Cell Biology and Anatomy

The eight human herpes viruses are endemic in the population and cause a spectrum of clinically significant   diseases.  In addition to acute infections, herpes viruses establish life-long latent infections of sensory neurons that reactivate periodically to cause recurrent disease. Using herpes simplex virus (HSV) as a model neurotropic herpes virus, current efforts ongoing in the Schaffer/Bringhurst lab are focused on identifying the mechanisms underlying the control of viral gene expression and viral DNA replication during productive infection and reactivation from neuronal latency.

We have shown that inhibitors of cyclin dependent kinases (cdks) block HSV replication efficiently and that during productive infection, cdks are required for transcription of viral genes, replication of viral DNA. Cdk inhibitors also block reactivation from neuronal latency.  Questions being asked in the laboratory include  “Why does HSV replication in non-dividing neurons require cell cycle-associated factors (cdks)? which cdks and cyclins are essential for HSV replication? what are the viral and cellular targets of cdks? and can cdk inhibitors serve as clinically useful antiviral drugs?”

ICP0 is an HSV-encoded immediate-early regulatory protein that modifies cells extensively to support viral replication and promote efficient reactivation from latency.  ICP0 regulates transcription of both viral and cellular genes and appears to achieve its trans-regulatory activity not by binding to DNA, but by disrupting nuclear structures called ND10 or pods. ICP0 functions at a pre-transcriptional level and can alter the nuclear environment of cells to resemble late G1/S or G2/M. ICP0’s trans-activating activity is blocked by cdk inhibitors and the reduction in this activity correlates with an increase in the electrophoretic mobility of ICP0. We hypothesize that cdks and other kinases affect the posttranslational modification of ICP0, which is required for its ability to regulate viral gene expression, disrupt ND10 and alter the state of the cell cycle.  Ongoing studies involve identification of the viral and cellular kinases that mediate the posttranslational modification of ICP0, identification of critical phosphorylation sites on ICP0 and analysis of the effects of phosphorylation on the multiple functions of this protein.

Of special interest is the role of ICP0 in reactivation from latency. ICP0 mutants replicate and reactivate from latency inefficiently in vitro and in vivo. We have shown, however, that a spectrum of treatments that induce cellular stress enhance both the replication and reactivation efficiencies of ICP0 mutants. A major objective of the lab is to identify cellular factors and pathways that result in the enhanced replication and reactivation of ICP0 mutant viruses.

Herpes viruses initiate viral DNA replication early during productive infection and reactivation. Unlike most viruses, however, they also have the capacity to inhibit viral DNA replication during the establishment of latency. For these reasons, HSV DNA replication and its regulation are central to the life cycle of the virus.  Origin-dependent viral DNA replication requires the function of the HSV origin binding protein (OBP) during the initial stage of viral DNA replication (stage I), yet OBP is inhibitory to replication during the later stage of replication (stage II). This observation implies that OBP function must be tightly regulated during the switch from stage I to stage II and vice versa. The mechanism by which OBP function is regulated is a central focus of research in he lab. Although much is known about the mechanism by which HSV DNA replication is initiated, little is known about how it is inhibited.  We have recently identified two C-terminal variants of OBP that together, serve to down-regulate viral DNA replication, and are in the process of identifying the mechanism by which inhibition is accomplished.

 HSV DNA replication begins at HSV origins, oriL and oriS, which share many structural features in common. Notably, initiation of viral DNA replication is equally efficient when begun at either origin during productive infection of cultured cells. In contrast, based on studies in animal and cell culture models of latency, reactivation of viruses with mutated origins indicate that oriL is the preferred origin during reactivation from latency and plays a major role in viral pathogenesis in vivo. The molecular basis for the distinct properties of oriL and oriS is being investigated.

Research in the Schaffer/Bringhurst lab is notable for its broad theoretical and technical approach to studying HSV.  Thus, most projects involve site-directed mutagenesis of cloned viral genes, isolation of mutant viruses, characterization of the effects of specific mutations on viral replication in cell culture, and characterization of the effects of these mutations on acute replication, latency and reactivation in animal models This approach permits identification of the roles of individual HSV genes, intra-genic domains and cis-acting elements in the pathogenesis of viral infection in animals.

Link MA Schaffer PA. Oct 2007. Herpes simplex virus type 1 C-terminal variants of the origin binding protein (OBP), OBPC-1 and OBPC-2, cooperatively regulate viral DNA levels in vitro, and OBPC-2 affects mortality in mice. J Virol, 81:10699-711

Link MA Silva LA Schaffer PA. Sep 2007. Cathepsin B mediates cleavage of herpes simplex virus type 1 origin binding protein (OBP) to yield OBPC-1, and cleavage is dependent upon viral DNA replication. J Virol, 81:9175-82

Balliet JW Kushnir AS Schaffer PA. May 2007. Construction and characterization of a herpes simplex virus type I recombinant expressing green fluorescent protein: acute phase replication and reactivation in mice. Virology, 361:372-83

Melroe GT Silva L Schaffer PA Knipe DM. Apr 2007. Recruitment of activated IRF-3 and CBP/p300 to herpes simplex virus ICP0 nuclear foci: Potential role in blocking IFN-beta induction. Virology, 360:305-21

Orlando JS Balliet JW Kushnir AS Astor TL Kosz-Vnenchak M Rice SA Knipe DM Schaffer PA. Oct 2006. ICP22 is required for wild-type composition and infectivity of herpes simplex virus type 1 virions. J Virol, 80:9381-90

Bringhurst RM Schaffer PA. May 2006. Cellular stress rather than stage of the cell cycle enhances the replication and plating efficiencies of herpes simplex virus type 1 ICP0- viruses. J Virol, 80:4528-37

Orlando JS Astor TL Rundle SA Schaffer PA. Apr 2006. The products of the herpes simplex virus type 1 immediate-early US1/US1.5 genes downregulate levels of S-phase-specific cyclins and facilitate virus replication in S-phase Vero cells. J Virol, 80:4005-16

Balliet JW Schaffer PA. Jan 2006. Point mutations in herpes simplex virus type 1 oriL, but not in oriS, reduce pathogenesis during acute infection of mice and impair reactivation from latency. J Virol, 80:440-50

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