The conserved set of host proteins incorporated into HIV-1 virions suggests a common egress pathway in multiple cell types.

HIV-1 incorporates a large array of host proteins into virions. Determining the host protein composition in HIV virions has technical difficulties, including copurification of microvesicles. We developed an alternative purification technique using cholesterol that differentially modulates the density of virions and microvesicles (density modification, DM) allowing for high-yield virion purification that is essential for tandem mass spectrometric and quantitative proteomic (iTRAQ) analysis. DM purified virions were analyzed using iTRAQ and validated against Optiprep (60% iodixanol) purified virions. We were able to characterize host protein incorporation in DM-purified HIV particles derived from CD4+ T-cell lines; we compared this data set to a reprocessed data set of monocyte-derived macrophages (MDM) derived HIV-1 using the same bioinformatics pipeline. Seventy-nine clustered proteins were shared between the MDM derived and T-cell derived data set. These clusters included an extensive collection of actin isoforms, HLA proteins, chaperones, and a handful of other proteins, many of which have previously been documented to interact with viral proteins. Other proteins of note were ERM proteins, the dynamin domain containing protein EH4, a phosphodiesterase, and cyclophilin A. As these proteins are incorporated in virions produced in both cell types, we hypothesize that these proteins may have direct interactions with viral proteins or may be important in the viral life cycle. Additionally, identified common set proteins are predicted to interact with >1000 related human proteins. Many of these secondary interacting proteins are reported to be incorporated into virions, including ERM proteins and adhesion molecules. Thus, only a few direct interactions between host and viral proteins may dictate the host protein composition in virions. Ultimately, interaction and expression differences in host proteins between cell types may drive virion phenotypic diversity, despite conserved viral protein-host protein interactions between cell types.

SIV/macaque model of HIV infection in cocaine users: minimal effects of cocaine on behavior, virus replication, and CNS inflammation.

Studies of the effects of drugs of abuse on HIV immune status, disease progression, and neuroAIDS have produced conflicting data and have not definitively shown whether this combination promotes cognitive impairment or disease progression. Using a consistent SIV-macaque model, we investigated the effects of cocaine on behavior, virologic parameters, and CNS inflammation. Macaques received either vehicle or chronic administration of behaviorally active doses of cocaine (1.7 or 3.2 mg/kg/day). Chronic cocaine administration reduced CD8+ T cell counts during acute and late stage infection but had no effect on CD4+ T cell counts. Low-dose cocaine-treated animals had lower CSF vRNA levels late in infection, but cocaine did not alter plasma viral load or vRNA or protein in brain. There were no differences in CSF CCL-2 or interleukin (IL)-6 levels or severity of encephalitis in cocaine-treated as compared to vehicle-treated macaques. There were no differences in brain inflammation or neurodegeneration markers, as determined by interferon (IFN)-ß, MxA, CCL2, IL-6, TNFα, IFNγ, and indolamine 2,3-deoxygenase mRNA levels. APP levels also were not altered. The executive function of inhibitory control was not impaired in cocaine-treated or control animals following SIV infection. However, animals receiving 3.2 mg/kg/day cocaine performed more slowly in a bimanual motor test. Thus, chronic administration of cocaine produced only minor changes in behavior, encephalitis severity, CNS inflammation/neurodegeneration, and virus replication in SIV-infected pigtailed macaques, suggesting that cocaine would have only modest effects on the progression of neuroAIDS in HIV-infected individuals.

Sterol regulatory element-binding protein 2 couples HIV-1 transcription to cholesterol homeostasis and T cell activation.

Cholesterol plays an essential role in the life cycle of several enveloped viruses. Many of these viruses manipulate host cholesterol metabolism to facilitate their replication. HIV-1 infection of CD4(+) T cells activates the sterol regulatory element-binding protein 2 (SREBP2) transcriptional program, which includes genes involved in cholesterol homeostasis. However, the role of SREBP2-dependent transcription in HIV-1 biology has not been fully examined. Here, we identify TFII-I, a gene critical for HIV-1 transcription in activated T cells, as a novel SREBP2 target gene. We found TFII-I expression increased after HIV-1 infection or activation of human primary CD4(+) T cells. We show that inhibition of SREBP2 activity reduced TFII-I induction in response to these stimuli. More importantly, small interfering RNA (siRNA)-mediated gene silencing of either SREBP2 or TFII-I significantly reduced HIV-1 production in CD4(+) T cells. We also found that TFII-I potentiates Tat-dependent viral gene expression, consistent with a role at the level of HIV-1 transcription. Collectively, our results demonstrate for the first time that HIV-1 transcription in T cells is linked to cholesterol homeostasis through control of TFII-I expression by SREBP2.

The role of positively charged amino acids and electrostatic interactions in the complex of U1A protein and U1 hairpin II RNA.

Previous kinetic investigations of the N-terminal RNA recognition motif (RRM) domain of spliceosomal protein U1A, interacting with its RNA target U1 hairpin II, provided experimental evidence for a 'lure and lock' model of binding in which electrostatic interactions first guide the RNA to the protein, and close range interactions then lock the two molecules together. To further investigate the 'lure' step, here we examined the electrostatic roles of two sets of positively charged amino acids in U1A that do not make hydrogen bonds to the RNA: Lys20, Lys22 and Lys23 close to the RNA-binding site, and Arg7, Lys60 and Arg70, located on 'top' of the RRM domain, away from the RNA. Surface plasmon resonance-based kinetic studies, supplemented with salt dependence experiments and molecular dynamics simulation, indicate that Lys20 predominantly plays a role in association, while nearby residues Lys22 and Lys23 appear to be at least as important for complex stability. In contrast, kinetic analyses of residues away from the RNA indicate that they have a minimal effect on association and stability. Thus, well-positioned positively charged residues can be important for both initial complex formation and complex maintenance, illustrating the multiple roles of electrostatic interactions in protein-RNA complexes.