DDX1 is an RNA-dependent ATPase involved in HIV-1 Rev function and virus replication.

The human immunodeficiency virus type 1 (HIV-1) Rev protein is essential for the virus because it promotes nuclear export of alternatively processed mRNAs, and Rev is also linked to translation of viral mRNAs and genome encapsidation. Previously, the human DEAD-box helicase DDX1 was suggested to be involved in Rev functions, but this relationship is not well understood. Biochemical studies of DDX1 and its interactions with Rev and model RNA oligonucleotides were carried out to investigate the molecular basis for association of these components. A combination of gel-filtration chromatography and circular dichroism spectroscopy demonstrated that recombinant DDX1 expressed in Escherichia coli is a well-behaved folded protein. Binding assays using fluorescently labeled Rev and cell-based immunoprecipitation analysis confirmed a specific RNA-independent DDX1-Rev interaction. Additionally, DDX1 was shown to be an RNA-activated ATPase, wherein Rev-bound RNA was equally effective at stimulating ATPase activity as protein-free RNA. Gel mobility shift assays further demonstrated that DDX1 forms complexes with Rev-bound RNA. RNA silencing of DDX1 provided strong evidence that DDX1 is required for both Rev activity and HIV production from infected cells. Collectively, these studies demonstrate a clear link between DDX1 and HIV-1 Rev in cell-based assays of HIV-1 production and provide the first demonstration that recombinant DDX1 binds Rev and RNA and has RNA-dependent catalytic activity.

Proteomic analysis of human immunodeficiency virus using liquid chromatography/tandem mass spectrometry effectively distinguishes specific incorporated host proteins.

A major challenge to studying virus-incorporated host proteins is the fact that they are not encoded by the viral genome. We used Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS) on whole virions to obtain a snapshot of the HIV-1 proteome. We identified known viral and host-cellular proteins and also identified novel components of HIV-1 and confirm these by traditional biochemical methods. Our comparison of wild-type and mutant viruses demonstrates that LC-MS/MS has the specificity to distinguish the presence/absence of a single host protein in intact virions.

Syndecan captures, protects, and transmits HIV to T lymphocytes.

This study demonstrates that syndecan functions as an in trans HIV receptor. We show that syndecan, when expressed in nonpermissive cells, becomes the major mediator for HIV adsorption. This adsorption is mediated by the binding of gp120 to the heparan sulfate chains of syndecan. Although syndecan does not substitute for HIV entry receptors, it enhances the in trans infectivity of a broad range of primate lentiviruses including primary viruses produced from PBMCs. Furthermore, syndecan preserves virus infectivity for a week, whereas unbound virus loses its infectivity in less than a day. Moreover, we obtain evidence suggesting that the vast syndecan-rich endothelial lining of the vasculature can provide a microenvironment which boosts HIV replication in T cells.

Cyclophilin a plays distinct roles in human immunodeficiency virus type 1 entry and postentry events, as revealed by spinoculation.

Cyclophilin A (CypA) is necessary for effective human immunodeficiency virus type 1 (HIV-1) replication. However, the functions of CypA and the precise steps at which CypA acts in the HIV-1 life cycle remain to be determined. By using a methodology that bypasses the need for attachment factors-spinoculation-we present evidence that CypA participates in both entry and postentry events.

trans-Complementation rescue of cyclophilin A-deficient viruses reveals that the requirement for cyclophilin A in human immunodeficiency virus type 1 replication is independent of its isomerase activity.

Human immunodeficiency virus type 1 (HIV-1) requires the incorporation of cyclophilin A (CypA) for replication. CypA is packaged by binding to the capsid (CA) region of Gag. This interaction is disrupted by cyclosporine (CsA). Preventing CypA incorporation, either by mutations in the binding region of CA or by the presence of CsA, abrogates virus infectivity. Given that CypA possesses an isomerase activity, it has been proposed that CypA acts as an uncoating factor by destabilizing the shell of CA that surrounds the viral genome. However, because the same domain of CypA is responsible for both its isomerase activity and its capacity to be packaged, it has been challenging to determine if isomerase activity is required for HIV-1 replication. To address this issue, we fused CypA to viral protein R (Vpr), creating a Vpr-CypA chimera. Because Vpr is packaged via the p6 region of Gag, this approach bypasses the interaction with CA and allows CypA incorporation even in the presence of CsA. Using this system, we found that Vpr-CypA rescues the infectivity of viruses lacking CypA, either produced in the presence of CsA or mutated in the CypA packaging signal of CA. Furthermore, a Vpr-CypA mutant which has no isomerase activity and no capacity to bind to CA also rescues HIV-1 replication. Thus, this study demonstrates that the isomerase activity of CypA is not required for HIV-1 replication and suggests that the interaction of the catalytic site of CypA with CA serves no other function than to incorporate CypA into viruses.