Requirement for Integrase during Reverse Transcription of Human Immunodeficiency Virus Type 1 and the Effect of Cysteine Mutations of Integrase on Its Interactions with Reverse Transcriptase

Retroviral integrase catalyzes the essential step of integrating a double-stranded DNA copy of the viral genome into a host cell chromosome. Mutational studies have revealed that integrase is involved in additional steps of viral replication, but the mechanism for the pleiotropic effect is not well characterized. Since Cys residues generally play crucial roles in protein structure and function, we introduced Cys-to-Ser substitutions at positions 56, 65, and 130 of human immunodeficiency virus type 1 (HIV-1) integrase to determine their effects on integration activity and viral replication. None of the substitutions significantly affected the enzymatic activities in vitro. When introduced into the NL4-3 molecular clone of HIV-1, mutant viruses encoding Cys mutations at positions 56 and 65 of integrase replicated similarly to the wild-type virus in CD4(+)-T-cell lines, whereas the C130S-containing virus was noninfectious. The entry and postintegration steps of the viral life cycle for all mutant viruses were normal, and all had particle-associated reverse transcriptase (RT) activity. However, early reverse-transcribed DNA products were absent in the lysate of cells infected with the C130S mutant virus, indicating that the mutation abolished the ability of the virus to initiate endogenous reverse transcription. Coimmunoprecipitation using purified integrase and RT showed that the C-terminal domain of wild-type HIV-1 integrase interacted with RT. The interaction between integrase and RT was not affected in the presence of a reducing or alkylating agent, suggesting that the interaction did not involve a disulfide linkage. The C130S substitution within the core region may disrupt the protein recognition interface of the C-terminal domain and abolish its ability to interact with RT. Our results indicate that integrase plays an important role during the reverse-transcription step of the viral life cycle, possibly through physical interactions with RT

Development of a repeat-exposure penile SHIV infection model in macaques to evaluate biomedical preventions against HIV.

Penile acquisition of HIV infection contributes substantially to the global epidemic. Our goal was to establish a preclinical macaque model of penile HIV infection for evaluating the efficacy of new HIV prevention modalities. Rhesus macaques were challenged once or twice weekly with consistent doses of SHIVsf162P3 (a chimeric simian-human immunodeficiency virus containing HIV env) ranging from 4-600 TCID50 (50% tissue culture infective dose), via two penile routes, until systemic SHIV infection was confirmed. One route exposed the inner foreskin, glans and urethral os to virus following deposition into the prepuce (foreskin) pouch. The second route introduced the virus non-traumatically into the distal urethra only. Single-route challenges resulted in dose-dependent rates of SHIV acquisition informing selection of optimal SHIV dosing. Concurrent SHIV challenges via the prepuce pouch (200 TCID50) and urethra (16 TCID50) resulted in infection of 100% (10/10) animals following a median of 2.5 virus exposures (range, 1-12). We describe the first rhesus macaque repeat-exposure SHIV challenge model of penile HIV acquisition. Utilization of the model should further our understanding of penile HIV infection and facilitate the development of new HIV prevention strategies for men

Infection of rhesus macaques following repeated SHIV challenges via the urethra.

<p>Beginning at week 0, rhesus macaques were challenged once weekly with 4 TCID₅₀ (A, B) or 16 TCID₅₀ (C, D) SHIV via the urethra using the ‘no contact’ technique, as described, until systemically infected (detectable plasma viremia) or until having received 12 challenges maximum. Viral RNA copies per ml plasma are shown as a function of study week (A, C). Percentages of animals remaining uninfected at seven days following administration of the indicated challenge number are shown (B, D). Dotted lines indicate the limit of quantitation of viral load assay (200 copies/ml). Identification codes represent individual animals. Arrowheads denote SHIV challenges.</p

Infection of rhesus macaques following repeated SHIV challenges via the prepuce pouch.

<p>Beginning at week 0, rhesus macaques were challenged twice weekly with 200 TCID₅₀ (A, B) or 600 TCID₅₀ (C, D) SHIV via the prepuce pouch with until systemically infected, as evidenced by detectable plasma viremia via RT-qPCR viral load assay. Viral RNA copies per ml plasma are shown as a function of study week (A, C). Percentages of animals remaining uninfected at seven days following administration of the indicated challenge number are shown (B, D). Dotted lines indicate the limit of quantitation of viral load assay (200 copies/ml). Identification codes represent individual animals. Arrowheads denote SHIV challenges.</p

Infection of rhesus macaques following repeated SHIV challenges via combined prepuce pouch and urethral routes.

<p>Ten rhesus macaques were challenged once weekly with 200 TCID₅₀ SHIV into the prepuce pouch and 16 TCID₅₀ SHIV into the urethra using the ‘no-contact’ method, as described, and monitored for the development of positive SHIV viremia by RT-qPCR viral load assay. Viral RNA copies per ml plasma (A) and number of SHIV challenges required to establish systemic infection (B) are presented as in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194837#pone.0194837.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194837#pone.0194837.g002" target="_blank">2</a>.</p

Complete Protection from Repeated Vaginal Simian-Human Immunodeficiency Virus Exposures in Macaques by a Topical Gel Containing Tenofovir Alone or with Emtricitabine▿

New-generation gels that deliver potent antiretroviral drugs against human immunodeficiency virus type 1 have renewed hopes for topical prophylaxis as a prevention strategy. Previous preclinical research with monkey models suggested that high concentrations and drug combinations are needed for high efficacy. We evaluated two long-acting reverse transcriptase inhibitors, tenofovir (TFV) and emtricitabine (FTC), by using a twice-weekly repeat challenge macaque model and showed that a preexposure vaginal application of gel with 1% TFV alone or in combination with 5% FTC fully protected macaques from a total of 20 exposures to simian-human immunodeficiency virus SF162p3. FTC and TFV were detected in plasma 30 min after vaginal application, suggesting rapid absorption. FTC was detected more frequently than TFV and showed higher levels, reflecting the fivefold-higher concentration of this drug than of TFV. Two of 12 repeatedly exposed but protected macaques showed limited T-cell priming, which did not induce resistance to infection when macaques were rechallenged. Thus, single drugs with durable antiviral activity can provide highly effective topical prophylaxis and overcome the need for noncoital use or for drug combinations which are more complex and costly to formulate and approve