Date of Award

2013-01-01

Degree Name

Doctor of Philosophy

Department

Biological Sciences

Advisor(s)

German Rosas-Acosta

Abstract

Influenza A virus is a negative sense single stranded RNA virus responsible for multiple devastating pandemics in the past. This virus is capable of entering multiple eukaryotic hosts, sequestering, and using their cellular machinery for its own replicative purposes. This plethora of molecular events is all orchestrated through a shockingly limited repertoire of only 11 viral proteins. All of which are all strictly dependent on cellular processes such as post-transcriptional regulation, protein translation, post-translational modifications, etc. This study is focused on the characterization of the interplay between Influenza A virus and the cellular SUMOylation system. The first part of this dissertation deals with the effects imposed by Influenza A virus on the cellular SUMOylation system. We demonstrate that SUMO-conjugation is upregulated in the absence of any change in the levels of the enzymatic components of the SUMO-cascade during viral infection with multiple influenza A strains. This event is independent of toll-like receptor mediated- or IFN-β signaling cascades as the increase in SUMOylation cannot be recapitulated by U.V. inactivated influenza viral particles nor by the stimulation of cells with recombinant IFN-β. Furthermore, we also determined that a transcriptionally active virus is required to trigger this increase in SUMOylation. This finding is highly indicative that the factor responsible for this phenomenon is of viral origin. Also, we show that both the early infected and non-infected cell populations adjacent to infected cells experience an increase in nuclear accumulation of SUMOylated proteins. In contrast, cells at late stages of infection experience a decrease in overall SUMOylation. Finally, by using a mass spectrometry approach we were able to identify multiple cellular as well as two viral proteins, namely M1 and NS1, as SUMO targets during infection. Moreover, we provided suggestive evidence where SUMOylation seems to interfere with dimer and trimer formation of the NS1 viral protein.

The second part of this dissertation deals with the identification of the viral protein Polymerase Basic 1 (PB1) as a bona fide SUMO target and the characterization of the effects imposed by SUMO conjugation. Our results demonstrate that the overexpression of the SUMO deconjugating enzyme SENP1 decreases the transcriptase activity of the viral RNA-dependent RNA-polymerase during viral ribonucleoprotein reconstitution assays. This decrease in transcriptase activity is not mediated by changing the cellular localization of the viral polymerase. Also, by evaluating the accumulation of seven viral proteins during infection we concluded that overexpressing SUMO1 leads to an increase in viral protein production.

Language

en

Provenance

Received from ProQuest

File Size

116 pages

File Format

application/pdf

Rights Holder

Andres Santos

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