Background
Individuals with nonsuppressible viremia (NSV) have persistent low-level viremia despite ART adherence and lack of drug resistance. A subset was reported to harbor deletions in the 5′ end of the HIV-1 genome, specifically within the 5′ PSI packaging element. We have previously reported three individuals with NSV and plasma variants harboring deletions in the 5’ leader region within otherwise genome-intact viruses. However, it is unclear whether these 5’ leader deletions lead to replication-defective virus. To investigate whether 5′Leader deletions affect HIV replication capacity fitness, we engineered a vector with a 5′Leader deletion and a unique enzyme site (pHIV-Δ5′Leader/PmeI) to study viral replication and infectivity.
Methods
We created an HIV-1 vector with 5′ Leader deletions and introduced a Pme I site with NL4-3 as the backbone. 5′leader amplicons from three NSV participants harboring 5’ deletions were either PCR-amplified or synthesized. Recombinant viruses carrying patient-derived or synthesized 5′Leader deletions of varying sizes were generated via co-transfection in 293T cells. Infectivity was measured using β-galactosidase assays in TZM-bl cells, while relative fitness was evaluated through kinetic assays in MT-2 cells. The sequence integrity of the 5′Leader was confirmed using NGS sequencing of culture supernatants.
Results
Recombinant HIV-1 viruses carrying the 5′Leader deletions from three NSV participants harboring 22, 15 and 41 bp deletions (LV4, 8, and 9, respectively) were replication-incompetent. Among viruses carrying synthesized 5′Leader amplicons, all variants containing the LV4, LV8 and LV9 deletions were replication-incompetent. In addition, using viruses harboring 5’ Leader deletions of varying sizes, 5’ Leader deletions as small as 6 nucleotides rendered the virus replication-defective, as long as that deletion encompassed a portion of the major splice donor site (MSD). The replication activity of the viruses from these participants could be rescued by the introduction of an intact 5’ Leader sequence.
Conclusions
Deletions in the 5′Leader region, especially within the major splice donor site, significantly impaired viral replication capacity, these results demonstrate that persistent low-level viremia may be comprised largely of replication-defective virions, with minimal transmission risk.
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