Human Immunodeficiency Virus cDNA Metabolism: Notable Stability of Two-Long Terminal Repeat Circles

Abstract

Early steps of retroviral replication involve reverse transcription of the viral RNA to yield a linear doublestranded cDNA copy and then integration of the viral cDNA into a chromosome of the host cell. A portion of the viral cDNA can also follow nonproductive pathways in which it becomes circularized. In one pathway, the ends of the linear cDNA become joined together by the cellular nonhomologous DNA end-joining system to form two-long terminal repeat (2-LTR) circles. It has been argued that 2-LTR circles are quickly degraded in human immunodeficiency virus (HIV)-infected cells, allowing the presence of 2-LTR circles to be used as a marker for ongoing de novo infection in patients. Following this idea, detection of 2-LTR circles in patients undergoing successful highly active antiretroviral therapy has led to the proposal that viral replication persists despite treatment. We have used fluorescence-monitored PCR (Taqman) to quantitate the metabolism of HIV cDNA early after infection. Contrary to previous work, we find that 2-LTR circles are actually quite stable in experiments where confounding variables are controlled. Thus, studies relying on the lability of 2-LTR circles are open to reinterpretation. We also used the quantitative PCR methods to analyze the effects of MG132, a proteasome inhibitor, which revealed that viral complexes containing mostly completed cDNAs are the primary substrates for proteasome-mediated degradation.

Tight Interplay among SAMHD1 Protein Level, Cellular dNTP Levels, and HIV-1 Proviral DNA Synthesis Kinetics in Human Primary Monocyte-derived Macrophages

“…2LTR circles are defective proviral DNA products that have failed to integrate into the host genome. Moreover, they are commonly used as a surrogate to monitor late proviral synthesis steps in the HIV life cycle (15). Previous studies have used pretreatment of target cell populations, macrophages, and dendritic cells before transducing them with HIV vectors (6 -8, 11, 16, 17).…”

Integrase, LEDGF/p75 and HIV replication

“…For example, PIC-associated IN could become vulnerable to the proteasome at some point in its nuclear trajectory unless complexed with p75. It has also been noted that inhibition of the proteasome can enhance HIV infection under certain conditions, suggesting that the proteasome may target incoming virions or host proteins involved in the early steps of infection [146,147]. Establishing a clear connection between the latter effect, the INstabilizing effect of p75, and the viral cofactor role requires further investigation.…”

Proteasome inhibitors uncouple rhesus TRIM5α restriction of HIV-1 reverse transcription and infection

“…[29][30][31]. In contrast, cyclohexamide treatment provided greater relief of rhTRIM5␣ restriction, increasing infection of the rhTRIM5␣ stable cells 6-fold compared with only a 1.3-fold increase in the control HeLa cells (Fig.…”