Stage-Dependent Inhibition of HIV-1 Replication by Antiretroviral Drugs in Cell Culture

Abstract

Upon the initiation of highly active antiretroviral therapy (HAART) for the treatment of HIV infection, the level of viral RNA in the blood rapidly decays in at least two distinct phases (12, 28), according to the death rates of cells that were already infected before treatment began. First-phase decay occurs during the first 7 to 10 days of treatment and has a half-life of 0.9 to 1.6 days (6,28,29), reflecting the death rate of productively infected CD4 ϩ T cells. The slower second phase occurs over a period of weeks and exhibits a half-life of approximately 14 days (28), corresponding to the turnover rate of long-lived infected cells. Second-phase sources of virus are believed to include cells of the monocyte/macrophage lineage and may also include infected resting CD4 ϩ T cells carrying unintegrated DNA (preintegration latent cells), follicular dendritic cells harboring intact virions, infected CD4 ϩ T cells that are not cleared by the host immune response, and an as-yet-unidentified cell type (4,12,17,28,30). An extremely slow third phase of decay is detectable by more sensitive assays and has a half-life estimated to range from months to years (7, 11). The viremia in this phase is believed to result from the reactivation of latently infected CD4 ϩ T cells (11, 40) or low-level ongoing replication (8, 14, 32), or both (35).Recent clinical trials with the first clinically approved HIV-1 integrase (IN) inhibitor, raltegravir (RAL), have yielded promising results. The phase II Merck protocol 004 part II trial (22, 23) and the phase III STARTMRK trial (20) compared the activity of RAL to that of the nonnucleoside reverse transcriptase inhibitor (NNRTI) efavirenz (EFV), each as part of standard combination therapy in drug-naïve HIV-1-infected individuals. While both drugs showed equal efficacy for the long-term suppression of the viral load, the limit of detection was reached more rapidly with RAL. This has been attributed to levels of virus production from second-phase sources that were 70% lower with RAL-based treatment than with EFV-based treatment (26).Several hypotheses may explain these unique viral load decay dynamics. They include differences in the time until drug bioavailability, differences in drug potency, a role for cells latently infected preintegration, the greater penetration of certain drugs into sanctuary sites, the stage of viral replication targeted, and the IN inhibitor-induced accumulation of unintegrated viral DNA (15,23,26,33,34). On the basis of the mathematical modeling of viral load decay, it has been proposed that the clinical observations can be explained by the effect of RAL on cells latently infected preintegration (26). These models also suggest that differences in drug potency might play a minor role but are inconsistent with a role for sanctuary sites (26). The mathematical models of viral load decay prepared by others suggest that the stage of viral replication targeted by RAL versus that targeted by EFV can explain the clinical trial results but are inconsistent with roles for ...

Timing of the Components of the HIV Life Cycle in Productively Infected CD4 + T Cells in a Population of HIV-Infected Individuals

“…After exposure to HIV, p24 appears in the cytoplasm of the infected cell after 1 h (38). Early stages of reverse transcription occur approximately 5 h after infection (4,18), and later transcripts appear 2 h afterwards. The addition of an NNRTI 10 h after infection sees an approximately 50% decrease in its ability to suppress productive infection (4), suggesting that by this time virus will be close to the last inhibitory point of an NNRTI.…”

“…Early stages of reverse transcription occur approximately 5 h after infection (4,18), and later transcripts appear 2 h afterwards. The addition of an NNRTI 10 h after infection sees an approximately 50% decrease in its ability to suppress productive infection (4), suggesting that by this time virus will be close to the last inhibitory point of an NNRTI. This timing is also consistent with HIV DNA peaking during single-round infections after approximately 9 h (2) and to the appearance between 9 and 12 h of cDNA species (18).…”

SUN2 Overexpression Deforms Nuclear Shape and Inhibits HIV

“…We next pretreated cells with nocodazole and infected them with single-round Env-deleted HIV. Nocodazole was maintained until 18 h p.i., at which time most viruses have entered the nucleus (as previously determined by qPCR-based studies of HIV infection kinetics in cell lines [53,68]). Long-term infections with replication-competent HIV in the presence of nocodazole were not possible, since prolonged treatment was toxic.…”

“…For integrated DNA, we used a nested Alu-gag PCR (52) that was modified as described previously (53), except that to avoid amplification of lentiviral vector sequences during the quantitative PCR (qPCR) step, we designed the following primers and probe that amplify a sequence near the 5= end of gag: 2nd-gag-F (5=-CAAGCAGCCATGCA AATGTTAA-3=), 2nd-gag-R (5=-GCACTGGATGCAATCTATCCCA-3=), and 2nd-gag-probe (5=-6-carboxy-4=,5=-dichloro-2=,7=-dimethoxyfluorescein (JOE)-AGACCATCAATGAGGAAGCTGCAGA-BHQ1-3=). Genomic DNA samples were normalized to their beta-globin content as described previously (53). All qPCRs were performed using Platinum qPCR supermix-UDG (Life Technologies) and amplified on an Eppendorf Mastercycler EP realplex 2 thermocycler.…”

Transcription of Preintegrated HIV-1 cDNA Modulates Cell Surface Expression of Major Histocompatibility Complex Class I via Nef

“…A previously described Alu-HIV PCR analysis was used (60), with the following modifications (12). The first-round reaction was performed on undiluted samples (100 ng template) and 1:10 dilutions of each sample (10 ng template diluted with uninfected DNA; 100 ng DNA total) in the presence of 2 mM MgCl 2 and 200 M deoxynucleoside triphosphates (dNTPs).…”