Nonsuppressible viremia during HIV-1 therapy meets molecular virology

We now know that it is possible for proviruses to produce virus particles with viral RNA even when the MSD site is gone. There are reported examples where the virus particles in elevated NSV are infectious, although not replicating given the presence of antiviral drugs (12, 13). White, Wu, et al. now provide examples of defective viral genomes producing particles at sufficient levels to require investigation (10).

The authors estimate that NSV occurs at a frequency of about 1 in 250 cases of people on suppressive therapy (10). Sequencing of the 5′-leader of virus in people with NSV will help round out our understanding of the mechanisms of NSV and may provide the impetus to follow more cases to get a precise estimate of its frequency.

Several mysteries remain about how these defective particles are made. First, it is unclear what is happening with splicing when the MSD site is missing and why transcripts that initiate splicing are skewed toward complete splicing, causing a reduction in the partially spliced Env mRNA. Second, why are the particles in the immature form? The viral Gag-Pro-Pol polyprotein precursor is made as an alternative translation product of Gag translation and thus independent of splicing. One would assume that if Gag is made, then Gag-Pro-Pol is also produced. Importantly, the Pro in Gag-Pro-Pol is the viral protease tasked with cleaving the Gag and Gag-Pro-Pol polyprotein precursors. White, Wu, and coauthors (10) showed that Gag was cleaved, which would be expected if Gag-Pro-Pol was made and assembled into the virion. However, the question of why the virion exists in an immature conformation remains unanswered. The findings imply that these particles lack a component, resulting in a failure of some heretofore-unknown nucleation event needed to initiate capsid cone formation.

What limits the rate for generating NSV? Viral DNA synthesis makes one mistake per three proviruses generated (19). The viral polymerase is error prone in making point mutations, and the generation of deletions is mechanistically related to recombination for retroviruses (20, 21). Given the large population of replicating virus, many mutations are generated daily, and the types of mutations identified in White, Wu, et al. are clearly incorporated into the latent reservoir and have been seen in LLV (22), indicating that the generation of such genomes is unlikely to limit viral production. Sufficient clonal expansion of a cell expressing this type of virus can clearly result in substantial viremia despite the presence of a defective genome. This possibility is consistent with observations from one of the patients in which the T cell clone with the NSV defective virus represented half of all cells containing viral DNA in the blood. However, in other cases, the expanded and expressing T cell clone giving rise to NSV was a minor component of the reservoir, and the size of these infected clones poorly correlated with levels of NSV. A driver of T cell clonal expansion may be the antigen specificity of the cell and frequent exposure to that antigen. Finally, the defective viral genome must have integrated into a site that allows expression in the context of the host cell chromatin. Some, and likely all, of these factors must happen to give rise to NSV.

It is also possible that the relatively infrequent detection of persistent NSV only occurs in people with inefficient immune surveillance of virus-producing cells that would normally limit the size of such a clone. This would be the opposite outcome of what is seen in elite controllers who can control viremia even in the absence of ART. Recent analysis (23) of viral genomes in elite controllers has shown that cells with intact viral genomes are under strong immune surveillance against expression, leaving only those proviruses that are in a state called “deep latency.” There are two points to consider regarding the possibility that people who experience this elevated NSV struggle to clear cells that are expressing viral proteins, allowing clonal superexpansion, expression of viral proteins, and particle production. First, mutations in the MSD site lead to reduced Env protein expression, both on the virus particle and on the cell surface. If immune surveillance depends on antibodies binding cell-surface Env protein (antibody-dependent cell killing), then these cells have a fortuitous escape mechanism mediated by the virus. The second possibility comes from the analysis of NSV in P2, who had a surprisingly complex NSV population: there were two populations of virus with different 5′-leader deletions, a population with an MSD site mutation, and wild-type virus. This pattern suggests that these viral genomes are capable of expressing as part of multiple expanded cellular clones. P2 also had a larger latent reservoir size, but it is unclear whether the larger reservoir created more opportunity to sample these types of virus-producing cells or whether an inability to efficiently clear virus-expressing cells resulted in a larger steady-state reservoir through more extensive clonal expansion. In this model, ART blocks viral replication, but the host immune selection struggles to clear virus-expressing cells. The opposite pattern occurs in elite controllers, where immune selection efficiently removes cells expressing virus (23). It will be important to understand whether there is an underlying reason why these NSV patients harbor expanded clones expressing viral proteins (10). If it is due to poor immunologic control of virus-expressing cells, this would emphasize the need to maintain potent regimens and possibly avoid drug-reducing regimens.

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