Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands.
Correspondence to Ben Berkhout, Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, Meibergdreef 15, 1105AZ Amsterdam, The Netherlands. E-mail: email@example.com
The HIV-1 RNA genome is rapidly evolving and, although its sequence heterogeneity is widely recognized, its biological origins and consequences are poorly understood. Sequence variability at the population (interhost) and the individual (intrahost) level stems mainly from the inherent infidelity of the reverse transcriptase enzyme that lacks a proofreading activity. The high HIV-1 replicative turnover, the relative large size of the viral quasispecies population in an infected individual, and the propensity for recombination all add to the viral capacity to adapt to immunological, pharmaceutical, or other selection pressures. Despite the high evolution rate of HIV-1, there are indications that its evolution is not unlimited but confined to a restricted sequence space. We tried to assess the actual sequence space available for HIV-1 in a novel experimental setting.
The evolutionary capacity of HIV-1 was probed by putting a strong selective pressure on highly conserved sequences within the HIV-1 genome and comparing the natural with the selected variation. We used RNAi by means of short hairpin RNA (shRNA) inhibitors to target highly conserved 19-nucleotide sequences within the HIV-1 RNA genome . Multiple cell culture infections were followed to select for viral escape variants. Details of this massive virus evolution study can be found in another article . We compiled the RNAi-resistant mutations that were selected (approximately 500 sequences) with four shRNA inhibitors (Fig. 1a) and compared this with the variation observed in 625 natural HIV-1 strains.
We grouped all escape data with respect to the position of the acquired mutations within the 19-nucleotide targets, which yields a typical pattern (Fig. 1b). In theory, 19 × 3 = 57 point mutations are possible per target, which means 228 escape possibilities for the four targets. However, we scored only a restricted set of 36 escape routes. Some target positions are highly preferred for acquisition of escape mutations, whereas others are strongly avoided, especially at both ends of the target (positions 1, 2, 18, 19). The lack of escape mutations at the target ends is most likely dictated by the RNAi mechanism, which is known to allow mismatches between the siRNA effector and the target sequence at these sites [3–8]. However, we also observed considerable variation in escape pattern for the central target domain (positions 3–17) that cannot be explained by properties of the RNAi mechanism. As we selected the most conserved viral target sequences for the RNAi-therapeutic approach [1,5,7], we checked whether the induced sequence variation would mimic the limited sequence variation present in natural HIV-1 strains in the Los Alamos database. We observed a striking similarity between the RNAi-induced sequence variation (Fig. 1b) and natural sequence variation (Fig. 1c). These results imply that only a selective set of HIV-1 variants are allowed to evolve, both in nature and under RNAi pressure. This also indicates that the HIV-1 variants that are not selected in vitro and in vivo represent viruses with suboptimal replication capacity and fitness. This idea is further supported by the preponderance of silent codon changes among the RNAi-resistant viruses . These results suggest that HIV-1 evolution already reached the outer limits of the allowed sequence space, at least for the highly conserved domains that were probed in this study.
Several studies previously suggested that HIV-1 evolution is restricted to a relatively small domain of sequence space. Most importantly, Lukashov and Goudsmit  compared HIV-1 subtype B sequences early in the Amsterdam HIV-1 epidemic versus 10 years later. They described that the high evolution rate of HIV-1 leads to a steady increase in synonymous distance from the consensus sequence, but the nonsynonymous distance remained constant over time. It was concluded that HIV-1 sequences fluctuate within a sequence space with fixed distance from the subtype consensus. This study focused on the V3 domain of the Envelope protein, which may be particularly constrained in function as it is a major determinant for receptor/co-receptor interactions and the key determinant of the CCR5-CXCR4 coreceptor switch.
In natural infections, cytotoxic T lymphocytes (CTLs) put pressure on different HIV-1 protein epitopes. There is accumulating evidence that some of the Gag epitopes tolerate only limited sequence variation because of a fitness cost . These fitness problems also explain the reversion of modified CTL target sequences to the consensus sequence upon HIV-1 transmission to a new host [11–13]. These in-vivo results strongly argue against unremitting adaptation of HIV-1 to host immune pressures and strongly support the idea of restricted HIV-1 evolution. Similar fitness losses have been described for drug-resistant variants of the Protease enzyme [14,15], variation within the Tat protein  and the Integrase enzyme . Protease sequences can, in fact, be subject to dual pressure from antiretroviral therapy and CTLs . A complicating factor is that compensatory mutations are able to mask the deleterious effects of another mutation. This has been described for CTL-escape mutations in the Gag protein  and drug-resistance mutations in the viral Protease enzyme [7,20]. Such compensatory changes can even be selected in vitro and in vivo in the absence of drug pressure, demonstrating that repair of enzyme function and viral fitness is the driving force .
Our current findings also have important implications for the design of improved antiviral RNAi strategies, in particular with respect to attempts to avoid viral escape. We previously used one simple criterion to select target sites: optimal conservation of the targeted sequence among virus strains . We now propose additional criteria to stipulate target selection. First, selection of the most conserved targets should focus on the center of the 19-nucleotide target domain (positions 3–17), as RNAi is tolerant for sequence variation at the termini of the target. Second, this 3–17 domain should be selected with the least number of silent codon positions (mostly third codon positions). Third, it seems beneficial to find targets enriched for AUG and UGG codons encoding methionine and tryptophan, respectively, because these are the only two codons that lack options for silent escape. Fourth, our data suggest that one can actually use the available information on natural HIV-1 sequence variation in the design of improved shRNA inhibitors, as this should define the available sequence space for HIV-1 that can be used to obtain RNAi resistance. Specifically, one could define targets that restrict sequence variation to only one or two positions, in which case one could anticipate these viral escape routes and design a limited set of second generation shRNAs to block these escape routes . These considerations are of importance for the development of a durable RNAi-based gene therapy for HIV-AIDS.
We thank Vladimir Lukashov for critical reading of the manuscript. RNAi research in the Berkhout laboratory is sponsored by ZonMw (Vici grant and Translational Gene Therapy grant).
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