Structural Investigations of Interactions between the Influenza a Virus NS1 and Host Cellular Proteins.

The Influenza A virus is a continuous threat to public health that causes yearly epidemics with the ever-present threat of the virus becoming the next pandemic. Due to increasing levels of resistance, several of our previously used antivirals have been rendered useless. There is a strong need for new antivirals that are less likely to be susceptible to mutations. One strategy to achieve this goal is structure-based drug development. By understanding the minute details of protein structure, we can develop antivirals that target the most conserved, crucial regions to yield the highest chances of long-lasting success. One promising IAV target is the virulence protein non-structural protein 1 (NS1). NS1 contributes to pathogenicity through interactions with numerous host proteins, and many of the resulting complexes have been shown to be crucial for virulence. In this review, we cover the NS1-host protein complexes that have been structurally characterized to date. By bringing these structures together in one place, we aim to highlight the strength of this field for drug discovery along with the gaps that remain to be filled.

The Effect of Inositol Hexakisphosphate on HIV-1 Particle Production and Infectivity can be Modulated by Mutations that Affect the Stability of the Immature Gag Lattice.

The assembly of an HIV-1 particle begins with the construction of a spherical lattice composed of hexamer subunits of the Gag polyprotein. The cellular metabolite inositol hexakisphosphate (IP6) binds and stabilizes the immature Gag lattice via an interaction with the six-helix bundle (6HB), a crucial structural feature of Gag hexamers that modulates both virus assembly and infectivity. The 6HB must be stable enough to promote immature Gag lattice formation, but also flexible enough to be accessible to the viral protease, which cleaves the 6HB during particle maturation. 6HB cleavage liberates the capsid (CA) domain of Gag from the adjacent spacer peptide 1 (SP1) and IP6 from its binding site. This pool of IP6 molecules then promotes the assembly of CA into the mature conical capsid that is required for infection. Depletion of IP6 in virus-producer cells results in severe defects in assembly and infectivity of wild-type (WT) virions. Here we show that in an SP1 double mutant (M4L/T8I) with a hyperstable 6HB, IP6 can block virion infectivity by preventing CA-SP1 processing. Thus, depletion of IP6 in virus-producer cells markedly increases M4L/T8I CA-SP1 processing and infectivity. We also show that the introduction of the M4L/T8I mutations partially rescues the assembly and infectivity defects induced by IP6 depletion on WT virions, likely by increasing the affinity of the immature lattice for limiting IP6. These findings reinforce the importance of the 6HB in virus assembly, maturation, and infection and highlight the ability of IP6 to modulate 6HB stability.

Structural basis of HIV-1 maturation inhibitor binding and activity.

HIV-1 maturation inhibitors (MIs), Bevirimat (BVM) and its analogs interfere with the catalytic cleavage of spacer peptide 1 (SP1) from the capsid protein C-terminal domain (CACTD), by binding to and stabilizing the CACTD-SP1 region. MIs are under development as alternative drugs to augment current antiretroviral therapies. Although promising, their mechanism of action and associated virus resistance pathways remain poorly understood at the molecular, biochemical, and structural levels. We report atomic-resolution magic-angle-spinning NMR structures of microcrystalline assemblies of CACTD-SP1 complexed with BVM and/or the assembly cofactor inositol hexakisphosphate (IP6). Our results reveal a mechanism by which BVM disrupts maturation, tightening the 6-helix bundle pore and quenching the motions of SP1 and the simultaneously bound IP6. In addition, BVM-resistant SP1-A1V and SP1-V7A variants exhibit distinct conformational and binding characteristics. Taken together, our study provides a structural explanation for BVM resistance as well as guidance for the design of new MIs.

HIV-1 is dependent on its immature lattice to recruit IP6 for mature capsid assembly.

