Human Immunodeficiency Virus Type 1: Resistance to Nucleoside Analogues and Replicative Capacity in Primary Human Macrophages

Abstract

Antiretroviral treatment failure is associated with the emergence of resistant human immunodeficiency virus type 1 (HIV-1) populations which often express altered replicative capacity (RC). The resistance and RC of clinical HIV-1 strains, however, are generally assayed using activated peripheral blood mononuclear cells (PBMC) or tumor cell lines. Because of their high proliferation rate and concurrent high deoxynucleoside triphosphate (dNTP) content, both resistance and RC alterations might be misestimated in these cell systems. We have evaluated the resistance of HIV-1 clones expressing a variety of RT resistance mutations in primary human macrophages using a single cycle system. Our experiments indicate that d4T, ddI, and 3TC are more potent in macrophages than in HeLa-derived P4 tumor cells. Mutant viruses bearing thymidine analogue mutations (TAMs) or the K65R mutation had similar resistance levels in the two cell types. Strikingly, however, the M184V mutant, although fully resistant to 3TC in P4 cells, maintained some susceptibility to 3TC in macrophages from 8 of 11 donors. Using the same system, we found that the impact of resistance mutations on HIV RC was minimal in activated PBMC and in P4 cells. In contrast, mutant viruses exhibited strongly impaired RC relative to the wild type (WT) in macrophages, with the following RC order: WT > two TAMs > four TAMs ‫؍‬ M184V > K65R. In undifferentiated monocytes, WT virus replication could be detected in three of six donors, but replication of all mutant viruses remained undetectable. Altogether, our results confirm that nucleoside reverse transcriptase inhibitors (NRTIs) are powerful antiviral agents in differentiated macrophages, reveal that HIV resistance to some NRTIs may be less efficient in these cells, and indicate that resistance-associated loss of RC is more pronounced in macrophages than in high-dNTP content cell systems.Nucleoside analogues, also referred to as nucleoside reverse transcriptase inhibitors (NRTIs) are one of the main drug classes used in the treatment of human immunodeficiency virus type 1 (HIV-1) infection. After phosphorylation into their active triphosphate form by cellular kinases, these compounds, which lack a 3Ј-hydroxyl group, inhibit reverse transcription through termination of viral DNA synthesis. HIV-1 resistance to NRTIs is achieved via two distinct and generally exclusive mechanisms: (i) decreased incorporation of the triphosphorylated NRTI or (ii) excision of the triphosphorylated NRTI and primer unblocking. Decreased NRTI incorporation is mediated by mutations such as the M184V substitution, which induces high-level resistance to ␤-L-2Ј,3Ј-dideoxy-3Ј-thiacytidine (3TC or lamivudine) (24,35,61,64), as well as other mutations such as K74V, K65R, or the Q151M complex. These mutations promote resistance through impairment of the correct positioning of triphosphate NRTIs within the RT active site (19,67). In contrast, thymidine analogue mutations (TAMs), such as the M41L, the D67N, the K70R, the L210W, the T215F/Y, and the K219Q...

Reduced dNTP Binding Affinity of 3TC-resistant M184I HIV-1 Reverse Transcriptase Variants Responsible for Viral Infection Failure in Macrophage

“…2B showing that M184I RT displays only 50% of Met-184 Clone 3 RT at 0.25 M dNTP. More interestingly, it was reported that, unlike M184I virus, M184V virus is able to infect macrophages (30,31), and this observation is consistent with our biochemical data illustrated in Fig. 2A that M184V RT still maintains RT activity at least 50% of the wildtype RT activity even at low dNTP concentrations found in macrophages.…”

Clinical Significance of Human Immunodeficiency Virus Type 1 Replication Fitness

“…Similar findings were also obtained using whole isolates obtained from patients failing tenofovir therapy (175). One study using single-cycle parallel cultures found a decreased fitness of K65R in primary macrophages but not in a cell line or primary human PBMCs (129). The replication defect of K65R correlates with a decrease in processivity of polymerization and in the polymerization rate, k pol , of purified reverse transcriptase (46,179).…”

“…M184V occurs frequently in viral isolates from patients failing lamivudine or emtricitabine therapy (63,153). M184V has a fitness similar to that of the wild type in most T-cell lines where nucleotide concentrations are high (10) but reduced fitness in primary cells that have limited nucleotide pools, such as PBMCs and macrophages (3,10,129). Of note is that there are some more-recent studies that have found reduced fitness of this mutant in cell lines (35,48).…”

“…A systematic study of HIV-1 with K65R or different numbers of thymidine analog mutations (TAMs) showed that the replication of each of these variants was less efficient in primary human macrophages than in T-cell lines (129). It is interesting that a mutant virus with the four TAMs D67N, K70R, T215Y, and K219Q actually had a replication advantage compared to wildtype virus in unstimulated PBMCs; this difference was not observed when the PBMCs were stimulated before infection (26).…”

Mutation V111I in HIV-2 Reverse Transcriptase Increases the Fitness of the Nucleoside Analogue-Resistant K65R and Q151M Viruses

“…In HIV-1, the mutations K65R, Q151M, and M184V were found to result in replication defects. Mutation K65R reduces the replication capacity of HIV-1 in T-cell lines and monocytes (41,42), whereas mutation M184V results in reduced fitness in monocytes (43,44). Mutation Q151M does not seem to reduce the HIV-1 replication capacity (45,46).…”