This study represents a comprehensive consideration of the replicative characteristics of 18 viral isolates derived from patients with primary HIV-1 infection, 9 of which encode some primary drug resistance-associated mutation in PR, RT, or both. The data described here suggest that drug-resistant HIV-1 isolates can bear adaptive mutations that allow for wild-type-level replicative function, thereby overcoming the potential defects associated with the genetic changes necessary for drug resistance.
Drug-resistant isolates displayed growth kinetics similar to those of drug-susceptible isolates in CD4+ T-lymphocyte cell culture. Furthermore, the single-cycle infectivity rates of these viruses were on average higher than those of drug-susceptible viruses. Additionally, the range of infectivity and growth rate values for the drug-resistant isolates was broader than that for the drug-susceptible isolates, with the latter group representing a more homogeneous set (Fig. and ).
The creation of chimeric viruses proved to be a useful tool for determining the contribution of certain viral regions towards the replicative fitness of the drug-resistant isolates (39
). Specifically, the introduction of the C-terminal region of Gag and the PR-RT regions of the resistant viruses into an NL4-3 molecular background resulted in chimeras with lower RC values than the drug-susceptible recombinants. Moreover, the majority of chimeric viruses with drug resistance had very similar RC values within a relatively narrow range (58 to 73%).
Interestingly, three drug-resistant isolates (MDR1, MDR2, and P1) were characterized by a significantly higher level of infectivity and better growth kinetics than the remaining drug-resistant viruses and all of the drug-susceptible viruses (Fig. and ). Indeed, the aforementioned differences between isolates and chimeras were most dramatic for these cases. Because the inserted regions here included the C-terminal region of Gag as well as PR and RT, we may surmise that mutations in these regions do not account for the high replication efficiency displayed by these isolates. Specifically, we can conclude that the well-characterized compensatory substitutions in the p7/p1 cleavage site and the PTAP duplication in p6 (e.g., MDR2), while they may have contributed to the restoration of fitness, were not sufficient for the high replication levels of these two MDR viruses. While further studies are needed to elucidate the viral regions essential for the observed phenotypes of MDR1 and MDR2 (e.g., the N-terminal portion of Gag or the C-terminal part of Pol or Env), we may speculate that in the case of isolate P1 the Y→L substitution in the p17/p24 cleavage site contributed to efficient replication (Table ). Although substitutions within this site were documented to take place under PI treatment (29
), this specific substitution is not known to be of compensatory relevance. Recent reports to the contrary (37
) demonstrate that wild-type PR cleaves p17/p24 less efficiently if Y is replaced by L in position P1 of the cleavage site. However, it is conceivable that the altered substrate specificity of drug-resistant PR (10
) results in better viral maturation and consequently restores viral replication to levels observed in isolate P1. Lastly, we need to take into consideration the possibility that other regions of the viral genome of isolate P1 contribute to the observed phenotype.
Also of interest was the difference in replication characteristics observed among the related resistant isolates (Fig. and ). Secondary transmission of the PI-resistant isolates occurred during primary infection. Isolate P3 showed a modest, but reproducible, twofold increase in infectivity relative to isolate P2. This difference was seen to be amplified (approximately fourfold) over multiple rounds of replication in PBMC culture in comparison to the single-cycle assay performed with the GHOST cell line. The chimeric viruses encoding the PR-RT sequences of these related viral isolates displayed similar RCs, however, suggesting that the PR functions in P2 and P3 are equivalent. The reversion to the consensus B p17/p24 cleavage site amino acid sequence in P3, therefore, may contribute to the increased replication efficiency of this isolate. Alternatively, the differences in replication efficiencies of P2 and P3 may reflect the differences in the duration of infection (P2, 30 days; P3, 70 days), since it has been proposed that replication efficiency increases over the time of infection (23
). If the latter is true, the relative adaptive changes could have taken place in regions such as env
, which are both highly variable and subject to the selective pressure of the immune system (14
The growth rates of the linked NNRTI-resistant viruses, on the other hand, remained stable after sequential transmission. The replication rates of these isolates were comparable to those of P3. The preservation of replication kinetics by transmitted NNRTI-resistant viruses observed here is not surprising, since the relevant mutations (e.g., K103N) are known to confer little RT impairment (11
Some have observed the persistence of transmitted drug-resistant variants within a host over time (e.g., 4 to 6 months) (3
), which might suggest intrinsic fitness. These results are in agreement with reports that some MDR isolates replicate to high levels both in vitro (3
) and in vivo (22
). We must consider, however, the likely homogeneity of the original inocula in these cases. In the setting of an emergent drug-resistant viral variant in chronic disease, for example, it is seen that discontinuation of antiretroviral treatment results in a rapid overgrowth by wild-type viruses. This suggests that these drug-resistant viruses are “fit” only in the presence of their respective drug pressures. The persistence of transmitted drug-resistant virus within a new host, therefore, may simply reflect an absence of competition.
Furthermore, in vitro observation may misrepresent the in vivo case. Indeed, a high level of replication in PBMC does not necessarily imply similar kinetics in other cell populations. Some PI-resistant viruses, for example, replicate well in PBMC but show profound replication defects in thymocytes (43
). Thus, it may be that the lower virological set point observed in patients infected with MDR viruses does not reflect a poor viral replication potential in general; rather, these viruses may simply be selectively incapable of replicating efficiently in certain cell populations (e.g., thymocytes) and therefore are less generally pathogenic to the host.
It is important, however, to emphasize that our data are based on isolates derived from PBMC collected within 10 to 79 days (median, 21 days) after the onset of symptoms. The virus populations were sampled before being subjected to a number of immunological and host cell-associated constraints (24
), but we cannot exclude the possibility that the viral characteristics analyzed in this study reflect but a snapshot of the viral replicative potential before the development of a mature HIV-1-specific immune response.
In conclusion, these findings indicate that the biological characteristics of newly transmitted viruses are similar, irrespective of the presence or absence of drug resistance-associated mutations. The specific compensatory mechanisms used to overcome defects imposed by primary drug resistance-associated mutations in PR and RT are likely to be complex and probably reflect an interplay between different viral regions. Elucidating how transmitted drug-resistant viruses regain infective potency can lead to the identification of new therapeutic targets which may someday allow us to prevent the emergence of such otherwise intractable variants.