Faculty Bibliography

Multiple herpes simplex virus type 1 functions control translation by regulating phosphorylation of the initiation factor eIF2 on its alpha subunit. Both of the two known regulators, the gamma(1)34.5 and Us11 gene products, are produced late in the viral life cycle, although the gamma(1)34.5 gene is expressed prior to the gamma(2) Us11 gene, as gamma(2) genes require viral DNA replication for their expression while gamma(1) genes do not. The gamma(1)34.5 protein, through a GADD34-related domain, binds a cellular phosphatase (PP1alpha), maintaining pools of active, unphosphorylated eIF2. Infection of a variety of cultured cells with a gamma(1)34.5 mutant virus results in the accumulation of phosphorylated eIF2alpha and the inhibition of translation prior to the completion of the viral lytic program. Ectopic, immediate-early Us11 expression prevents eIF2alpha phosphorylation and the inhibition of translation observed in cells infected with a gamma(1)34.5 mutant by inhibiting activation of the cellular kinase PKR and the subsequent phosphorylation of eIF2alpha; however, a requirement for the Us11 protein, produced in its natural context as a gamma(2) polypeptide, remains to be demonstrated. To determine if Us11 regulates late translation, we generated two Us11 null viruses. In cells infected with a Us11 mutant, elevated levels of activated PKR and phosphorylated eIF2alpha were detected, viral translation rates were reduced 6- to 7-fold, and viral replication was reduced 13-fold compared to replication in cells infected with either wild-type virus or a virus in which the Us11 mutation was repaired. This establishes that the Us11 protein is critical for proper late translation rates. Moreover, it demonstrates that the shutoff of protein synthesis observed in cells infected with a gamma(1)34.5 mutant virus, previously ascribed solely to the gamma(1)34.5 mutation, actually results from the combined loss of gamma(1)34.5 and Us11 functions, as the gamma(2) Us11 mRNA is not translated in cells infected with a gamma(1)34.5 mutant

The herpes simplex virus type 1 (HSV-1) Us11 gene encodes a multifunctional double-stranded RNA (dsRNA)-binding protein that is expressed late in infection and packaged into the tegument layer of the virus particle. As a tegument component, Us11 associates with nascent capsids after its synthesis late in the infectious cycle and is delivered into newly infected cells at times prior to the expression of viral genes. Us11 is also an abundant late protein that regulates translation through its association with host components and contains overlapping nucleolar retention and nuclear export signals, allowing its accumulation in both nucleoli and the cytosol. Thus, at various times during the viral life cycle and in different intracellular compartments, Us11 has the potential to execute discrete tasks. The analysis of these functions, however, is complicated by the fact that Us11 is not essential for viral replication in cultured cells. To discover new host targets for the Us11 protein, we searched for cellular proteins that interact with Us11 and have identified PAT1 as a Us11-binding protein according to multiple, independent experimental criteria. PAT1 binds microtubules, participates in amyloid precursor protein trafficking, and has homology to the kinesin light chain (KLC) in its carboxyl terminus. The carboxyl-terminal dsRNA-binding domain of Us11, which also contains the nucleolar retention and nuclear export signals, binds PAT1, whereas 149 residues derived from the KLC homology region of PAT1 are important for binding to Us11. Both PAT1 and Us11 colocalize within a perinuclear area in transiently transfected and HSV-1-infected cells. The 149 amino acids derived from the KLC homology region are required for colocalization of the two polypeptides. Furthermore, although PAT1 normally accumulates in the nuclear compartment, Us11 expression results in the exclusion of PAT1 from the nucleus and its accumulation in the perinuclear space. Similarly, Us11 does not accumulate in the nucleoli of infected cells that overexpress PAT1. These results establish that Us11 and PAT1 can associate, resulting in an altered subcellular distribution of both polypeptides. The association between PAT1, a cellular trafficking protein with homology to KLC, and Us11, along with a recent report demonstrating an interaction between Us11 and the ubiquitous kinesin heavy chain (R. J. Diefenbach et al., J. Virol. 76:3282-3291, 2002), suggests that these associations may be important for the intracellular movement of viral components

