Session 36Poster Presentations Accessory Genes Session Day and Time: Tuesday 1:30 - 3:30 pm Room: Hall D
208 Nef Expressed from Integrase Defective HIV-1 Down-regulates CD4 Expression L. Gillim*, M. Klotman Mt Sinai Sch of Med, New York, NY
Background: We, as well as other groups, previously demonstrated that HIV-1 extrachromosomal 2-LTR circular DNA is long-lived in primary non-dividing cells. HIV-1 extrachromosomal DNA (E-DNA) has been shown to be transcriptionally active although at much lower levels than integrated proviral DNA. With the advent of integrase inhibitors that shunt towards the formation of E-DNA in the absence of integration, a better understanding of E-DNA transcriptional activity, as well as the functional activity of the expressed proteins, is essential.
Methods: To better understand the relevance of HIV-1 E-DNA, we studied both the transcriptional activity of E-DNA as well as the functional activity of proteins expressed from E-DNA in primary macrophages, primary CD4 T-cells, and CEM cells. The pattern of transcription of viral messages from E-DNA was determined by RT-PCR analysis of RNA for specific splice-site utilization. To assess the activity of proteins expressed from E-DNA, we quantitated the ability of Nef expressed from IN defective virus to down-regulate CD4 expression. Surface CD4 expression was determined by flow cytometry of cells infected with integrase (IN) defective or IN defective/Nef deleted recombinant HIV-1.
Results: The pattern of expression of multiply-spliced RNA (Tat, Rev, Nef) from E-DNA was identical to that from integrated HIV-1 DNA. Nef was expressed from IN defective recombinant HIV-1 at levels sufficient to down-regulate CD4 expression to a greater extent than down-regulation of CD4 in cells infected with IN-defective/Nef-deleted recombinant virus.
Conclusions: Extrachromosomal HIV-1 DNA generates the full spectrum of spliced mRNAs. Nef expressed from IN-defective, recombinant HIV-1 is sufficient to down-regulate CD4 expression. HIV-1 E-DNA which has been demonstrated to be long-lived in non-dividing cells, may serve as a reservoir of viral proteins during HIV-1 infection. IN inhibitors that block integration, and subsequently viral replication, do not prevent the formation of E-DNA. E-DNA generated in the presence of IN inhibitors may produce functional viral protein in the absence of integration.
