Herpes Simplex Virus Type 1 (HSV-1) is a human pathogen that is estimated to have infected 60–80% of the population worldwide, causing oral lesions, encephalitis, and blindness. Infection results in life-long latency of the virus, with periods of reactivation and symptomatic disease. Early in infection, HSV-1 induces the formation of replication compartments and VICE (virus induced chaperone enriched) domains within the nucleus. Adjacent to VICE domains, we observed foci which contained Mss1, a cellular protein normally associated with the proteasome. Mss1 belongs to the AAA-ATPase family of proteins, which have been shown to function in DNA repair, transcription, translation, and the stress response. In ECP19 cells, which are naturally reduced in Mss1 protein and mRNA levels, HSV-1 does not replicate. Based on Mss1's accessory functions and observed localization, we sought to determine if Mss1 played a role in HSV-1 infection. Addition of Mss1 to non-permissive ECP19 cells which have endogenously lower levels of Mss1 did not alter HSV-1 replication. Furthermore, siRNA knockdown of Mss1 in normally permissive cells did not alter HSV-1 replication. These results indicated that Mss1 was likely a dispensable accessory factor incorporated to VICE domains during HSV-1 replication.
Interferon gamma induces the expression of a number of genes. Among them are three alternate subunits of the 20S core of the proteasome, β1i, β2i, and β5i. Expression and incorporation of these subunits alters the functionality of the proteasome, thereby creating the immunoproteasome. While it is known that proteasomal function is required for HSV-1 replication, the functional capacity of immunoproteasomes during HSV-1 infection is not known. We found that immunoproteasomes persist throughout infection and that the activity of immunoproteasomes purified from infected cells is higher than that of uninfected controls.
MicroRNAs (miRNAs) are small ∼20 nt regulatory RNAs that alter gene expression by binding to target mRNAs, causing translational repression. Viruses have evolved the use of miRNAs which regulate cellular and/or viral gene expression. HSV-1 miRNAs have exhibited functionality during latency, but little is known about viral miRNA function during lytic infection. We evaluated the production of viral miRNAs from HSV-1 infected cells from 2–24 hours post infection. We found 19 novel RNA species, 6 of which formed predicted stem-loop structures indicative of microRNAs. Using RT-PCR and stem-loop PCR we were able to determine the kinetic profile and mature 5' and 3' ends of the novel HSV-1 miRNA miR-92944. We identified the precursor structures using Northern blotting. Mutational studies revealed a 3–4 log decrease in viral titer in both single and multi-step growth analyses, as well as a significant reduction in plaque size. Through these studies we discovered a novel HSV-1 miRNA produced during latency that appears to control a global regulation pathway for HSV-1 replication.
|Advisor:||Burch, April D.|
|Commitee:||Masters, Paul, McDonough, Kathleen, Tenenbaum, Scott, Wade, Joseph|
|School:||State University of New York at Albany|
|School Location:||United States -- New York|
|Source:||DAI-B 73/04, Dissertation Abstracts International|
|Keywords:||Cellular proteasome, Hsv-1, MicroRNA, Mirna, Vice|
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