In animal models of herpes simplex virus type 1 (HSV-1) infection, ICP34.5-null viruses are avirulent and also fail to grow in a variety of cultured cells due to their inability to prevent RNA-dependent protein kinase (PKR)-mediated inhibition of protein synthesis. We show here that the inability of ICP34.5 mutants to grow in vitro is due specifically to the accumulation of phosphorylated eIF2 alpha. Mutations suppressing the in vitro phenotype of ICP34.5-null mutants have been described which map to the unique short region of the HSV-1 genome, resulting in dysregulated expression of the US11 gene. Despite the inability of the suppressor mutation to suppress the avirulent phenotype of the ICP34.5-null parental virus following intracranial inoculation, the suppressor mutation enhanced virus growth in the cornea, trigeminal ganglia, and periocular skin following corneal infection compared to that with the ICP34.5-null virus. The phosphorylation state of eIF2 alpha following in vitro infection with the suppressor virus was examined to determine if in vivo differences could be attributed to differential regulation of eIF2 alpha phosphorylation. The suppressor virus prevented accumulation of phosphorylated eIF2 alpha, while the wild-type virus substantially reduced eIF2 alpha phosphorylation levels. These data suggest that US11 functions as a PKR antagonist in vivo, although its activity may be modulated by tissue-specific differences in translation regulation

A variety of viral strategies are utilized for dominance of the host-cell protein synthetic machinery, optimization of viral mRNA translation and evasion of host-cell antiviral responses that act at the translational level. Many viruses exploit regulated steps in the initiation of cellular protein synthesis to their own advantage. They have developed some rather unconventional means for mRNA translation, which were probably adapted from specialized cellular mRNA translation systems. Regardless of the type of translational tricks exploited, viruses typically ensure efficient viral translation, often at the expense of host-cell protein synthesis

Over the past decade, rapid progress has been made in engineering safe, replicating herpes simplex virus-1 (HSV-1) mutants for use as biological oncolytic agents in the treatment of human cancer. While initial efforts demonstrated the potential of HSV-1 mutants as antitumour agents, they relied on viruses that were not sufficiently attenuated. Following its identification as the major viral neurovirulence determinant, mutations in the gamma34.5 gene were subsequently incorporated into oncolytic strains. Despite the fact that gamma34.5 mutant derivatives can be safely administered to mice, non-human primates and humans, their efficacy is limited because, like many weakened viral strains, they replicate poorly in a number of cell types, including cancer cells. Strategies to improve the oncolytic properties of gamma34.5 mutant derivatives through further genetic manipulation are reviewed. In addition, traditional treatment modalities that incorporate viral inoculation, along with efforts to elicit an antitumour immune response following treatment with gamma34.5 derivatives, are discussed

The Epstein-Barr virus (EBV) SM protein is a posttranscriptional regulator of viral gene expression. Like many transactivators encoded by herpesviruses, SM transports predominantly unspliced viral mRNA cargo from the nucleus to the cytosol, where it is subsequently translated. This activity likely involves a region of the protein that has homology to the herpes simplex virus type 1 (HSV-1) ICP27 gene product, the first member of this class of regulators to be discovered. However, SM also contains a repetitive segment rich in arginine and proline residues that is dispensable for its effects on RNA transport and splicing. This portion of SM, comprised of RXP triplet repeats, shows homology to the carboxyl-terminal domain of Us11, a double-stranded RNA (dsRNA) binding protein encoded by HSV-1 that inhibits activation of the cellular PKR kinase. To evaluate the intrinsic ability of SM to regulate PKR, we expressed and purified several SM protein derivatives and examined their activity in a variety of biochemical assays. The full-length SM protein bound dsRNA, associated physically with PKR, and prevented PKR activation. Removal of the 37-residue RXP domain significantly compromised all of these activities. Furthermore, the SM RXP domain was itself sufficient to inhibit PKR activation and interact with the kinase. Relative to its Us11 counterpart, the SM RXP segment bound dsRNA with reduced affinity and responded differently to single-stranded competitor polynucleotides. Thus, SM represents the first EBV gene product expressed during the lytic cycle that can prevent PKR activation. In addition, the RXP repeat segment appears to be a conserved herpesvirus motif capable of associating with dsRNA and modulating activation of the PKR kinase, a molecule important for the control of translation and the cellular antiviral response

