The viral capsid of HIV-1 interacts with a number of host factors to orchestrate uncoating and regulate downstream events, such as reverse transcription, nuclear entry, and integration site targeting. PF-3450074 (PF74), an HIV-1 capsid-targeting low-molecular-weight antiviral compound, directly binds to the capsid (CA) protein at a site also utilized by host cell proteins CPSF6 and NUP153. Here, we found that the dose-response curve of PF74 is triphasic, consisting of a plateau and two inhibitory phases of different slope values, consistent with a bimodal mechanism of drug action. High PF74 concentrations yielded a steep curve with the highest slope value among different classes of known antiretrovirals, suggesting a dose-dependent, cooperative mechanism of action. CA interactions with both CPSF6 and cyclophilin A (CypA) were essential for the unique dose-response curve. A shift of the steep curve at lower drug concentrations upon blocking the CA-CypA interaction suggests a protective role for CypA against high concentrations of PF74. These findings, highlighting the unique characteristics of PF74, provide a model in which its multimodal mechanism of action of both noncooperative and cooperative inhibition by PF74 is regulated by interactions of cellular proteins with incoming viral capsids. IMPORTANCEPF74, a novel capsid-targeting antiviral against HIV-1, shares its binding site in the viral capsid protein (CA) with the host factors CPSF6 and NUP153. This work reveals that the dose-response curve of PF74 consists of two distinct inhibitory phases that are differentially regulated by CA-interacting host proteins. PF74's potency depended on these CA-binding factors at low doses. In contrast, the antiviral activity of high PF74 concentrations was attenuated by cyclophilin A. These observations provide novel insights into both the mechanism of action of PF74 and the roles of host factors during the early steps of HIV-1 infection.T he emergence of HIV-1 variants resistant to currently approved antiretrovirals necessitates the development of novel classes of inhibitors that possess high levels of genetic barriers to resistance (1). Among viral proteins that are not exploited as an antiviral target, the viral capsid (CA) protein is an attractive target for antiviral interventions (2, 3). CA, a genetically fragile protein (4), exhibits limited tolerance to genetic changes and, hence, would predictably temper the evolution of drug resistance (5). The mutational intolerance of CA is caused by structural and functional constraints (6-8). CA, which is the major virion core structural protein, generated by protease-mediated cleavage of the precursor Gag Pr55 protein, plays essential roles during both particle assembly and disassembly (9, 10). Perhaps it is this genetic fragility that makes CA highly vulnerable to host immune responses, such as CD8-specific adaptive immunity (11) and TRIM5␣-mediated intrinsic immunity (12), both of which target highly conserved portions of CA.Recent work discovered several novel smal...

Cleavage and polyadenylation specificity factor subunit 6 (CPSF6), a host factor that interacts with the HIV-1 capsid (CA) protein, is implicated in diverse functions during the early part of the HIV-1 life cycle, including uncoating, nuclear entry, and integration targeting. Preservation of CA binding to CPSF6 in vivo suggests that this interaction is fine-tuned for efficient HIV-1 replication in physiologically relevant settings. Nevertheless, this possibility has not been formally examined. To assess the requirement for optimal CPSF6-CA binding during infection of primary cells and in vivo, we utilized a novel CA mutation, A77V, that significantly reduced CA binding to CPSF6. The A77V mutation rendered HIV-1 largely independent from TNPO3, NUP358, and NUP153 for infection and altered the integration site preference of HIV-1 without any discernible effects during the late steps of the virus life cycle. Surprisingly, the A77V mutant virus maintained the ability to replicate in monocyte-derived macrophages, primary CD4 ؉ T cells, and humanized mice at a level comparable to that for the wild-type (WT) virus. Nonetheless, revertant viruses that restored the WT CA sequence and hence CA binding to CPSF6 emerged in three out of four A77V-infected animals. These results suggest that the optimal interaction of CA with CPSF6, though not absolutely essential for HIV-1 replication in physiologically relevant settings, confers a significant fitness advantage to the virus and thus is strictly conserved among naturally circulating HIV-1 strains. IMPORTANCECPSF6 interacts with the HIV-1 capsid (CA) protein and has been implicated in nuclear entry and integration targeting. Preservation of CPSF6-CA binding across various HIV-1 strains suggested that the optimal interaction between CA and CPSF6 is critical during HIV-1 replication in vivo. Here, we identified a novel HIV-1 capsid mutant that reduces binding to CPSF6, is largely independent from the known cofactors for nuclear entry, and alters integration site preference. Despite these changes, virus carrying this mutation replicated in humanized mice at levels indistinguishable from those of the wild-type virus. However, in the majority of the animals, the mutant virus reverted back to the wild-type sequence, hence restoring the wild-type level of CA-CPSF6 interactions. These results suggest that optimal binding of CA to CPSF6 is not absolutely essential for HIV-1 replication in vivo but provides a fitness advantage that leads to the widespread usage of CPSF6 by HIV-1 in vivo.T he HIV-1 capsid (CA) protein forms the protein core that envelopes viral enzymes and genetic material following fusion with the plasma membrane (1-3). Uncoating of the viral core is a fine-tuned process that regulates downstream events, including reverse transcription, nuclear entry, and integration targeting (4-9). It is likely that HIV-1 exploits multiple host factors that can directly interact with the viral CA protein for proper uncoating in virus-infected cells. These host factors likely in...

