weeks, surprisingly HSV-1-infected cells survived beyond 10 weeks, the time limit of the study. The detachment of uninfected cells was not the result of cell death or apoptosis, as determined by viability assays performed on cells after detachment. Expression of the HSV-1 latency associated transcript (LAT) gene and virus replication was not necessary for the virus to suppress the 'detachment' phenomenon, since NGF-differentiated PC12 cells infected with either wild-type, DNA polymerase mutant or LAT null mutant virus survived in culture for similar lengths of time. Viral gene expression does appear to be necessary for the suppression, however, since cells infected with UV-inactivated virus were lost from culture with kinetics similar to those of uninfected cells. These findings indicate that de novo viral gene synthesis mediates changes to the host NGF-differentiated PC12 cells, which results in prevention of detachment.

Animals↗

PubMed2002

The 2-kilobase intron of the herpes simplex virus type 1 latency-associated transcript has a half-life of approximately 24 hours in SY5Y and COS-1 cells.

The herpes simplex virus type 1 (HSV-1) 2-kb latency-associated transcript (LAT) is a stable intron, which accumulates in cells both lytically and latently infected with HSV-1. We have used a tetracycline-repressible expression system to determine the half-life of the 2-kb LAT RNA intron in the human neuroblastoma cell line SY5Y. Using Northern hybridization analyses of RNA isolated from transiently transfected SY5Y cells over time after repression of LAT expression, we measured the half-life of the 2-kb LAT to be approximately 24 h. Thus, unlike typical introns that are rapidly degraded in a matter of seconds following excision, the 2-kb LAT intron has a half-life similar to those of some of the more stable cellular mRNAs. Furthermore, a similar half-life was measured for the 2-kb LAT in transiently transfected nonneuronal monkey COS-1 cells, suggesting that the stability of the 2-kb LAT is neither cell type nor species specific. Previously, we found that the determinant responsible for the unusual stability of the 2-kb LAT maps to the 3' terminus of the intron. At this site is a nonconsensus intron branch point located adjacent to a predicted stem-loop structure that is hypothesized to prevent debranching by cellular enzymes. Here we show that mutations which alter the predicted stem-loop structure, such that branching is redirected, either reduce or abolish the stability of the 2-kb LAT intron.

Animals↗

PubMed2002

Regions of the herpes simplex virus type 1 latency-associated transcript that protect cells from apoptosis in vitro and protect neuronal cells in vivo.

Recent studies have suggested that the latency-associated transcript (LAT) region of herpes simplex virus type 1 (HSV-1) is effective at blocking virus-induced apoptosis both in vitro and in the trigeminal ganglia of acutely infected rabbits (Inman et al., J. Virol. 75:3636-3646, 2001; Perng et al., Science 287:1500-1503, 2000). By transfecting cells with a construct expressing the Pst-Mlu segment of the LAT, encompassing the LAT exon 1, the stable 2.0-kb intron, and 5' part of exon 2, we confirmed that this region was able to diminish the onset of programmed cell death initiated by anti-Fas and camptothecin treatment. In addition, caspase 8-induced apoptosis was specifically inhibited in cells expressing the Pst-Mlu LAT fragment. To further delineate the minimal region of LAT that is necessary for this antiapoptotic function, LAT mutants were used in our cotransfection assays. In HeLa cells, the plasmids lacking exon sequences were the least effective at blocking apoptosis. However, similar to previous work (Inman et al., op. cit.), our data also indicated that the 5' end of the stable 2.0-kb LAT intron appeared to contribute to the promotion of cell survival. Furthermore, cells productively infected with the 17N/H LAT mutant virus, a virus deleted in the LAT promoter, exon 1, and about half of the intron, exhibited a greater degree of DNA fragmentation than cells infected with wild-type HSV-1. These data support the finding that the exon 1 and 2.0-kb intron region of the LAT transcription unit display an antiapoptotic function both in transfected cells and in the context of the virus infection in vitro. In trigeminal ganglia of mice acutely infected with the wild-type virus, 17, and 17DeltaSty, a virus lacking most of exon 1, apoptosis was not detected in cells that were positive for virus particles. However, dual staining was observed in cells from mice infected with 17N/H virus, indicating that the LAT antiapoptotic function demonstrated in cells transfected by LAT-expressing constructs may also play a role in protecting cells from virus-induced apoptosis during acute viral infection in vivo.

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