Identification of novel cellular components associated with early HIV-1 nucleoprotein complexes

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Authors
Schweitzer, Cameron J.
Issue Date
2012-04-05
Type
Dissertation
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en_US
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Abstract
The human immunodeficiency virus type 1 (HIV-1) is a member of the subfamily lentiviridae and the etiological agent of acquired immune deficiency syndrome (AIDS). Treatment of HIV-1 infection includes a multi-drug regimen that severely limits active replication but is unable to completely eradicate the virus. Current drugs target several viral proteins to inhibit critical functions; however HIV-1 multi-drug resistance continues to rise. Several genomic screens have highlighted the importance of numerous cellular proteins in HIV-1 replication. The viral nucleoprotein complexes (NPCs) formed during early HIV-1 infection represent potential targets for antiviral therapy. Despite extensive research the structure and composition of these structures remain poorly characterized. To better characterize these structures, two methods were developed to purify HIV-1 complexes for proteomic analysis and a potential co-factor was extensively studied. The first purification method used biotin tagging of two HIV-1 proteins, IN and MA. When the HIV-1 BAS-containing proteins IN and MA were expressed in conjunction with the biotin ligase BirA, each protein was efficiently and specifically biotinylated. The MA virus was fully competent for infectivity and replication both with and without biotin. However, the IN virus was approximately 50% less infectious without biotin and the addition of biotin rendered this virus noninfectious. The insertion and addition of biotin severely disrupted integration activity of this virus. HIV-1 IN and MA protein complexes were affinity purified at 4 hours post infection and candidate cellular proteins were identified by mass spectrometry. Several known HIV-1 interacting proteins were identified as well as previously undescribed proteins. The cellular protein leucine rich PPR-motif containing (LRPPRC) was further examined in HIV-1 infection. The next study aimed to determine the importance of LRPPRC in HIV-1 replication. During early HIV-1 infection LRPPRC was associated with both viral RNA and DNA. To ascertain whether LRPPRC expression was required for HIV-1 infection, endogenous protein levels were depleted in cells by short hairpin RNA interference. Both transient and stable depletion of LRPPRC significantly reduced HIV-1 infectivity. Furthermore, LRPPRC expression was required for nuclear import and preintegration complex stability, but not reverse transcription. Lastly, LRPPRC expression was not essential for viral release and vRNA incorporation. Combined, these data identified LRPPRC as a HIV-1 factor that is critical for HIV-1 replication through two different mechanisms. In the final study, functional HIV-1 RTCs and PICs were partially purified by velocity gradient centrifugation and fractionation, concentrated, trypsin digested, and analyzed by LC-MS/MS. A comparison of the HIV-1 samples to parallel uninfected control samples was used to identify associated cellular factors. A total of seven parallel infected and control biological replicates were completed, producing a data set of 8340 proteins. Several previously discovered HIV-1 factors were identified in our screen including GANAB, CD58, VPS37B, BTRC, and CD28. The database searches identified 99 proteins unique to the viral samples. Identification of two proteins by Western blot and immunoprecipitation confirmed the MS results. These data identify a number of additional candidate proteins that may regulate the early events of HIV-1 replication.
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Creighton University
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