Background: Autophagy is induced by stress, starvation, and viral infections; however, the role of RNase L in autophagy has not been investigated. Results: Activation of RNase L induces autophagy involving JNK and PKR. Conclusion: Autophagy induced by activation of RNase L modulates viral growth. Significance: Our findings identify a novel role of RNase L in inducing autophagy affecting outcomes of viral infections.

Virus infection leads to activation of the interferon (IFN)-induced endoribonuclease RNase L, which results in degradation of viral and cellular RNAs. Both cellular and viral RNA cleavage products of RNase L bind pattern recognition receptors (PRRs), like retinoic acid-inducible I (Rig-I) and melanoma differentiation-associated protein 5 (MDA5), to further amplify IFN production and antiviral response. Although much is known about the mechanics of ligand binding and PRR activation, how cells coordinate RNA sensing with signaling response and interferon production remains unclear. We show that RNA cleavage products of RNase L activity induce the formation of antiviral stress granules (avSGs) by regulating activation of double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) and recruit the antiviral proteins Rig-I, PKR, OAS, and RNase L to avSGs. Biochemical analysis of purified avSGs showed interaction of a key stress granule protein, G3BP1, with only PKR and Rig-I and not with OAS or RNase L. AvSG assembly during RNase L activation is required for IRF3-mediated IFN production, but not IFN signaling or proinflammatory cytokine induction. Consequently, cells lacking avSG formation or RNase L signaling produced less IFN and showed higher susceptibility during Sendai virus infection, demonstrating the importance of avSGs in RNase L-mediated host defense. We propose a role during viral infection for RNase L-cleaved RNAs in inducing avSGs containing antiviral proteins to provide a platform for efficient interaction of RNA ligands with pattern recognition receptors to enhance IFN production to mount an effective antiviral response. IMPORTANCE Double-stranded RNAs produced during viral infections serve as pathogen-associated molecular patterns (PAMPs) and bind pattern recognition receptors to stimulate IFN production. RNase L is an IFN-regulated endoribonuclease that is activated in virus-infected cells and cleaves single-stranded viral and cellular RNAs. The RNase L-cleaved dsRNAs signal to Rig-like helicases to amplify IFN production. This study identifies a novel role of antiviral stress granules induced by RNase L as an antiviral signaling hub to coordinate the RNA ligands with cognate receptors to mount an effective host response during viral infections.

The HIV-1 capsid executes essential functions that are regulated by capsid stability and host factors. In contrast to increasing knowledge on functional roles of capsid-interacting host proteins during postentry steps, less is known about capsid stability and its impact on intracellular events. Here, using the antiviral compound PF-3450074 (PF74) as a probe for capsid function, we uncovered a novel phenotype of capsid stability that has a profound effect on innate sensing of viral DNA by the DNA sensor cGAS. A single mutation, R143A, in the capsid protein conferred resistance to high concentrations of PF74, without affecting capsid binding to PF74. A cell-free assay showed that the R143A mutant partially counteracted the capsid-destabilizing activity of PF74, pointing to capsid stabilization as a resistance mechanism for the R143A mutant. In monocytic THP-1 cells, the R143A virus, but not the wild-type virus, suppressed cGAS-dependent innate immune activation. These results suggest that capsid stabilization improves the shielding of viral DNA from innate sensing. We found that a naturally occurring transmitted founder (T/F) variant shares the same properties as the R143A mutant with respect to PF74 resistance and DNA sensing. Imaging assays revealed delayed uncoating kinetics of this T/F variant and the R143A mutant. All these phenotypes of this T/F variant were controlled by a genetic polymorphism located at the trimeric interface between capsid hexamers, thus linking these capsid-dependent properties. Overall, this work functionally connects capsid stability to innate sensing of viral DNA and reveals naturally occurring phenotypic variation in HIV-1 capsid stability. IMPORTANCE The HIV-1 capsid, which is made from individual viral capsid proteins (CA), is a target for a number of antiviral compounds, including the small-molecule inhibitor PF74. In the present study, we utilized PF74 to identify a transmitted/founder (T/F) strain that shows increased capsid stability. Interestingly, PF74-resistant variants prevented cGAS-dependent innate immune activation under a condition where the other T/F strains induced type I interferon. These observations thus reveal a new CA-specific phenotype that couples capsid stability to viral DNA recognition by cytosolic DNA sensors.