The herpes simplex virus Us11 gene product inhibits activation of the cellular PKR kinase and associates with a limited number of unrelated viral and cellular RNA molecules via a carboxyl-terminal 68-amino-acid segment rich in arginine and proline. To characterize the determinants underlying the recognition of an RNA target by Us11, we employed an in vitro selection technique to isolate RNA ligands that bind Us11 with high affinity from a population of molecules containing an internal randomized segment. Binding of Us11 to these RNA ligands is specific and appears to occur preferentially on conformational isoforms that possess a higher-order structure. While the addition of unlabeled poly(I. C) reduced binding of Us11 to a selected radiolabeled RNA, single-stranded homopolymers were not effective competitors. Us11 directly associates with poly(I. C), and inclusion of an unlabeled selected RNA in the reaction reduces poly(I. C) binding, while single-stranded RNA homopolymers have no effect. Finally, Us11 binds to defined, double-stranded RNA (dsRNA) molecules that exhibit greater sequence complexity. Binding to these dsRNA perfect duplexes displays a striking dependence on length, as 39-bp or shorter duplexes do not bind efficiently. Furthermore, this interaction is specific for dsRNA as opposed to dsDNA, implying that the Us11 RNA binding domain can distinguish nucleic acid duplexes containing 2' hydroxyl groups from those that do not. These results establish that Us11 is a dsRNA binding protein. The arginine- and proline-rich Us11 RNA binding domain is unrelated to known dsRNA binding elements and thus constitutes a unique recognition motif that interacts with dsRNA. The ability of Us11 to bind dsRNA may be important for inhibiting activation of the cellular PKR kinase in response to dsRNA

PACT, a protein activator of PKR, can cause inhibition of cellular protein synthesis and apoptosis. Here, we report that the Us11 protein of herpes simplex virus type 1 can block PKR activation by PACT both in vitro and in vivo. Although Us11 can bind to both PKR and PACT, mutational analyses revealed that the binding of Us11 to PKR, and not to PACT, was essential for its inhibitory action. Similar analyses also revealed that the inhibitory effect was mediated by an interaction between the C-terminal half of Us11 and the N-terminal domain of PKR. The binding of Us11 to PKR did not block the binding of PKR to PACT but prevented its activation. Us11 is the first example of a viral protein that can inhibit the action of PACT on PKR

Replication-competent, attenuated herpes simplex virus-1 (HSV-1) derivatives that contain engineered mutations into the viral gamma34.5 virulence gene have been used as oncolytic agents. However, as attenuated mutants often grow poorly, they may not completely destroy some tumors and surviving cancer cells simply regrow. Thus, although HSV-1 gamma34.5 mutants can reduce the growth of human tumor xenografts in mice and have passed phase I safety studies, their efficacy is limited because they replicate poorly in many human tumor cells. Previously, we selected for a gamma34.5 deletion mutant variant that regained the ability to replicate efficiently in tumor cells. Although this virus contains an extragenic suppressor mutation that confers enhanced growth in tumor cells, it remains attenuated. Here, we demonstrate that the suppressor virus replicates to greater levels in prostate carcinoma cells and, importantly, is a more potent inhibitor of tumor growth in an animal model of human prostate cancer than the gamma34.5 parent virus. Thus, genetic selection in cancer cells can be used as a tool to enhance the antitumor activity of a replication-competent virus. The increased therapeutic potency of this oncolytic virus may be useful in the treatment of a wide variety of cancers