HIV-1 Gag metamorphoses inside each virion, from an immature lattice that forms during viral production to a mature capsid that drives infection. Here we show that the immature lattice is required to concentrate the cellular metabolite inositol hexakisphosphate (IP6) into virions to catalyze mature capsid assembly. Disabling the ability of HIV-1 to enrich IP6 does not prevent immature lattice formation or production of the virus. However, without sufficient IP6 molecules inside each virion, HIV-1 can no longer build a stable capsid and fails to become infectious. IP6 cannot be replaced by other inositol phosphate (IP) molecules, as substitution with other IPs profoundly slows mature assembly kinetics and results in virions with gross morphological defects. Our results demonstrate that while HIV-1 can become independent of IP6 for immature assembly, it remains dependent upon the metabolite for mature capsid formation.

Enhanced Transmissibility and Decreased Virulence of HIV-1 CRF07_BC May Explain Its Rapid Expansion in China.

HIV-1 CRF07_BC is one of the most common circulating recombinant forms (CRFs) in China and is becoming increasingly prevalent especially in HIV-infected men who have sex with men (MSM). The reason why this strain expanded so quickly in China remains to be defined. We previously observed that individuals infected with HIV-1 CRF07_BC showed slower disease progression than those infected with HIV-1 subtype B or CRF01_AE. CRF07_BC viruses carry two unique mutations in the p6Gag protein: insertion of PTAPPE sequences downstream of the original Tsg101 binding domain, and deletion of a seven-amino-acid sequence (30PIDKELY36) that partially overlaps with the Alix binding domain. In this study, we confirmed the enhanced transmission capability of CRF07_BC over HIV-1 subtype B or CRF01_AE by constructing HIV-1 transmission networks to quantitatively evaluate the growth rate of transmission clusters of different HIV-1 genotypes. We further determined lower virus infectivity and slower replication of CRF07_BC with aforementioned PTAPPE insertion (insPTAP) and/or PIDKELY deletion (Δ7) in the p6Gag protein, which in turn may increase the pool of people infected with CRF07_BC and the risk of HIV-1 transmission. These new features of CRF07_BC may explain its quick spread and will help adjust prevention strategy of HIV-1 epidemic. IMPORTANCE HIV-1 CRF07_BC is one of the most common circulating recombinant forms (CRFs) in China. The question is why and how CRF07_BC expanded so rapidly remains unknown. To address the question, we explored the transmission capability of CRF07_BC by constructing HIV-1 transmission networks to quantitatively evaluate the growth rate of transmission clusters of different HIV-1 genotypes. We further characterized the role of two unique mutations in CRF07_BC, PTAPPE insertion (insPTAP) and/or PIDKELY deletion (Δ7) in the p6Gag in virus replication. Our results help define the molecular mechanism regarding the association between the unique mutations and the slower disease progression of CRF07_BC as well as the quick spread of CRF07_BC in China.

CryoET structures of immature HIV Gag reveal six-helix bundle.

Gag is the HIV structural precursor protein which is cleaved by viral protease to produce mature infectious viruses. Gag is a polyprotein composed of MA (matrix), CA (capsid), SP1, NC (nucleocapsid), SP2 and p6 domains. SP1, together with the last eight residues of CA, have been hypothesized to form a six-helix bundle responsible for the higher-order multimerization of Gag necessary for HIV particle assembly. However, the structure of the complete six-helix bundle has been elusive. Here, we determined the structures of both Gag in vitro assemblies and Gag viral-like particles (VLPs) to 4.2 Å and 4.5 Å resolutions using cryo-electron tomography and subtomogram averaging by emClarity. A single amino acid mutation (T8I) in SP1 stabilizes the six-helix bundle, allowing to discern the entire CA-SP1 helix connecting to the NC domain. These structures provide a blueprint for future development of small molecule inhibitors that can lock SP1 in a stable helical conformation, interfere with virus maturation, and thus block HIV-1 infection.

A stable immature lattice packages IP6 for HIV capsid maturation.

