James Smiley, PhD
Dept. of Medical Microbiology & Immunology
University of Alberta
Faculty of Medicine & Dentistry
6-32 Heritage Medical Research Building
T6G 2S2 Edmonton, AB
Ph : (780) 492-4070
Fx : (780) 492-9828
- Professor, Dept. of Medical Microbiology & Immunology
Virus infections remain the single most common reason that Canadians seek medical attention. Although impressive progress has been made in developing anti-viral drugs, drug resistant variants often arise and many virus infections remain untreatable. The innate immune system is our first line of defense against virus infection. Unfortunately, most viruses produce proteins that serve as effective countermeasures. My laboratory is focused on how viral regulatory proteins function at the molecular level, and how cellular antiviral responses inhibit viral replication. The hope is that increased understanding of host antiviral defenses and viral immune evasion strategies will open up new approaches to controlling virus infections. Most of our work focuses on herpes simplex virus (HSV), a ubiquitous human pathogen and the prototypical member of the herpesviridae, a large family of enveloped DNA viruses that replicate in the nuclei of host cells. Recently we have also begun similar studies with HIV-1, the retrovirus that causes AIDS.
Here is a snap-shot summary of the current research in my lab.
1. HSV-encoded host shutoff functions. HSV rapidly shuts off cellular gene expression, thereby dampening host responses to infection. We are currently studying three HSV proteins that contribute to shutoff: the virion host shutoff protein (vhs) encoded by gene UL41, the immediate-early protein ICP27 encoded by gene UL54, and the alkaline exonuclease encoded by gene UL12.
Vhs is an endoribonuclease that accelerates the decay cellular cytoplasmic mRNAs, and serves as a critical virulence determinant in animal models of infection. Vhs mutants are attenuated largely because they are unable to disarm the host innate immune response. We are currently focused on two aspects of vhs function. First, vhs binds to host translation initiation factors and we would like to determine if this is the mechanism that vhs uses to selectively target mRNAs over other cytoplasmic RNA species. Second, although vhs inhibits the expression of most cellular mRNAs, in unpublished work we have unexpectedly found that it is also capable of activating gene expression under certain conditions. Specifically, we find that it is able to strongly stimulate the activity of certain cellular internal ribosome entry sites (IRESs). IRESs are present in the 5’ untranslated regions of cellular mRNAs involved with a variety of stress responses, where they serve to modulate translation in response to external signals. Our major effort here is to determine exactly how vhs stimulates IRES-dependent translation, and clarify the biological relevance of this activity during virus infection.
ICP27 is an essential RNA binding protein and post-transcriptional regulator that down regulates cellular gene expression and activates expression of viral genes. We have recently shown that ICP27 acts at least in part at the translational level, and that it is also capable of modulating the stability of cellular mRNAs that bear AU-rich instability elements. Such mRNAs include those encoding most cytokines and immediate-early response proteins. Current efforts focus on determining how ICP27 stimulates translation and regulates the stability of ARE mRNAs.
UL12 is a virallycoded nuclease involved in processing of mature HSV genomes from larger concatameric precursors. In recent studies we discovered that HSV infection rapidly eliminates mitochondrial DNA and mitochondrial mRNAs from infected cells and that the HSV UL12 gene is required for this activity. UL12 encodes two related proteins, UL12 and UL12.5, and we have shown that UL12.5 localizes to mitochondria and is able to deplete mitochondrial DNA in the absence of other HSV proteins. We currently seek to determine the precise mechanisms involved, and delineate if and how this activity stimulates virus replication and contributes to pathogenesis.
2. Viral inhibition of the cellular interferon-based antiviral response. The host type I interferon response is the first line of defense against virus infection. Interferons are secreted by virus-infected cells, and serve to warn neighboring uninfected cells that they are likely to be infected. Interferons act by increasing the expression of an array of cellular proteins that collectively serve to inhibit virus replication. Such interferon-induced proteins have widely varied activities, and in combination appear to block essentially every step in diverse viral life cycles. Studying how such proteins work and how viruses counteract their effects is a major focus of our research. We have shown that the HSV immediate-early protein ICP0 (an E3 ubiquitin ligase encoded by gene RL2) overcomes an interferon-induced intranuclear antiviral defense mechanism that blocks HSV gene expression at the transcriptional or pre-transcriptional level. We currently seek to define components of this cellular interferon-induced gene silencing system, and determine how it is inactivated by ICP0. We have shown that the U2OS osteosarcoma cell line lacks this repression mechanism, providing a novel means of moving this project forward. One of the surprises to recently emerge from this work is the discovery that the HSV tegument protein VP16, a well-characterized transcriptional activator of viral gene expression encoded by gene UL48, acts in part by overcoming these host defense mechanisms. Other ongoing studies in this area focus on the mechanisms of action host interferon-induced proteins, including HERC5 and TRIM22, that block HSV and HIV replication.
