Previous studies have shown that in addition to its function in specific RNA encapsidation, the human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) is required for efficient virus particle assembly. However, the mechanism by which NC facilitates the assembly process is not clearly established. Formally, NC could act by constraining the Pr55 ^gag polyprotein into an assembly-competent conformation or by masking residues which block the assembly process. Alternatively, the capacity of NC to bind RNA or make interprotein contacts might affect particle assembly. To examine its role in the assembly process, we replaced the NC domain in Pr55 ^gag with polypeptide domains of known function, and the chimeric proteins were analyzed for their abilities to direct the release of virus-like particles. Our results indicate that NC does not mask inhibitory domains and does not act passively, by simply providing a stable folded monomeric structure. However, replacement of NC by polypeptides which form interprotein contacts permitted efficient virus particle assembly and release, even when RNA was not detected in the particles. These results suggest that formation of interprotein contacts by NC is essential to the normal HIV-1 assembly process.

The human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein targets HIV-1 precursor Gag (PrGag) proteins to assembly sites at plasma membrane (PM) sites that are enriched in cholesterol and phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P ₂ ]. MA is myristoylated, which enhances membrane binding, and specifically binds PI(4,5)P ₂ through headgroup and 2′ acyl chain contacts. MA also binds nucleic acids, although the significance of this association with regard to the viral life cycle is unclear. We have devised a novel MA binding assay and used it to examine MA interactions with membranes and nucleic acids. Our results indicate that cholesterol increases the selectivity of MA for PI(4,5)P ₂ -containing membranes, that PI(4,5)P ₂ binding tolerates 2′ acyl chain variation, and that the MA myristate enhances membrane binding efficiency but not selectivity. We also observed that soluble PI(4,5)P ₂ analogues do not compete effectively with PI(4,5)P ₂ -containing liposomes for MA binding but surprisingly do increase nonspecific binding to liposomes. Finally, we have demonstrated that PI(4,5)P ₂ -containing liposomes successfully outcompete nucleic acids for MA binding, whereas other liposomes do not. These results support a model in which RNA binding protects MA from associating with inappropriate cellular membranes prior to PrGag delivery to PM assembly sites.

Background In 1999 North Carolina initiated STI screening programs in seven county jails as part of the CDC Syphilis Elimination Effort (SEE). All jails were willing to permit screening programs for syphilis but only two also screened for HIV. In 2007 the SEE funding was depleted and the 7 SEE jail projects were converted to HIV screening under the CDC Expanded Testing Initiative (ETI). New jails were added (n=21) and several have added screening for other STIs. Testing is supported by 10 local health departments and 5 non-traditional testing sites which assure treatment for STDs and make referrals for HIV care services. Methods From January to June 2010, STI screening data for 28 jails was analysed. All jails tested for HIV and syphilis, one jail added HCV screening and another jail added NAAT testing for gonorrhoea and chlamydial infection. Reactive syphilis tests and positive HIV tests were verified against surveillance records to determine whether or not they were previous cases or newly diagnosed infections. Positive tests for Neisseria gonorrhoeae and Chlamydia trachomatis were considered to be new infections. Positive tests for HCV are considered prevalent infections. All data were analysed using SAS 9.1.3. Results Under SEE, only two jails screened for both HIV and syphilis. From 2002 to 2005, 3626 inmates were screened. There were 46 (1.3%) HIV-positive inmates and 158 (4.4%) with reactive syphilis tests. Of those 158, 30 were confirmed new syphilis cases (0.83% case rate). Under ETI, 28 jails screened 4688 inmates for syphilis and 4662 for HIV in just 6 months (January–June, 2010, see Abstract P1-S6.28 table 1). HIV positivity rates were similar for men and women (p=0.90) but women were more likely than men to be both a syphilis reactor (OR 4.8 95% CI 2.8 to 8.2) and a new case (OR 5.9 95% CI 1.9 to 17.9). Among all positive tests, there were no comorbid patients. Conclusions The jail setting proves to be an important setting for detecting new syphilis cases, particularly among women, and for identifying HIV-positive inmates. Many of these HIV positives were not new diagnoses but may represent persons who need referrals for care and treatment. Screening for HCV and combined Ct/Gc screening was also productive and should be expanded to other jails. Due to the high concentration of persons who trade sex for drugs or money in jail settings, it is especially important that these HIV and STI cases be identified and referred for treatment and partner services to prevent ongoing transmission.

We have examined the interactions of wild-type (WT) and matrix protein-deleted (ΔMA) HIV-1 precursor Gag (PrGag) proteins in virus-producing cells using a biotin ligase-tagging approach. To do so, WT and ΔMA PrGag proteins were tagged with the Escherichia coli promiscuous biotin ligase (BirA*), expressed in cells, and examined. Localization patterns of PrGag proteins and biotinylated proteins overlapped, consistent with observations that BirA*-tagged proteins biotinylate neighbor proteins that are in close proximity. Results indicate that BirA*-tagged PrGag proteins biotinylated themselves as well as WT PrGag proteins in trans . Previous data have shown that the HIV-1 Envelope (Env) protein requires an interaction with MA for assembly into virions. Unexpectedly, ΔMA proteins biotinylated Env, whereas WT BirA*-tagged proteins did not, suggesting that the presence of MA made Env inaccessible to biotinylation. We also identified over 50 cellular proteins that were biotinylated by BirA*-tagged PrGag proteins. These included membrane proteins, cytoskeleton-associated proteins, nuclear transport factors, lipid metabolism regulators, translation factors, and RNA-processing proteins. The identification of these biotinylated proteins offers new insights into HIV-1 Gag protein trafficking and activities and provides new potential targets for antiviral interference. IMPORTANCE We have employed a novel strategy to analyze the interactions of the HIV-1 structural Gag proteins, which involved tagging wild-type and mutant Gag proteins with a biotin ligase. Expression of the tagged proteins in cells allowed us to analyze proteins that came in close proximity to the Gag proteins as they were synthesized, transported, assembled, and released from cells. The tagged proteins biotinylated proteins encoded by the HIV-1 pol gene and neighbor Gag proteins, but, surprisingly, only the mutant Gag protein biotinylated the HIV-1 Envelope protein. We also identified over 50 cellular proteins that were biotinylated, including membrane and cytoskeletal proteins and proteins involved in lipid metabolism, nuclear import, translation, and RNA processing. Our results offer new insights into HIV-1 Gag protein trafficking and activities and provide new potential targets for antiviral interference.