Faculty Bibliography

Viral pathogenesis depends on a suitable milieu in target host cells permitting viral gene expression, propagation, and spread. In many instances, viral genomes can be manipulated to select for propagation in certain tissues or cell types. This has been achieved for the neurotropic poliovirus (PV) by exchange of the internal ribosomal entry site (IRES), which is responsible for translation of the uncapped plus-strand RNA genome. The IRES of human rhinovirus type 2 (HRV2) confers neuron-specific replication deficits to PV but has no effect on viral propagation in malignant glioma cells. We report here that placing the critical gamma(1)34.5 virulence genes of herpes simplex virus type 1 (HSV) under translation control of the HRV2 IRES results in neuroattenuation in mice. In contrast, IRES insertion permits HSV propagation in malignant glioma cell lines that do not support replication of HSV recombinants carrying gamma(1)34.5 deletions. Our observations indicate that the conditions for alternative translation initiation at the HRV2 IRES in malignant glioma cells differ from those in normal central nervous system (CNS) cells. Picornavirus regulatory sequences mediating cell type-specific gene expression in the CNS can be utilized to target cancerous cells at the level of translation regulation outside their natural context

Via careful control of multiple kinases that inactivate the critical translation initiation factor eIF2 by phosphorylation of its alpha subunit, the cellular translation machinery can rapidly respond to a spectrum of environmental stresses, including viral infection. Indeed, virus replication produces a battery of stresses, such as endoplasmic reticulum (ER) stress resulting from misfolded proteins accumulating within the lumen of this organelle, which could potentially result in eIF2alpha phosphorylation and inhibit translation. While cellular translation is exquisitely sensitive to ER stress-inducing agents, protein synthesis in herpes simplex virus type 1 (HSV-1)-infected cells is notably resistant. Sustained translation in HSV-1-infected cells exposed to acute ER stress does not involve the interferon-induced, double-stranded RNA-responsive eIF2alpha kinase PKR, and it does not require either the PKR inhibitor encoded by the Us11 gene or the eIF2alpha phosphatase component specified by the gamma(1)34.5 gene, the two viral functions known to regulate eIF2alpha phosphorylation. In addition, although ER stress potently induced the GADD34 cellular eIF2alpha phosphatase subunit in uninfected cells, it did not accumulate to detectable levels in HSV-1-infected cells under identical exposure conditions. Significantly, resistance of translation to the acute ER stress observed in infected cells requires HSV-1 gene expression. Whereas blocking entry into the true late phase of the viral developmental program does not abrogate ER stress-resistant translation, the presence of viral immediate-early proteins is sufficient to establish a state permissive of continued polypeptide synthesis in the presence of ER stress-inducing agents. Thus, one or more previously uncharacterized viral functions exist to counteract the accumulation of phosphorylated eIF2alpha in response to ER stress in HSV-1-infected cells

As they are completely dependent upon the protein synthesis machinery resident in the cells of their host to translate their mRNAs, it is imperative that viruses are able to effectively manipulate the elaborate cellular regulatory network that controls translation. Indeed, this exquisite dependence on host functions has made viral models attractive systems to explore translational regulatory mechanisms operative in eukaryotic cells. Central among these are an intricate array of phosphorylation and dephosphorylation events that have far reaching consequences on the activity of cellular translation factors. Not only do these modulate the activity of a given factor, but they can also determine if the translation of host proteins persists in infected cells, the efficiency with which viral mRNAs are translated and the outcome of a systemic host anti-viral response. In this review, we discuss how various viruses manipulate the phosphorylation state of key cellular translation factors, illustrating the critical nature these interactions play in virus replication, pathogenesis and innate host defense

Recruitment of the 40S ribosome to the 5' end of a eukaryotic mRNA requires assembly of translation initiation factors eIF4E, the cap-binding protein, together with eIF4A and eIF4G into a complex termed eIF4F. While the translational repressor 4E-BP1 regulates binding of eIF4E to eIF4G, the forces required to construct an eIF4F complex remain unidentified. Here, we establish that the herpes simplex virus-1 (HSV-1) ICP6 polypeptide associates with eIF4G to promote eIF4F complex assembly. Strikingly, release of eIF4E from the 4E-BP1 repressor is insufficient to drive complex formation, suggesting that ICP6 is an eIF4F-assembly chaperone. This is the first example of a translation initiation factor-associated protein that promotes active complex assembly and defines a new, controllable step in the initiation of translation. Homology of the N-terminal, eIF4G-binding segment of ICP6 with cellular chaperones suggest that factors capable of interacting with eIF4G and promoting eIF4F complex assembly may play important roles in a variety of processes where translation complexes need to be remodeled or assembled on populations of newly synthesized or derepressed mRNAs, including development, differentiation, and the response to a broad spectrum of environmental cues