HIV virion assembly begins with the construction of an immature lattice consisting of Gag hexamers. Upon virion release, protease-mediated Gag cleavage leads to a maturation event in which the immature lattice disassembles and the mature capsid assembles. The cellular metabolite inositiol hexakisphosphate (IP6) and maturation inhibitors (MIs) both bind and stabilize immature Gag hexamers, but whereas IP6 promotes virus maturation, MIs inhibit it. Here we show that HIV is evolutionarily constrained to maintain an immature lattice stability that ensures IP6 packaging without preventing maturation. Replication-deficient mutant viruses with reduced IP6 recruitment display increased infectivity upon treatment with the MI PF46396 (PF96) or the acquisition of second-site compensatory mutations. Both PF96 and second-site mutations stabilise the immature lattice and restore IP6 incorporation, suggesting that immature lattice stability and IP6 binding are interdependent. This IP6 dependence suggests that modifying MIs to compete with IP6 for Gag hexamer binding could substantially improve MI antiviral potency.

How to package the RNA of HIV-1.

Interactions between viral RNA and the integrase enzyme are required for HIV-1 particles to become infectious, a process that can be disrupted through multiple mechanisms.

HIV-1 Maturation: Lessons Learned from Inhibitors.

Since the emergence of HIV and AIDS in the early 1980s, the development of safe and effective therapies has accompanied a massive increase in our understanding of the fundamental processes that drive HIV biology. As basic HIV research has informed the development of novel therapies, HIV inhibitors have been used as probes for investigating basic mechanisms of HIV-1 replication, transmission, and pathogenesis. This positive feedback cycle has led to the development of highly effective combination antiretroviral therapy (cART), which has helped stall the progression to AIDS, prolong lives, and reduce transmission of the virus. However, to combat the growing rates of virologic failure and toxicity associated with long-term therapy, it is important to diversify our repertoire of HIV-1 treatments by identifying compounds that block additional steps not targeted by current drugs. Most of the available therapeutics disrupt early events in the replication cycle, with the exception of the protease (PR) inhibitors, which act at the virus maturation step. HIV-1 maturation consists of a series of biochemical changes that facilitate the conversion of an immature, noninfectious particle to a mature infectious virion. These changes include proteolytic processing of the Gag polyprotein by the viral protease (PR), structural rearrangement of the capsid (CA) protein, and assembly of individual CA monomers into hexamers and pentamers that ultimately form the capsid. Here, we review the development and therapeutic potential of maturation inhibitors (MIs), an experimental class of anti-HIV-1 compounds with mechanisms of action distinct from those of the PR inhibitors. We emphasize the key insights into HIV-1 biology and structure that the study of MIs has provided. We will focus on three distinct groups of inhibitors that block HIV-1 maturation: (1) compounds that block the processing of the CA-spacer peptide 1 (SP1) cleavage intermediate, the original class of compounds to which the term MI was applied; (2) CA-binding inhibitors that disrupt capsid condensation; and (3) allosteric integrase inhibitors (ALLINIs) that block the packaging of the viral RNA genome into the condensing capsid during maturation. Although these three classes of compounds have distinct structures and mechanisms of action, they share the ability to block the formation of the condensed conical capsid, thereby blocking particle infectivity.

The influenza NS1 protein modulates RIG-I activation via a strain-specific direct interaction with the second CARD of RIG-I.

A critical role of influenza A virus nonstructural protein 1 (NS1) is to antagonize the host cellular antiviral response. NS1 accomplishes this role through numerous interactions with host proteins, including the cytoplasmic pathogen recognition receptor, retinoic acid-inducible gene I (RIG-I). Although the consequences of this interaction have been studied, the complete mechanism by which NS1 antagonizes RIG-I signaling remains unclear. We demonstrated previously that the NS1 RNA-binding domain (NS1RBD) interacts directly with the second caspase activation and recruitment domain (CARD) of RIG-I. We also identified that a single strain-specific polymorphism in the NS1RBD (R21Q) completely abrogates this interaction. Here we investigate the functional consequences of an R21Q mutation on NS1's ability to antagonize RIG-I signaling. We observed that an influenza virus harboring the R21Q mutation in NS1 results in significant up-regulation of RIG-I signaling. In support of this, we determined that an R21Q mutation in NS1 results in a marked deficit in NS1's ability to antagonize TRIM25-mediated ubiquitination of the RIG-I CARDs, a critical step in RIG-I activation. We also observed that WT NS1 is capable of binding directly to the tandem RIG-I CARDs, whereas the R21Q mutation in NS1 significantly inhibits this interaction. Furthermore, we determined that the R21Q mutation does not impede the interaction between NS1 and TRIM25 or NS1RBD's ability to bind RNA. The data presented here offer significant insights into NS1 antagonism of RIG-I and illustrate the importance of understanding the role of strain-specific polymorphisms in the context of this specific NS1 function.