3. Virus-induced inactivation of host natural killer cells and cytotoxic T lymphocytes. Natural killer (NK) cells and cytotoxic T lymphocytes (CTL) are key components of the innate and adaptive immune system respectively. Both cell types are critical for antiviral defense, and HSV is able to disarm these cells. This virus-induced inactivation requires cell-cell contact, and is accompanied by the appearance of an abundant tyrosine-phosphorylated protein in the inactivated lymphocytes. In unpublished work we have shown that this tyrosine-phosphoryated protein is VP11/12, an HSV tegument protein encoded by gene UL46. Remarkably, the VP11/12 present in fibroblasts is not tyrosine phosphorylated, suggesting that it is phosphorylated by a lymphocte-specific tyrosine kinase. Indeed, phosphorylation of VP11/12 is severely impaired in T cells lacking p56(lck). We currently seek to determine the role, if any, of VP11/12 in the lymphocyte inactivation process, and document its mechanism of action. Additional studies focus on the mechanism of spread of HSV from infected fibroblasts to NK cells.
Figure 1. A UL12.5-GFP fusion protein (green) localizes to mitochondria (red)
Figure 2. Herpes simplex virus UL12.5 protein degrades mammalian mitochondrial DNA.
A. Mitochondrial DNA in untreated cells can be visualized by staining with PicoGreen, which stains both mitochondrial and nuclear DNA green. Mitochondria have been counterstained red with MitoTracker.
B. Expression of the HSV UL12.5 protein depletes mitochondrial DNA, giving rise to viable colonies of cells completely lacking mitochondrial genomes (panels iii and vi).
Figure 3. U2OS cells lack a repression system that blocks HSV gene expression in other cell types (part 1). Human embryonic lung cells (HEL) and U2OS osteosarcoma cells (blue) were infected with an HSV-1 mutant lacking functional VP16 and ICP0. Note that only the blue U2OS cells express the HSV ICP4 protein (green). Additional somatic cell fusion experiments demonstrated that the non-permissive phenotype of the HEL cells is dominant in HEL-U2OS hybrids (see figure 4).
Figure 4. U2OS cells lack a repression system that blocks HSV gene expression in other cell types (part 2). A heterokaryon formed by fusing HEL (red) and U2OS (blue) cells was infected with an HSV-1 mutant lacking functional VP16 and ICP0. Note that no viral gene expression was observed (A) unless the viral ICP0 protein was provided in trans (B), indicating that the non-permissive phenotype of HEL cells is dominant.
- Bianca Dauber (Postdoctoral Fellow)
- Brett Duguay (PDF)
- Holly Saffran (Technologist)
- Ulrike Strunk (Graduate Student)
- Mossman KL, Macgregor PF, Rozmus JJ, Goryachev AB, Edwards AM, Smiley JR. Herpes simplex virus triggers and then disarms a host antiviral response. J Virol 75:750-8. 2001
- Lu P, Saffran HA, Smiley JR. The vhs1 mutant form of herpes simplex virus virion host shutoff protein retains significant internal ribosome entry site-directed RNA cleavage activity. J Virol 75:1072-6. 2001
- Lu P, Jones FE, Saffran HA, Smiley JR. Herpes Simplex Virus Virion Host Shutoff Protein Requires a Mammalian Factor for Efficient In Vitro Endoribonuclease Activity. J Virol 75:1172-1185.2001
- Smiley JR, Elgadi MM, Saffran HA. Herpes simplex virus vhs protein. Methods Enzymol 342:440-51 2001
- Mossman KL, Smiley JR. Herpes Simplex Virus ICP0 and ICP34.5 Counteract Distinct Interferon-Induced Barriers to Virus Replication. J Virol 76:1995-8 2002