Although the frequency of neurological and neurobehavioral dysfunction in HIV-1-infected individuals has been reduced by HAART, nonetheless, the CNS may represent an important reservoir or sanctuary site for HIV-1 during virally-suppressive therapy.
In Specific Aim I, unique molecular mechanism(s) of HIV-1 persistence in astrocytes will be examined. We have recently demonstrated that HIV-1 Rev binds to, and functionally interacts with, a DEAD-box helicase, DDX1. This moiety binds to a specific motif in Rev (entitled NTS), which is involved with proper subcellular localization of Rev (i.e., nuclear greater than cytoplasmic). Recent data suggest that Rev is hyper-localized in primary astrocytes within the cytoplasm. Thus, we hypothesize that the DDX1 cellular co-factor may be involved with low-level residual HIV-1 replication in astrocytes. Presence and quantitation of DDX1 in human astrocytes, as well as potential co-localization with Rev, will be explored. Direct interactions between Rev plus selected Rev mutants and DDX1 will be assessed with a mammalian two-hybrid system. As well, over-expression of DDX1 and anti-sense and RNAi inhibition of DDX1 mRNA expression in astrocytes will be used to evaluate this potentially unique CNS-cell specific mechanism inducing HIV-1 persistence in human astrocytes.). A translation component will utilize single cell microdissection technology to directly probe the Rev:DDXl axis in vivo in the brains of diverse HIV-1-infected individuals.
In Specific Aim II, we will evaluate, in a complementary fashion with Specific Aim I, the potential additional and/or alternative mechanisms for alterations of Rev subcellular compartmentalization in low-level viral production in astrocytes. This will include analysis of potential splice variants of DDX1, and a hypothesized cellular inhibitor of DDX1 binding to NTS which is astrocyte-specific. As well, identification and purification of astrocyte-specific NIS binding cellular proteins which may alter subcellular compartmentalization will be analyzed by affinity chromatography and/or phage-peptide display libraries. Thus, these complementary approaches will be utilized to analyze, on molecular levels, the mechanisms of HIV-1 reservoirs and persistence in human astrocytes. ? ?
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