Cellular IP6 Levels Limit HIV Production while Viruses that Cannot Efficiently Package IP6 Are Attenuated for Infection and Replication.

HIV-1 hijacks host proteins to promote infection. Here we show that HIV is also dependent upon the host metabolite inositol hexakisphosphate (IP6) for viral production and primary cell replication. HIV-1 recruits IP6 into virions using two lysine rings in its immature hexamers. Mutation of either ring inhibits IP6 packaging and reduces viral production. Loss of IP6 also results in virions with highly unstable capsids, leading to a profound loss of reverse transcription and cell infection. Replacement of one ring with a hydrophobic isoleucine core restores viral production, but IP6 incorporation and infection remain impaired, consistent with an independent role for IP6 in stable capsid assembly. Genetic knockout of biosynthetic kinases IPMK and IPPK reveals that cellular IP6 availability limits the production of diverse lentiviruses, but in the absence of IP6, HIV-1 packages IP5 without loss of infectivity. Together, these data suggest that IP6 is a critical cofactor for HIV-1 replication.

Structural analyses reveal the mechanism of inhibition of influenza virus NS1 by two antiviral compounds.

The influenza virus is a significant public health concern causing 250,000-500,000 deaths worldwide each year. Its ability to change quickly results in the potential for rapid generation of pandemic strains for which most individuals would have no antibody protection. This pandemic potential highlights the need for the continuous development of new drugs against influenza virus. As an essential component and well established virulence determinant, NS1 (nonstructural protein 1) of influenza virus is a highly prioritized target for the development of anti-influenza compounds. Here, we used NMR to determine that the NS1 effector domain (NS1ED) derived from the A/Brevig Mission/1/1918 (H1N1) strain of influenza (1918H1N1) binds to two previously described anti-influenza compounds A9 (JJ3297) and A22. We then used X-ray crystallography to determine the three-dimensional structure of the 1918H1N1 NS1ED Furthermore, we mapped the A9/A22-binding site onto our 1918H1N1 NS1ED structure and determined that A9 and A22 interact with the NS1ED in the hydrophobic pocket known to facilitate binding to the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), suggesting that the two compounds likely attenuate influenza replication by inhibiting the NS1ED-CPSF30 interaction. Finally, our structure revealed that NS1ED could dimerize via an interface that we termed the α3-α3 dimer. Taken together, the findings presented here provide strong evidence for the mechanism of action of two anti-influenza compounds that target NS1 and contribute significant structural insights into NS1 that we hope will promote and inform the development and optimization of influenza therapies based on A9/A22.

Structural Basis for a Novel Interaction between the NS1 Protein Derived from the 1918 Influenza Virus and RIG-I.

The influenza non-structural protein 1 (NS1) plays a critical role in antagonizing the innate immune response to infection. One interaction that facilitates this function is between NS1 and RIG-I, one of the main sensors of influenza virus infection. While NS1 and RIG-I are known to interact, it is currently unclear whether this interaction is direct or if it is mediated by other biomolecules. Here we demonstrate a direct, strain-dependent interaction between the NS1 RNA binding domain (NS1(RBD)) of the influenza A/Brevig Mission/1918 H1N1 (1918(H1N1)) virus and the second caspase activation and recruitment domain of RIG-I. Solving the solution structure of the 1918(H1N1) NS1(RBD) revealed features in a functionally novel region that may facilitate the observed interaction. The biophysical and structural data herein suggest a possible mechanism by which strain-specific differences in NS1 modulate influenza virulence.