Molecular Characterization of Preintegration Latency in Human Immunodeficiency Virus Type 1 Infection

Abstract

Most current evidence suggests that the infection of resting CD4؉ T cells by human immunodeficiency virus type 1 (HIV-1) is not productive due to partial or complete blocks in the viral life cycle at steps prior to integration of the viral genome into the host cell chromosome. However, stimulation of an infected resting T cell by antigen, cytokines, or microenvironmental factors can overcome these blocks and allow for the production of progeny virions. In this study, we sought to understand the structure and fate of the virus in unstimulated resting CD4 ؉ T cells. Using a novel linker-mediated PCR assay designed to detect and characterize linear unintegrated forms of the HIV-1 genome, we demonstrate that reverse transcription can proceed to completion following the infection of resting T cells, generating the substrate for the retroviral integration reaction. However, reverse transcription in resting T cells is far slower than in activated T cells, requiring 2 to 3 days to complete. The delay in completing reverse transcription may make the viral DNA genome more susceptible to competing decay processes. To explore the relationship between the formation of the linear viral genome and the stability of the preintegration state, we employed a recombinant HIV-1 virus expressing the enhanced green fluorescent protein to measure the rate at which HIV-1 decays in the preintegration state. Our results demonstrate that the preintegration state is labile and decays rapidly (half-life ‫؍‬ 1 day) following the entry of HIV-1 into a resting T cell, with significant decay occurring during the slow process of reverse transcription.

Characterization of Restrictions to Human Immunodeficiency Virus Type 1 Infection of Monocytes

“…However, although exogenous stimulation of dNTP levels enhanced reverse transcription and generation of full-length transcripts, this did not overcome the block to infection. These and other studies (22,26) support the notion that a block subsequent to reverse transcription prevents productive infection of quiescent lymphocytes. Upon infection of monocytes, reverse transcription was highly inefficient when analyzed up to 66 h postinfection and was not improved by normalization of dNTP levels (Fig.…”

Effects of cell cycle status on early events in retroviral replication

“…The majority of CD4þ T-cells are non-cycling and are resting in the G 0 phase. This phase can restrict early steps in HIV-1 replication including reverse transcription, nuclear import, and integration [Stevenson et al, 1990;Zack et al, 1990;Bukrinsky et al, 1991;Pierson et al, 2002;Swiggard et al, 2004]. However, intact unintegrated DNA may be a significant ''latent'' form of HIV in the resting T-cell [Bukrinsky et al, 1991].…”

“…It is possible that the ability to infect a cell outside of S-phase depends on the availability of threshold levels of dNTP precursors or other factors in such cells, and different retroviruses may display different sensitivities to such conditions. In a cellular environment where dNTPs are limiting, reverse transcription may be initiated, but not completed [Pierson et al, 2002;Swiggard et al, 2004]. The half-life of the incomplete viral DNA may be a critical determinant for successful infection, as stable replication intermediates may be completed at a later time, when cell cycle conditions are more favorable.…”

Surgical Pathology in HIV Infection in the Era of Antiretroviral Therapy

“…One explanation for this activity in the gastrointestinal tract could be because cells in this area have the co-receptor CCR5, which is used by HIV to infect cells. (Pierson et al 2002) The second period is the chronic asymptomatic infection, the average duration of this phase is 10 years, it is characterized by stable levels of CD4 + with a tendency to decrease progressively, the viral load drops and may be undetectable, but the virus continues its replication in the lymphoid tissue. The causes of decline in CD4 + cells during this phase of infection are only partially known.…”