Secondary mutations in viruses resistant to HIV-1 integrase inhibitors that restore viral infectivity and replication kinetics

158

206

The human immunodeficiency virus type 1 (HIV-1) integrase mutations N155H and Q148R(H)(K) that reduce susceptibility to the integrase inhibitor raltegravir have been identified in patients failing treatment regimens containing raltegravir. Whether these resistance mutations occur individually or in combination within a single virus genome has not been defined, nor do we fully understand the impact of these primary mutations and other secondary mutations on raltegravir susceptibility and viral replication capacity. To address these important questions, we investigated the raltegravir susceptibility and replication capacity of viruses containing mutations at positions 155 and 148 separately or in combination with secondary mutations selected in subjects failing treatment regimens containing raltegravir. Clonal analysis demonstrated that N155H and Q148R(H)(K) occur independently, not in combination. Viruses containing a Q148R(H)(K) mutation generally displayed larger reductions in raltegravir susceptibility than viruses with an N155H mutation. Analysis of site-directed mutants indicated that E92Q in combination with N155H resulted in a higher level of resistance to raltegravir than N155H alone. Viruses containing a Q148R(H) mutation together with a G140S mutation were more resistant to raltegravir than viruses containing a Q148R(H) mutation alone; however, viruses containing G140S and Q148K were more susceptible to raltegravir than viruses containing a Q148K mutation alone. Both N155H and Q148R(H)(K) mutations reduced the replication capacity, while the addition of secondary mutations either improved or reduced the replication capacity depending on the primary mutation. This study demonstrates distinct genetic pathways to resistance in subjects failing raltegravir regimens and defines the effects of primary and secondary resistance mutations on raltegravir susceptibility and replication capacity.

Section: Discussionsupporting

confidence: 82%

Loss of Raltegravir Susceptibility by Human Immunodeficiency Virus Type 1 Is Conferred via Multiple Nonoverlapping Genetic Pathways

Fransen¹,

Gupta²,

Danovich

et al. 2009

158

206

“…We observed that each of G118R, Y143R, Q148R, R263K, and G140S/Q148R mutations in IN impaired viral infectivity, while H51Y and N155H were relatively innocuous in this regard. These infectivity data correlate well with previous reports that Y143R, Q148R, and G140S/Q148R in HIV-1 significantly decreased viral fitness in the HeLa-P4 reporter cell system at 48 h postinfection, while N155H exhibited similar viral fitness to WT viruses (50,51). The relative infectivities of H51Y and R263K SIV reported here compared to WT are also in agreement with results reported for HIV-1 that docu- mented an effect of R263K on infectiousness of Ϸ20% (21,27,28), although the effect in SIVmac239 was more pronounced, i.e., ϳ90%.…”

Section: Discussionsupporting

confidence: 81%

Effect of HIV-1 Integrase Resistance Mutations When Introduced into SIVmac239 on Susceptibility to Integrase Strand Transfer Inhibitors

Hassounah

Mésplède

Quashie

et al. 2014

Studies on the in vitro susceptibility of SIV to integrase strand transfer inhibitors (INSTIs) have been rare. In order to determine the susceptibility of SIVmac239 to INSTIs and characterize the genetic pathways that might lead to drug resistance, we inserted various integrase (IN) mutations that had been selected with HIV under drug pressure with raltegravir (RAL), elvitegravir (EVG), and dolutegravir (DTG) into the IN gene of SIV. We evaluated the effects of these mutations on SIV susceptibility to INSTIs and on viral infectivity. Sequence alignments of SIVmac239 IN with various HIV-1 isolates showed a high degree of homology and conservation of each of the catalytic triad and the key residues involved in drug resistance. Each of the G118R, Y143R, Q148R, R263K, and G140S/Q148R mutations, when introduced into SIV, impaired infectiousness and replication fitness compared to wild-type virus. Using TZM-bl cells, we demonstrated that the Q148R and N155H mutational pathways conferred resistance to EVG (36-and 62-fold, respectively), whereas R263K also displayed moderate resistance to EVG (12-fold). In contrast, Y143R, Q148R, and N155H all yielded low levels of resistance to RAL. The combination of G140S/Q148R conferred highlevel resistance to both RAL and EVG (>300-and 286-fold, respectively). DTG remained fully effective against all site-directed mutants except G118R and R263K. Thus, HIV INSTI mutations, when inserted into SIV, resulted in a similar phenotype. These findings suggest that SIV and HIV may share similar resistance pathways profiles and that SIVmac239 could be a useful nonhuman primate model for studies of HIV resistance to INSTIs. IMPORTANCEThe goal of our project was to establish whether drug resistance against integrase inhibitors in SIV are likely to be the same as those responsible for drug resistance in HIV. Our data answer this question in the affirmative and show that SIV can probably serve as a good animal model for studies of INSTIs and as an early indicator for possible emergent mutations that may cause treatment failure. An SIV-primate model remains an invaluable tool for investigating questions related to the potential role of INSTIs in HIV therapy, transmission, and pathogenesis, and the present study will facilitate each of the above.

