Research Interests
The Interactions Between Herpes Viruses and Their Host Cells
Figure 1.
My lab is interested in the interactions between herpesviruses and their host cells. Over the past several years, we have concentrated on those interactions important for the regulation of protein synthesis.
The lytic replication of many viruses requires that large quantities of viral proteins be produced to assemble the next generation of viral progeny. To accomplish this, viral functions must, i) prevent a cellular antiviral response designed to globally inhibit cellular and viral protein synthesis; and ii) ensure that viral mRNAs are translated efficiently.
In the course of many different viral infections, copious amounts of double stranded RNA are produced, activating the cellular kinase PKR. Unchecked, activated PKR would phosphorylate eIF2 on its alpha subunit, inactivating this critical translation initiation factor and hindering virus assembly and replication by inhibiting protein synthesis. To counteract this cellular response and prevent the cessation of protein synthesis prior to the completion of the viral lifecycle, many viruses encode factors that prevent the accumulation of phosphorylated eIF2 alpha. We have taken a molecular genetic and biochemical approach to investigate herpesvirus encoded proteins that inhibit this antiviral response. One of these proteins, the herpes simplex virus ¡V1 gamma 34.5 gene product, binds a cellular phosphatase and dephosphorylates the inactivated pools of phosphorylated eIF2. Another protein encoded by the Us11 gene binds RNA via a novel RNA binding motif rich in arginine and prolines (fig. 1). This RNA binding motif has a high affinity for double stranded or structured RNA molecules. Currently, we are exploring the molecular mechanisms by which these viral proteins exert their effects. Significantly, both the Us11 and gamma 34.5 gene products are important determinants of interferon resistance in primary human cells infected with HSV-1.
Figure 2.
To ensure that its mRNAs are translated, viruses must effectively capture key, potentially limiting components of the host translational machinery. One of these components is the multi-subunit eIF4F complex. Composed of the cap binding protein eIF4E, the large molecular scaffold eIF4G, and the eIF4A RNA unwinding enzyme, eIF4F recognizes the 7-methyl GTP cap structure at the 5¡¦ end of the mRNA and recruits the 40S ribosome via an association between eIF3 and eIF4G (fig 2). Finally, the cellular polyA binding protein (PABP), while itself not a component of eIF4F, physically interacts with eIF4G through the PABP interacting protein PAIP-1, bridging the 5¡¦ and 3¡¦ ends of the mRNA and generating a circular topology which possibly serves as a checkpoint to ensure the mRNA is both capped and polyadenylated prior to translation initiation.
Figure 3.
The activity of eIF4F is regulated in part by the 4E-BP1 translational repressors that bind eIF4E and prevent it from associating with eIF4G. Phosphorylation of 4E-BP1 by the cellular kinase mTOR results in the release of free eIF4E (fig. 3), which can now in turn associate with eIF4G and assemble the eIF4F complex. In addition, an eIF4F associated kinase, mnk-1, binds eIF4G and phosphorylates eIF4E, stimulating translation. Strikingly, phosphorylation of eIF4E together with 4E-BP1 is stimulated in resting cells infected with HSV-1, and 4E-BP1 appears to be degraded by the cellular proteasome. Moreover, blocking eIF4E phosphorylation with a specific mnk inhibitor dramatically reduces viral replication. Finally, the assembly of eIF4F complexes is enhanced considerably upon infection with HSV-1. All of these events appear to be dependent upon the HSV-1 ICP0 gene product. Current work in the lab is focused on understanding how this is achieved. It is likely that the mobilization of eIF4F by viral functions is important for the virus to replicate in quiescent, non-dividing cells, such as neurons.
A second area of interest concerns the design and use of attenuated viruses as anti-tumor agents. Ideally, such a virus would be able to replicate in and destroy cancer cells without destroying normal terminally differentiated cells. Unfortunately, the attenuation process, which is necessary to ensure safety, often substantially impairs the ability of the virus to replicate in a variety of cells in culture and in animals. We have modified an attenuated HSV-1 mutant by genetic selection in cancer cells and isolated an attenuated variant with enhanced ability to replicate in cultured tumor cells. Our modified HSV-1 is an extremely potent anti - tumor agent in an animal model of human cancer. Thus, additional mutations can be introduced into the genome of weakened, attenuated viruses that confer upon them enhanced anti ¡Vtumor activity.