In the past three decades, various steps of the human immunodeficiency virus type 1 (HIV-1) life cycle have been thoroughly studied. Many of these steps, such as viral entry, reverse transcription and proteolysis have been targets of antiretroviral therapy. Retroviral genomic RNA (gRNA) dimerization appears essential for viral infectivity and this process appears to be chaperoned by the nucleocapsid (NC) protein of HIV-1. In this dissertation, the role of NC in genome dimerization and other aspects of the viral life cycle have been thoroughly studied. Various positions of the NC protein have been mutated through site-directed mutagenesis and relevant and dispensable positions of NC have been identified through this method. 34 of its 55 residues were mutated, individually or in small groups, in a panel of 40 HIV-1 mutants. It was found that the amino-terminus, the proximal zinc finger, the linker, and the distal zinc finger of NC each contributed roughly equally to efficient HIV-1 gRNA dimerization. The various mutations introduced into NC show the first evidence that gRNA dimerization can be inhibited by: 1) mutations in the N-terminus or the linker of retroviral NC; 2) mutations in the proximal or distal zinc finger of lentiviral NC; 3) mutations in the hydrophobic patch (plateau) or the conserved glycines of the proximal or the distal retroviral zinc finger. Some NC mutations impaired gRNA dimerization more than mutations inactivating the viral protease, indicating that gRNA dimerization may be stimulated by the NC component of the Gag polyprotein (Pr55gag). In the second section of my work, I studied the effect of Pr55gag processing on gRNA dimerization by introducing rate alternating mutants into Pr55gag protein cleavage sites. I showed that Maturation ofNCp15 into NCp9 is essential for fast rates of genomic RNA dimerization and maturation of NCp9 into NCp7 has no incidence on genomic RNA dimerization but is essential for viral replication. In order to delineate the amount of viral protease activity needed to produce mature virus 48 hours post transfection, we also studied, by cotransfection studies, the effect of various ratios of wild-type (BH10) and protease-inactive (PR- ) plasmids and found that HIV-1 reaches its full genomic RNA dimerization despite 75% unprocessed Pr55gag polyproteins. We have also shown that wild type BH10 plasmid can rescue those mutations in NCp7 protein that have an effect on gRNA dimerization through rescue experiments. Overall, this thesis sheds light on the role of NC in HIV-1 genome dimerization and other aspects of the viral life cycle and identifies the importance of each component of NC during these processes.

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Human immunodeficiency virus (HIV) is the causing agent of the acquired immune deficiency syndrome (AIDS). Like all retroviruses, HIV carries two copies of viral genomic RNA in each virion. HIV genome encodes three structural genes, including gag, pol and env, as well as two regulatory genes (rev and tat) and four accessory genes (vif, vpr, vpu and nef). It is noted that none of these nine viral proteins bears the helicase activity. Helicases are able to unwind RNA duplex and remodel the structure of RNA-protein (RNP) complexes using energy derived from hydrolysis of nucleotide triphosphates (NTPs). They are involved in every step of cellular RNA metabolisms. It is conceivable that HIV needs to exploit cellular RNA helicases to promote the replication of its RNA at various steps such as transcription, folding and transport. / In this study, we found that a DEAD-box protein named DDX24 associates with HIV-1 Gag in an RNA-dependent manner but is not found within virus particles. Knockdown of DDX24 inhibits the packaging of HIV-1 RNA and thus diminishes viral infectivity. The decreased viral RNA packaging as a result of DDX24-knockdown is observed only in the context of the Rev/RRE (Rev response element)-dependent but not the CTE (constitutive transport element)-mediated nuclear export of viral RNA, which is explained by the specific interaction of DDX24 with the Rev protein. We propose that DDX24 acts at the early phase of HIV-1 RNA metabolism prior to nuclear export and the consequence of this action extends to the viral RNA packaging stage during virus assembly.

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