The internal -methyladenosine (mA) modification of cellular mRNA regulates post-transcriptional gene expression. The YTH domain family proteins (YTHDF1-3 or Y1-3) bind to mA-modified cellular mRNAs and modulate their metabolism and processing, thereby affecting cellular protein translation. We previously reported that HIV-1 RNA contains the mA modification and that Y1-3 proteins inhibit HIV-1 infection by decreasing HIV-1 reverse transcription activity. Here, we investigated the mechanisms of Y1-3-mediated inhibition of HIV-1 infection in target cells and the effect of Y1-3 on viral production levels in virus-producing cells. We found that Y1-3 protein overexpression in HIV-1 target cells decreases viral genomic RNA (gRNA) levels and inhibits both early and late reverse transcription. Purified recombinant Y1-3 proteins preferentially bound to the mA-modified 5' leader sequence of gRNA compared with its unmodified RNA counterpart, consistent with the strong binding of Y1-3 proteins to HIV-1 gRNA in infected cells. HIV-1 mutants with two altered mA modification sites in the 5' leader sequence of gRNA exhibited significantly lower infectivity than WT, replication-competent HIV-1, confirming that these sites alter viral infection. HIV-1 produced from cells in which endogenous Y1, Y3, or Y1-3 proteins were knocked down singly or together had increased viral infectivity compared with HIV-1 produced in control cells. Interestingly, we found that Y1-3 proteins and HIV-1 Gag protein formed a complex with RNA in HIV-1-producing cells. Overall, these results indicate that Y1-3 proteins inhibit HIV-1 infection and provide new insights into the mechanisms by which the mA modification of HIV-1 RNA affects viral replication.

A number of human immunodeficiency virus 1 integrase (IN) alterations, referred to as class II substitutions, exhibit pleotropic effects during virus replication. However, the underlying mechanism for the class II phenotype is not known. Here we demonstrate that all tested class II IN substitutions compromised IN-RNA binding in virions by one of three distinct mechanisms: i) markedly reducing IN levels thus precluding formation of IN complexes with viral RNA; ii) adversely affecting functional IN multimerization and consequently impairing IN binding to viral RNA; iii) directly compromising IN-RNA interactions without substantially affecting IN levels or functional IN multimerization. Inhibition of IN-RNA interactions resulted in mislocalization of the viral ribonucleoprotein complexes outside the capsid lattice, which led to premature degradation of the viral genome and IN in target cells. Collectively, our studies uncover causal mechanisms for the class II phenotype and highlight an essential role of IN-RNA interactions for accurate virion maturation.

Recent evidence indicates that inhibition of HIV-1 integrase (IN) binding to the viral RNA genome by allosteric integrase inhibitors (ALLINIs) or through mutations within IN yields aberrant particles in which the viral ribonucleoprotein complexes (vRNPs) are eccentrically localized outside the capsid lattice. These particles are noninfectious and are blocked at an early reverse transcription stage in target cells. However, the basis of this reverse transcription defect is unknown. Here, we show that the viral RNA genome and IN from ALLINI-treated virions are prematurely degraded in target cells, whereas reverse transcriptase remains active and stably associated with the capsid lattice. The aberrantly shaped cores in ALLINI-treated particles can efficiently saturate and be degraded by a restricting TRIM5 protein, indicating that they are still composed of capsid proteins arranged in a hexagonal lattice. Notably, the fates of viral core components follow a similar pattern in cells infected with eccentric particles generated by mutations within IN that inhibit its binding to the viral RNA genome. We propose that IN-RNA interactions allow packaging of both the viral RNA genome and IN within the protective capsid lattice to ensure subsequent reverse transcription and productive infection in target cells. Conversely, disruption of these interactions by ALLINIs or mutations in IN leads to premature degradation of both the viral RNA genome and IN, as well as the spatial separation of reverse transcriptase from the viral genome during early steps of infection.IMPORTANCE Recent evidence indicates that HIV-1 integrase (IN) plays a key role during particle maturation by binding to the viral RNA genome. Inhibition of IN-RNA interactions yields aberrant particles with the viral ribonucleoprotein complexes (vRNPs) eccentrically localized outside the conical capsid lattice. Although these particles contain all of the components necessary for reverse transcription, they are blocked at an early reverse transcription stage in target cells. To explain the basis of this defect, we tracked the fates of multiple viral components in infected cells. Here, we show that the viral RNA genome and IN in eccentric particles are prematurely degraded, whereas reverse transcriptase remains active and stably associated within the capsid lattice. We propose that IN-RNA interactions ensure the packaging of both vRNPs and IN within the protective capsid cores to facilitate subsequent reverse transcription and productive infection in target cells.KEYWORDS ALLINIs, capsid, HIV-1, integrase, maturation, protein-RNA interaction, RNA packaging, TRIM5, reverse transcriptase

