The RNase H (RNH) function of HIV-1 reverse transcriptase (RT) plays an essential part in the viral life cycle. We report the characterization of YLC2-155, a 2-hydroxyisoquinoline-1,3-dione (HID)-based active-site RNH inhibitor. YLC2-155 inhibits both polymerase (50% inhibitory concentration [IC 50 ] ϭ 2.6 M) and RNH functions (IC 50 ϭ 0.65 M) of RT but is more effective against RNH. X-ray crystallography, nuclear magnetic resonance (NMR) analysis, and molecular modeling were used to show that YLC2-155 binds at the RNH-active site in multiple conformations.KEYWORDS RNase H, human immunodeficiency virus, inhibitor, reverse transcriptase H IV-1 reverse transcriptase (RT) plays a critical role in virus replication. It has multiple functions, including RNA-dependent DNA synthesis, RNase H (RNH) activity, and DNA-dependent DNA synthesis to convert the viral single-stranded RNA genome into double-stranded DNA for downstream incorporation into the host cell genome (1). At least one component of highly active antiretroviral therapy (HAART) administered to patients includes RT polymerase inhibitors, either a nucleoside RT inhibitor(s) or a nonnucleoside RT inhibitor, or both. Prolonged use of antivirals can lead to side effects or drug resistance (2, 3). Hence, new antivirals that act by novel mechanisms of action are needed. No currently approved therapeutics target the RNH function of HIV-1 RT. Hence, RNH is an attractive target for future antiviral therapies.RNH inhibitors of several different chemotypes have been identified and characterized for their effectiveness against HIV-1 (4). These include acylhydrazones (5-7), diketo acids (8, 9), ␣-hydroxytropolones (10), vinylogous ureas (11), naphthyridinones (12), pyridopyrimidinones (13,14), pyrimidinol carboxylic acids (15), hydroxypyridonecarboxylic acids (16), 3-hydroxypyrimidine-2,4-diones (17, 18), and 2-hydroxyisoquinoline-1,3-diones (HIDs) (19). Notably, many 2-hydroxyisoquinoline-1,3-diones inhibit both RT polymerase and RNH functions of RT (19). In order to further understand the mechanism of RT inhibition by HIDs, we further characterized YLC2-155, which is substituted at the C7 position of the 2-hydroxyisoquinoline-1,3-dione with a furan ring (Fig. 1).

Human immunodeficiency virus type 1 subtype C (HIV-1C) has a natural deletion of a YPxL motif in its Gag-p6 late domain. This domain mediates the binding of Gag to host cell protein ALIX and subsequently facilitates viral budding. In a subset of HIV-1C-infected individuals, the tetrapeptide insertion PYxE has been identified at the deleted YPxL motif site. Here, we report the consequences of PYxE insertion on the interaction with ALIX and the relevance regarding replication fitness and drug sensitivity. In our three HIV-1C cohorts, PYKE and PYQE were most prevalent among PYxE variants. Through in silico predictions and in vitro experiments, we showed that HIV-1C Gag has an increased binding to ALIX when the PYxE motif is present. To go more into the clinical relevance of the PYxE insertion, we obtained patient-derived gag-pol sequences from HIV-1CPYxEi viruses and inserted them in a reference HIV-1 sequence. Viral growth was increased, and the sensitivity to the protease inhibitor (PI) lopinavir (LPV) and nucleoside reverse transcriptase inhibitor tenofovir alafenamide (TAF) was decreased for some of the HIV-1C PYxE variants compared to that of wild-type variants. Our data suggest that PYxE insertion in Gag restores the ability of Gag to bind ALIX and correlates with enhanced viral fitness in the absence or presence of LPV and TAF. The high prevalence and increased replication fitness of the HIV-1C virus with PYxE insertion indicates the clinical importance of these viral variants. IMPORTANCE Genomic differences within HIV-1 subtypes is associated with various degrees of viral spread, disease progression, and clinical outcome. Viral budding is essential in the HIV-1 life cycle and mainly mediated through the interaction of Gag with host proteins. Two motifs within Gag-p6 mediate binding of host cell proteins and facilitate budding. HIV-1C has a natural deletion of one of these two motifs, resulting in an inability to bind to host cell protein ALIX. Previously, we have identified a tetrapeptide (PYxE) insertion at this deleted motif site in a subset of HIV-1C patients. Here, we report the incidence of PYxE insertions in three different HIV-1C cohorts, and the insertion restores the binding of Gag to ALIX. It also increases viral growth even in the presence of the antiretroviral drugs lopinavir and tenofovir alafenamide. Hence, PYxE insertion in HIV-1C might be biologically relevant for viruses and clinically significant among patients.

Our combined virological and biochemical data suggest that EFdA inhibits both WT and reverse transcriptase inhibitor-resistant viruses efficiently in a subtype-independent manner. In contrast, HIV-1C is least susceptible to etravirine and rilpivirine.

Human immunodeficiency virus type 1 subtype C (HIV-1C) has a natural deletion of a YPxL motif in its Gag-p6 late domain. This domain mediates the binding of Gag to host cell protein ALIX and subsequently facilitates viral budding. In a subset of HIV-1C infected individuals, the tetrapeptide insertion PYxE has been identified at the deleted YPxL motif site. Here, we report the consequences of PYxE insertion on the interaction with ALIX and the relevance regarding replication fitness and drug sensitivity. In our three HIV-1C cohorts, PYKE and PYQE were most prevalent among PYxE variants. Through in silico predictions and in vitro experiments, we showed that HIV-1C Gag has an increased binding to ALIX when PYxE motif is present. To go more into the clinical relevance of the PYxE insertion, we obtained patient-derived gag-pol sequences from HIV-1C PYxEi viruses and inserted them in a reference HIV-1. Viral growth was increased, and the sensitivity to protease inhibitor (PI) lopinavir (LPV) and nucleoside reverse transcriptase inhibitor tenofovir alafenamide (TAF) was decreased for some of the HIV-1C PYxE variants compared to wild-type variants. Our data suggest that PYxE insertion in Gag restores the ability of Gag to bind ALIX and correlates with enhanced viral fitness in the absence or presence of LPV and TAF. The high prevalence and increased replication fitness of the HIV-1C virus with PYxE insertion could indicate the clinical importance of these viral variants.