Investigating the Roles of NEDD4.2s and Nef in the Release and Replication of HIV-1: A Dissertation

Abstract

Replication of HIV-1 requires the assembly and release of mature and infectious viral particles. In order to accomplish this goal, HIV-1 has evolved multiple methods to interact with the host cell. HIV-1 recruits the host cell ESCRT machinery to facilitate the release of nascent viral particles from the host cell membrane. Recruitment of these cellular factors is dependent on the presence of short motifs in Gag referred to as Late-domains. Deletion or mutation of these domains results in substantial decrease in the release of infectious virions. However, previously published work has indicated that over-expression of the E3 ubiquitin ligase, NEDD4.2s is able to robustly rescue release of otherwise budding-defective HIV-1 particles. This rescue is specific to the NEDD4.2s isoform as related E3 ubiquitin ligases display no ability to rescue particle release. In addition, rescue of particle release is dependent on the presence of the partial C2 domain and a catalytically active HECT domain of NEDD4.2s. Here I provide evidence supporting the hypothesis that a partial C2 domain of NEDD4.2s constitutes a Gag interacting module capable of targeting the HECT domains of other E3 ubiquitin ligases to HIV-1 Gag. Also, by generating chimeras between HECT domains shown to form poly-ubiquitin chains linked through either K48 or K63 of ubiquitin, I demonstrate that the ability of NEDD4.2s to catalyze the formation of K63-polyubiquitin chains is required for its stimulation of HIV-1 L-domain mutant particle release. In addition, I present findings from on-going research into the role of the HIV-1 accessory protein Nef during viral replication using the culture T-cell line, MOLT3. My current findings indicate that downregulation of CD4 from the host cell membrane does not solely account for the dramatic dependence of HIV-1 replication on Nef expression in this system. In addition, I present evidence indicating that Nef proteins from diverse HIV-1 Groups and strains are capable of enhancing HIV-1 replication in this system. Analysis of a range of mutations in Nef known to impact interaction with cellular proteins suggest that the observed replication enhancement requires Nef targeting to the host cell membrane and may also require the ability to interact with select Src-kinases. Lastly, we find that the ability of Nef to enhance replication in this system is separate from any increase in viral particle infectivity, in agreement with current literature.