“…This finding is consistent with the notion that a fitness-compromising mutation such as G118R might then require HIV-1 to accumulate secondary mutations in order to partially restore replication capacity. Previous work has documented that this is also true with regard to each of three different primary mutations that are associated with resistance to both RAL and EVG: i.e., 143, 148, and 155 (5,6,10,23). Whether the removal of MK-2048 from culture medium would likewise lead to reversions and the possible reemergence of more replication-fit wild-type virus is currently under examination.…”

Section: Discussionmentioning

confidence: 99%

“…6) shows that a change from the flexible glycine (G) at position 118 to a positively charged arginine (R) and a change from a negatively charged glutamate (E) at position 138 to a positively charged lysine (K) might lead to alterations in the organization and structure of this domain. The location of amino acid 138 in proximity to a flexible loop makes it a candidate for a role in interactions with the target DNA (2,23). In addition, these changes have the potential to alter the charge distribution across the catalytic core of the protein (Fig.…”

Section: Assessment Of Reverse Transcription and Impairment Of Integrmentioning

confidence: 99%

Identification of Novel Mutations Responsible for Resistance to MK-2048, a Second-Generation HIV-1 Integrase Inhibitor

Bar-Magen

Sloan

Donahue

et al. 2010

MK-2048 represents a prototype second-generation integrase strand transfer inhibitor (INSTI) developed with the goal of retaining activity against viruses containing mutations associated with resistance to firstgeneration INSTIs, raltegravir (RAL) and elvitegravir (EVG). Here, we report the identification of mutations (G118R and E138K) which confer resistance to MK-2048 and not to RAL or EVG. These mutations were selected in vitro and confirmed by site-specific mutagenesis. G118R, which appeared first in cell culture, conferred low levels of resistance to MK-2048. G118R also reduced viral replication capacity to approximately 1% that of the isogenic wild-type (wt) virus. The subsequent selection of E138K partially restored replication capacity to Ϸ13% of wt levels and increased resistance to MK-2048 to Ϸ8-fold. Viruses containing G118R and E138K remained largely susceptible to both RAL and EVG, suggesting a unique interaction between this second-generation INSTI and the enzyme may be defined by these residues as a potential basis for the increased intrinsic affinity and longer "off" rate of MK-2048. In silico structural analysis suggests that the introduction of a positively charged arginine at position 118, near the catalytic amino acid 116, might decrease Mg 2؉ binding, compromising enzyme function and thus leading to the significant reduction in both integration and viral replication capacity observed with these mutations.Selective pressure exerted by antiretroviral drugs, in conjunction with high viral mutation rates, promotes the inevitable emergence of drug-resistant HIV-1 variants. This necessitates an ongoing search for novel antiretroviral compounds that either have novel mechanisms and inhibit different stages of viral replication or inhibit targets that have acquired resistance to existing drugs. In the latter case, such newer next-generation agents should ideally display resistance profiles which are distinct and nonoverlapping with those of the first-generation drugs.Integration of viral cDNA into the host cell genome is a distinct feature of retroviral replication, and inhibitors of HIV-1 integrase have recently been added to the arsenal of clinically approved antiretroviral drugs. Raltegravir (RAL) was the first integrase strand transfer inhibitor (INSTI) to be approved by the U.S. Food and Drug Administration (FDA) after clinical trials showed that this drug promoted a rapid and sustained antiviral effect (13). Elvitegravir (EVG), another integrase inhibitor, is currently in phase III clinical trials (27). Resistance mutations common to both of these first-generation integrase inhibitors have been reported and can result in high levels of drug resistance (26). Mutations which engender crossresistance between RAL and EVG have been reported in clinical trials, cell culture studies, and biochemical assays (9,26). This has prompted the search for second-generation integrase inhibitors that might display novel patterns of resistance, allowing their use in patients who have failed therapy with RAL or E...