To ensure that their mRNAs are translated and that the viral proteins necessary for assembling the next generation of infectious progeny are produced, viruses must effectively seize control of the translational machinery within their host cells. In many cases, the ability to productively engage host translational components can determine if a given cell type can support viral replication, illustrating the critical importance of this task in the viral life cycle. Failure to interface properly with the host translational apparatus can compromise the productive growth cycle, resulting in an abortive infection and radically restricting viral replication. Not only have viruses become facile at commandeering this machinery, they are also particularly adept at manipulating cellular translation control pathways for their own ends. In this review, the mechanisms by which numerous viruses manipulate host translational control circuits are discussed. Furthermore, particular attention is devoted to understanding how interfering with the ability of a virus to properly regulate translation in its host can be exploited to generate oncolytic strains that selectively replicate in cancer cells

As a viral opportunistic pathogen associated with serious disease among the immunocompromised and congenital defects in newborns, human cytomegalovirus (HCMV) must engage the translational machinery within its host cell to synthesize the viral proteins required for its productive growth. However, unlike many viruses, HCMV does not suppress the translation of host polypeptides. Here, we examine how HCMV regulates the cellular cap recognition complex eIF4F, a critical component of the cellular translation initiation apparatus that recruits the 40S ribosome to the 5' end of the mRNA. This study establishes that the cap binding protein eIF4E, together with the translational repressor 4E-BP1, are both phosphorylated early in the productive viral growth cycle and that the activity of the cellular eIF4E kinase, mnk, is critical for efficient viral replication. Furthermore, HCMV replication also induces an increase in the overall abundance of eIF4F components and promotes assembly of eIF4F complexes. Notably, increasing the abundance of select eIF4F core components and associated factors alters the ratio of active eIF4F complexes in relation to the 4E-BP1 translational repressor, illustrating a new strategy through which members of the herpesvirus family enhance eIF4F activity during their replicative cycle

The gamma(1)34.5 gene product is important for the resistance of herpes simplex virus type 1 (HSV-1) to interferon. However, since the inhibition of protein synthesis observed in cells infected with a gamma(1)34.5 mutant virus results from the combined loss of the gamma(1)34.5 gene product and the failure to translate the late Us11 mRNA, we sought to characterize the relative interferon sensitivity of mutants unable to produce either the Us11 or the gamma(1)34.5 polypeptide. We now demonstrate that primary human cells infected with a Us11 mutant virus are hypersensitive to alpha interferon, arresting translation upon entry into the late phase of the viral life cycle. Furthermore, immediate-early expression of Us11 by a gamma(1)34.5 deletion mutant is sufficient to render translation resistant to alpha interferon. Finally, we establish that the Us11 gene product is required for wild-type levels of replication in alpha interferon-treated cells and, along with the gamma(1)34.5 gene, is an HSV-1-encoded interferon resistance determinant

Although the activity of the translation initiation factor eIF4F is regulated in part by translational repressors (4E-BPs) that prevent incorporation of eIF4E, the cap-binding protein, into the initiation complex, the contribution of eIF4E phosphorylation to translational control remains controversial. Here, we demonstrate that the herpes simplex virus-1 (HSV-1) ICP0 gene product, a multifunctional transactivator of viral gene expression with ubiquitin E3 ligase activity that is important for vegetative replication and reactivation of latent infections, is required to stimulate phosphorylation of eIF4E as well as 4E-BP1, and promote assembly of eIF4F complexes in infected cells. Furthermore, 4E-BP1 is degraded by the proteasome in an ICP0-dependent manner, establishing that the proteasome can control 4E-BP1 steady-state levels. Preventing eIF4E phosphorylation by inhibiting the eIF4E kinase mnk-1 dramatically reduced viral replication and the translation of viral polypeptides in quiescent cells, providing the first evidence that phosphorylation of eIF4E by mnk-1 is critical for viral protein synthesis and replication. Thus, in marked contrast to many viruses that inactivate eIF4F, HSV-1 stimulates eIF4F complex assembly in quiescent, differentiated cells; moreover, this is important for viral replication, and may be crucial for HSV-1 to initiate its productive growth cycle in resting cells, such as latently infected neurons

Lytic replication of many viruses activates an innate host response designed to prevent the completion of the viral lifecycle, thus impeding the spread of the infection. One branch of the host's complex reaction functions to incapacitate the cellular translational machinery on which the synthesis of viral polypeptides completely depends. This is achieved through the activation of specific protein kinases that phosphorylate eIF2 on its alpha subunit and inactivate this critical translation initiation factor. However, as continued synthesis of viral proteins is required to assemble the viral progeny necessary to transmit the infection to neighboring cells, viruses have developed a variety of strategies to counter this cellular response. Genetic and biochemical studies with herpes simplex virus type 1 (HSV-1) have revealed that the virus produces at least two discrete products at different times during its replicative program that act to prevent the accumulation of phosphorylated eIF2alpha. The gamma(1)34.5 gene product is expressed first, encoding a regulatory subunit that binds the cellular protein phosphatase 1alpha and regenerates pools of active eIF2 by removing the inhibitory phosphate from the alpha subunit. The second function, encoded by the product of the Us11 gene, specifies a double-stranded RNA-binding protein that prevents activation of PKR, a cellular eIF2alpha kinase. Together, both proteins cooperate to overcome the antiviral response of the host and properly regulate translation in HSV-1-infected cells