To reliably infect a primate model for human immunodeficiency virus (HIV), ϳ10,000-fold more virus must be delivered vaginally than intravenously. However, the vaginal mechanisms that help protect against HIV are poorly understood. Here, we report that human cervicovaginal mucus (CVM), obtained from donors with normal lactobacillus-dominated vaginal flora, efficiently traps HIV, causing it to diffuse more than 1,000-fold more slowly than it does in water. Lactobacilli acidify CVM to pH ϳ4 by continuously producing lactic acid. At this acidic pH, we found that lactic acid, but not HCl, abolished the negative surface charge on HIV without lysing the virus membrane. In contrast, in CVM neutralized to pH 6 to 7, as occurs when semen temporarily neutralizes the vagina, HIV maintained its native surface charge and diffused only 15-fold more slowly than it would in water. Thus, methods that can maintain both a high lactic acid content and acidity for CVM during coitus may contribute to both vaginal and penile protection by trapping HIV before it can reach target cells. Our results reveal that CVM likely plays an important but currently unappreciated role in decreasing the rate of HIV sexual transmission.Cervicovaginal mucus (CVM) and semen from human immunodeficiency virus (HIV)-infected individuals contain cellfree and cell-associated HIV (8,25,26). Both forms of virions are plausible mediators of infection, and both, to be infectious, must penetrate the mucus barrier that coats and adheres to vaginal and penile epithelia during coitus. To the extent that mucus can limit the amount of virus that contacts the epithelium, the mucus layer can reduce the probability of infection. Leukocytes can migrate through neutral mucus (24) but are rapidly immobilized and then killed by mild acidity (pH Յ 6) (22); leukocytes do not survive in the acidic vagina. However, prior research has not revealed whether cell-free HIV can penetrate human CVM.To directly determine whether cell-free HIV can diffuse through CVM, we used a HIV virus-like particle (VLP) that was fluorescently labeled internally by incorporation of a green fluorescent protein (GFP)-Vpr fusion (5). For biosafety considerations, the derivative was replication defective and pseudotyped with X4-tropic HIV envelope. We mixed the labeled HIV at minimal dilution (ϳ3% [vol/vol]) into fresh, undiluted CVM obtained from donors with normal lactobacillus-dominated vaginal flora and observed the translational movements of hundreds of individual HIV virions in each sample using high-resolution multiple-particle tracking (15,29). CVM from women with healthy vaginal microflora is acidified to pH ϳ4 by lactic acid produced continuously by anaerobic metabolism of the lactobacilli (4, 21). However, during coitus, vaginal secretions are temporarily neutralized by the alkaline pH of semen (9, 30). In addition, women with bacterial vaginosis (BV), a condition that leads to a more neutral vaginal pH of ϳ5 to 6 (4), are at significantly increased risk of acquiring HIV infection (1, 28). Ther...
Nonnucleoside reverse transcriptase inhibitors (NNRTIs) target HIV-1 reverse transcriptase (RT) by binding to a pocket in RT that is close to, but distinct, from the DNA polymerase active site and prevent the synthesis of viral cDNA. NNRTIs, in particular, those that are potent inhibitors of RT polymerase activity, can also act as chemical enhancers of the enzyme's inter-subunit interactions. However, the consequences of this chemical enhancement effect on HIV-1 replication are not understood. Here, we show that the potent NNRTIs efavirenz, TMC120, and TMC125, but not nevirapine or delavirdine, inhibit the late stages of HIV-1 replication. These potent NNRTIs enhanced the intracellular processing of Gag and Gag-Pol polyproteins, and this was associated with a decrease in viral particle production from HIV-1-transfected cells. The increased polyprotein processing is consistent with premature activation of the HIV-1 protease by NNRTI-enhanced Gag-Pol multimerization through the embedded RT sequence. These findings support the view that Gag-Pol multimerization is an important step in viral assembly and demonstrate that regulation of Gag-Pol/Gag-Pol interactions is a novel target for small molecule inhibitors of HIV-1 production. Furthermore, these drugs can serve as useful probes to further understand processes involved in HIV-1 particle assembly and maturation.
Anti-N-methyl-d-aspartate receptor (anti-NMDAR) encephalitis usually presents with behavioral manifestations and subsequently emerge by an acute or subacute movement disorders. Although in adults the movement disorders seem to appear after a prodromal period with psychiatric features, in children diagnosed with anti-NMDAR encephalitis they may be the initial manifestation. We report a case of a 7 years old male with history of choreiform movements and two isolated seizures episodes.
Previous studies have demonstrated that nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) act as chemical enhancers of human immunodeficiency virus type 1 (HIV-1) RT dimerization. In the current study, we sought to define the role of key residues (101, 103, 108, 181, 188, 190, 225 and 318) in the NNRTI-binding pocket on HIV-1 RT heterodimer stability. Thirteen mutant RTs were constructed and evaluated for p66/p51 RT heterodimer formation using the well-established yeast two-hybrid assay. We found that the mutations K101A, P225H, Y318F and Y318W decreased RT heterodimer stability whereas K103N, V108I, V108W, Y181C, Y188L, G190A, G190E, G190W and P225W increased RT heterodimer stability. While these results demonstrate that residues that comprise the NNRTI-binding pocket contribute to the stability of p66/p51 HIV-1 RT, they did not suggest any obvious correlation between RT dimer stability and the extent of NNRTI resistance. Remarkably, mutations at residue G190 (A, E, W) in the p66 RT subunit were found to dramatically increase heterodimer stability. Notably, the G190W mutation increased RT dimer stability almost to the same extent as did 5 microM efavirenz. In light of these findings, we characterized the in vitro activity of recombinant RT expressing mutations at G190 in the p66 subunit only and compared them with a wild-type enzyme complexed with efavirenz. We found that while mutations at G190 had a significant effect on both the DNA polymerase and ribonuclease H activity of the enzyme, their phenotypic effects did not mirror those induced by efavirenz-binding to RT.
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