부산대학교 도서관

Antiretroviral therapy (ART) effectively inhibits HIV-1 replication but is not curative due to the persistence of a latent viral reservoir in resting CD4+ T cells. This reservoir is a major barrier to cure. Sequencing studies have revealed that the population of proviruses persisting in ART-treated individuals is dominated by defective proviruses that cannot give rise to viral rebound due to fatal defects including large deletions and APOBEC3-mediated hypermutation. Near full genome sequencing (nFGS) of individual proviruses is used in reservoir assays to provide an estimate of the fraction of proviruses that are intact. nFGS methods rely on a long-distance outer PCR capturing most (~9 kb) of the genome, followed by nested inner PCRs. The outer PCR is carried out at limit dilution, and interpretation of the results is based on the assumption that all proviruses are quantitatively captured. Here, we evaluate nFGS methods using the intact proviral DNA assay (IPDA), a multiplex digital droplet PCR assay that quantitates intact and defective proviruses with single molecule sensitivity using only short, highly efficient amplicons. We analyzed proviral templates of known sequence to avoid the additional complication of sequence polymorphism. With the IPDA, we quantitated molecular yields at each step of nFGS methods. We demonstrate that nFGS methods are inefficient and miss ~70% of full-length proviruses due to amplification failure at the initial outer PCR step. In contrast, proviruses with large internal deletions encompassing 70% of the genome can be quantitatively amplified under the same conditions. Accurate measurement of the latent reservoir of HIV-1 is essential for evaluating the efficacy of cure strategies, and the bias against full length proviruses in nFGS methods must be considered. Author summary: Despite antiretroviral therapy, HIV-1 persists in a small population of resting memory CD4+ T cells carrying a latent viral genome. This latent reservoir is the major barrier to cure. Accurate reservoir assays are critical for evaluating therapies aimed at reducing the reservoir. Sequencing studies have shown defective proviruses greatly outnumbered the intact, replication-competent proviruses responsible for viral rebound, and thus reservoir assays that rely on near full-genome sequencing (nFGS) have provided important qualitative information about intact and defective proviruses. However, it is assumed that all proviruses are amplified with equal efficiency in nFGS methods, regardless of sequence length. Here, we rigorously measure the efficiency with which nFGS methods detect intact and defective proviruses using a highly efficient multiplex digital droplet PCR assay, the intact proviral DNA assay. This assay allows direct counting of input proviral template molecules as well as PCR amplified products generated with different nFGS methods. We determined that nFGS methods do not provide an accurate quantitative measure of intact proviruses. Only ~30% of intact proviruses were detected, while proviruses with large internal deletions were amplified at expected frequencies. Our study demonstrates that nFGS methods do not provide accurate quantitative information about the size and composition of the latent reservoir. [ABSTRACT FROM AUTHOR]