Several interesting presentations were delivered in session 3 of the 5^th International Workshop on HIV Drug Resistance and Treatment Strategies on possible mechanisms of NRTI and PI resistance.

NRTI resistance
In abstracts 41 and 42, the group of E. Arnold presented structural data that contribute to our understanding of nucleoside drug resistance [1,2]. These findings showed that the fingers sub-domain of RT may not be fully folded down to interact with AZT monophosphate but, in contrast, may remain in only a half-closed position that mimics the structure of an intermediate compound after catalysis and prior to translocation. The results of crystallographic analysis also demonstrated that the acyclic nature of PMPA (tenofovir) may help to explain its favorable resistance profile. Among other considerations, the M184V substitution may discriminate against the modified ribose ring of 3TC by steric hindrance, something that would not occur with a relatively small acyclic phosphonate moity. In the same context, tenofovir may not provide a substrate that can be easily excised following its incorporation as a chain terminator into a growing DNA strand.

In abstract 46, M. Gotte et al demonstrated that a series of RT mutations, notably both L74V and M184V, lead to diminished fitness and reduced enzymatic activity of RT [3]. Both of these substitutions, which occur frequently in cases of drug resistance, led to significant reductions in rates of tRNA-primed DNA synthesis; and the highest rates of impaired enzymatic fitness were observed with enzymes containing both of these mutations. In contrast, neither M184V nor L74V, when present in a background of thymidine-associated mutations, notably M41L and T215Y, did not appear to contribute to significant reductions in these reactions. Both L74V and M184V were also shown to lead to severe diminutions in rates of tRNA-primed synthesis of (+) strand DNA. These data suggest that the efficiency of initiation of reverse transcription is crucial as a determinant of viral replication fitness.

In abstract 47, B.A. Larder presented data that suggest resistance to ddI may be much rarer than commonly assumed, and that the L74V substitution, while conferring resistance to ddI, is able to suppress the emergence of resistance to AZT as efficiently as the M184V substitution in reverse transcriptase [4]. However, the greatest effect was seen when both L74V and M184V were simultaneously present together with a variety of thymidine-associated mutations. In general, phenotypic resistance to ddI was seen less commonly than that to any of AZT, 3TC or abacavir. High-level resistance to ddI was only attributable to the Q151M substitution, and modest resistance to ddI could be explained by a combination of AZT-associated mutations, but was not significantly associated with the M184V substitution.

Novel mechanisms of resistance to PI
A series of papers were presented that dealt with changes in cleavage sites within HIV-1 with gag-associated altered sensitivity to PI. In abstract 29, K. Suzuki et al presented data on the role of amino acid insertions in the p6 protein in patients who failed a variety of antiretroviral regimens [5]. These amino acid insertions in the p6 region were associated with low levels of RT activity and a relative deficit of vpr to be incorporated into virions. It was speculated that p6 insertions may therefore lead to a problem with regard to the assembly of viral particles, concomitant with the appearance of resistance to PI. Hence, viruses that develop resistance to PI as a consequence of these insertions may simultaneously suffer from significant reductions in fitness.

In abstract 59, D. Kempf et al showed that an A431V mutation in the gag gene at the NC-p1 junction was associated with resistance to PI as well as to mutations in HIV-1 protease in PI-experienced patients [6]. However, this cleavage site mutation did not necessarily impact on responsiveness to antiviral therapy.

And lastly, E-Y Kim et al presented data by on the topic of insertion mutations within the HIV-1 pol gene in the secondary structure of RNA molecules that encode protease and RT (abstract 60) [7]. This team provided evidence that such insertions might cause the RT of HIV to pause and that this, in turn, might lead to weaker base pairing and additional pause sites. This could encourage dissociation of nascent DNA from the RNA template. Re-annealing of nascent cDNA might then occur from the point at which dissociation had taken place and this might be followed by reinsertion of the same bases, resulting in duplication. These results help to explain the mechanisms whereby insertions occur in the pol gene as well as the increased RT processivity associated with pol gene inserts.

References
Abstracts can be accessed by hyperlink after registering for the 5th International Workshop on HIV Drug Resistance & Treatment Strategies Webcast at Mediscover.net

Sarafianos SG, Clark AD, Das K, Ilankwnaran P, Sayer JM, Jerina DM, Boyer PL, Hughes SH, Arnold E. Structural basis of HIV resistance to zidovudine: crystal structure of HIV-1 reverse transcriptase with template:zidovudine monophosphate-blocked primer bound at the dNTP-binding site. Antiviral Ther. 2001;6(Suppl 1):41. Abstract 41
Tuske S, Sarafianos S, Clark AD, Ding J, Naeger LK, Miller MD, Gibbs C, Jerina DM, Hughes S, Arnold E. Crystal structure of HIV-1 reverse transcriptase with template-primer terminated with the acyclic nucleotide RT inhibitor tenofovir. Antiviral Ther. 2001;6(Suppl 1):42. Abstract 42
Gotte M, Wei X, Diallo K, Marchand B, Schaffer A, Wainberg MA. Blockage of tRNA-primed initiation of reverse transcription provides a mechanism for the diminished fitness of viruses containing L74V and M184V mutations. Antiviral Ther. 2001;6(Suppl 1):46. Abstract 46
Larder BA, Bloor S. Analysis of clinical isolates and site-directed mutatnts reveals the genetic determinants of didanosine resistance. Antiviral Ther. 2001;6(Suppl 1):47. Abstract 47
Suzuki K, Kaufmann G, Jones D, Piller S, Leas L, Harris C, Cunningham P, Mallon P, Mukaide M, Kelleher A, Cooper DA. Impact of amino acid insertions in p6 identified in patients with failure of combination antiretroviral therapy: alterations in Vpr incorporation and virus maturation. Antiviral Ther. 2001;6(Suppl 1):29. Abstract 29
Kempf D, King M, Brun S, Sylte J, Marsh T, Stewart K, Sun E. Association of the nucleocapsid-p1 (NC-p1) gag cleavage site alanine to valine mutation with mutational patterns in HIV protease producing protease inhibitor resistance. Antiviral Ther. 2001;6(Suppl 1):59. Abstract 59
Kim E-Y, Winters MA, Lobato RL, Harrison GP, Kagan R, Merigan TC. Mechanisms and frequencies of insertion mutations in the protease and RT gene of HIV-1 isolates. Antiviral Ther. 2001;6(Suppl 1):60. Abstract 60