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are a promising new class of antiretroviral agents that disrupt proper viral maturation by inducing hyper-multimerization of IN. Here we show that lead pyridine-based ALLINI KF116 exhibits striking selectivity for IN tetramers versus lower order protein oligomers. IN structural features that are essential for its functional tetramerization and HIV-1 replication are also critically important for KF116 mediated higher-order IN multimerization. Live cell imaging of single viral particles revealed that KF116 treatment during virion production compromises the tight association of IN with capsid cores during subsequent infection of target cells. We have synthesized the highly active (-)-KF116 enantiomer, which displayed EC50 of ~7 nM against wild type HIV-1 and ~10 fold higher, sub-nM activity against a clinically relevant dolutegravir resistant mutant virus suggesting potential clinical benefits for complementing dolutegravir therapy with pyridine-based ALLINIs.

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Allosteric integrase inhibitors (ALLINIs) are a class of experimental anti-HIV agents that target the noncatalytic sites of the viral integrase (IN) and interfere with the IN-viral RNA interaction during viral maturation. Here, we report a highly potent and safe pyrrolopyridine-based ALLINI, STP0404, displaying picomolar IC50 in human PBMCs with a >24,000 therapeutic index against HIV-1. X-ray structural and biochemical analyses revealed that STP0404 binds to the host LEDGF/p75 protein binding pocket of the IN dimer, which induces aberrant IN oligomerization and blocks the IN-RNA interaction. Consequently, STP0404 inhibits proper localization of HIV-1 RNA genomes in viral particles during viral maturation. Y99H and A128T mutations at the LEDGF/p75 binding pocket render resistance to STP0404. Extensive in vivo pharmacological and toxicity investigations demonstrate that STP0404 harbors outstanding therapeutic and safety properties. Overall, STP0404 is a potent and first-in-class ALLINI that targets LEDGF/p75 binding site and has advanced to a human trial.

27A large number of HIV-1 integrase (IN) alterations, referred to as class II substitutions, exhibit 28 pleotropic effects during virus replication. However, the underlying mechanism for the class II 29 phenotype is not known. Here we demonstrate that all tested class II IN substitutions 30 compromised IN-RNA binding in virions by one of three distinct mechanisms: i) markedly 31 reducing IN levels thus precluding formation of IN complexes with viral RNA; ii) adversely 32 affecting functional IN multimerization and consequently impairing IN binding to viral RNA; iii) 33 directly compromising IN-RNA interactions without substantially affecting IN levels or functional 34 IN multimerization. Inhibition of IN-RNA interactions resulted in mislocalization of the viral 35 ribonucleoprotein complexes outside the capsid lattice, which led to premature degradation of 36 the viral genome and IN in target cells. Collectively, our studies uncover causal mechanisms for 37 the class II phenotype and highlight an essential role of IN-RNA interactions for accurate virion 38 maturation. 39 40 42 between the HIV-1 Gag and Gag-Pol polyproteins, and the viral RNA (vRNA) genome. At the 43 plasma membrane of an infected cell, Gag and Gag-Pol molecules assemble around a vRNA 44 dimer and bud from the cell as a spherical immature virion, in which the Gag proteins are 45 radially arranged [1-3]. As the immature virion buds, the viral protease enzyme is activated and 46 cleaves Gag and Gag-Pol into their constituent domains triggering virion maturation [1, 2].47 During maturation the cleaved nucleocapsid (NC) domain of Gag condenses with the RNA 48 genome and pol-encoded viral enzymes [reverse transcriptase (RT) and integrase (IN)] inside 49 the conical capsid lattice, composed of the cleaved capsid (CA) protein, which together form the 50 core [1-3]. 51 3 After infection of a target cell, RT in the confines of the reverse transcription 52 complex (RTC) synthesizes linear double stranded DNA from vRNA [4]. The vDNA is 53 subsequently imported into the nucleus, where the IN enzyme catalyzes its insertion into the 54 host cell chromosome [5, 6]. Integration is mediated by the intasome nucleoprotein complex that 55 consists of a multimer of IN engaging both ends of linear vDNA [7]. While the number of IN 56 protomers required for intasome function varies across Retroviridae, single particle cryogenic 57 electron microscopy (cryo-EM) structures of HIV-1 and Maedi-visna virus indicate that lentivirus 58 integration proceeds via respective higher-order dodecamer and hexadecamer IN arrangements 59 [8, 9], though a lower-order intasome comprised of an HIV-1 IN tetramer was also resolvable by 60 cryo-EM [9].61 A number of IN substitutions which specifically arrest HIV-1 replication at the integration 62 step have been described [10]. These substitutions are grouped into class I to delineate them 63 from a variety of other IN substitutions, which exhibit pleiotropic effects and are collectively 64 referred to as class II substitutions [10-12]. Class ...

Edited by Norma Allewell HIV-1 integrase (IN) is essential for virus replication andCollectively, our findings provide a plausible path for structurebased development of second-generation ALLINIs.