Cone-shaped HIV-1 capsids are transported through intact nuclear pores.

Human immunodeficiency virus (HIV-1) remains a major health threat. Viral capsid uncoating and nuclear import of the viral genome are critical for productive infection. The size of the HIV-1 capsid is generally believed to exceed the diameter of the nuclear pore complex (NPC), indicating that capsid uncoating has to occur prior to nuclear import. Here, we combined correlative light and electron microscopy with subtomogram averaging to capture the structural status of reverse transcription-competent HIV-1 complexes in infected T cells. We demonstrated that the diameter of the NPC in cellulo is sufficient for the import of apparently intact, cone-shaped capsids. Subsequent to nuclear import, we detected disrupted and empty capsid fragments, indicating that uncoating of the replication complex occurs by breaking the capsid open, and not by disassembly into individual subunits. Our data directly visualize a key step in HIV-1 replication and enhance our mechanistic understanding of the viral life cycle.

Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B.

Enterovirus B (EV-B), a major proportion of the genus Enterovirus in the family Picornaviridae, is the causative agent of severe human infectious diseases. Although cellular receptors for coxsackievirus B in EV-B have been identified, receptors mediating virus entry, especially the uncoating process of echovirus and other EV-B remain obscure. Here, we found that human neonatal Fc receptor (FcRn) is the uncoating receptor for major EV-B. FcRn binds to the virus particles in the "canyon" through its FCGRT subunit. By obtaining multiple cryo-electron microscopy structures at different stages of virus entry at atomic or near-atomic resolution, we deciphered the underlying mechanisms of enterovirus attachment and uncoating. These structures revealed that different from the attachment receptor CD55, binding of FcRn to the virions induces efficient release of "pocket factor" under acidic conditions and initiates the conformational changes in viral particle, providing a structural basis for understanding the mechanisms of enterovirus entry.

Recognition of HIV-1 capsid by PQBP1 licenses an innate immune sensing of nascent HIV-1 DNA.

We have previously described polyglutamine-binding protein 1 (PQBP1) as an adapter required for the cyclic GMP-AMP synthase (cGAS)-mediated innate response to the human immunodeficiency virus 1 (HIV-1) and other lentiviruses. Cytoplasmic HIV-1 DNA is a transient and low-abundance pathogen-associated molecular pattern (PAMP), and the mechanism for its detection and verification is not fully understood. Here, we show a two-factor authentication strategy by the innate surveillance machinery to selectively respond to the low concentration of HIV-1 DNA, while distinguishing these species from extranuclear DNA molecules. We find that, upon HIV-1 infection, PQBP1 decorates the intact viral capsid, and this serves as a primary verification step for the viral nucleic acid cargo. As reverse transcription and capsid disassembly initiate, cGAS is recruited to the capsid in a PQBP1-dependent manner. This positions cGAS at the site of PAMP generation and sanctions its response to a low-abundance DNA PAMP.

The Ebola virus VP40 matrix layer undergoes endosomal disassembly essential for membrane fusion.

Ebola viruses (EBOVs) assemble into filamentous virions, whose shape and stability are determined by the matrix viral protein 40 (VP40). Virus entry into host cells occurs via membrane fusion in late endosomes; however, the mechanism of how the remarkably long virions undergo uncoating, including virion disassembly and nucleocapsid release into the cytosol, remains unknown. Here, we investigate the structural architecture of EBOVs entering host cells and discover that the VP40 matrix disassembles prior to membrane fusion. We reveal that VP40 disassembly is caused by the weakening of VP40-lipid interactions driven by low endosomal pH that equilibrates passively across the viral envelope without a dedicated ion channel. We further show that viral membrane fusion depends on VP40 matrix integrity, and its disassembly reduces the energy barrier for fusion stalk formation. Thus, pH-driven structural remodeling of the VP40 matrix acts as a molecular switch coupling viral matrix uncoating to membrane fusion during EBOV entry.

Strain and rupture of HIV-1 capsids during uncoating.

SignificanceThe mature capsids of HIV-1 are transiently stable complexes that self-assemble around the viral genome during maturation, and uncoat to release preintegration complexes that archive a double-stranded DNA copy of the virus in the host cell genome. However, a detailed view of how HIV cores rupture remains lacking. Here, we elucidate the physical properties involved in capsid rupture using a combination of large-scale all-atom molecular dynamics simulations and cryo-electron tomography. We find that intrinsic strain on the capsid forms highly correlated patterns along the capsid surface, along which cracks propagate. Capsid rigidity also increases with high strain. Our findings provide fundamental insight into viral capsid uncoating.

Molecular basis of differential receptor usage for naturally occurring CD55-binding and -nonbinding coxsackievirus B3 strains.

Receptor usage defines cell tropism and contributes to cell entry and infection. Coxsackievirus B (CVB) engages coxsackievirus and adenovirus receptor (CAR), and selectively utilizes the decay-accelerating factor (DAF; CD55) to infect cells. However, the differential receptor usage mechanism for CVB remains elusive. This study identified VP3-234 residues (234Q/N/V/D/E) as critical population selection determinants during CVB3 virus evolution, contributing to diverse binding affinities to CD55. Cryoelectron microscopy (cryo-EM) structures of CD55-binding/nonbinding isolates and their complexes with CD55 or CAR were obtained under both neutral and acidic conditions, and the molecular mechanism of VP3-234 residues determining CD55 affinity/specificity for naturally occurring CVB3 strains was elucidated. Structural and biochemical studies in vitro revealed the dynamic entry process of CVB3 and the function of the uncoating receptor CAR with different pH preferences. This work provides detailed insight into the molecular mechanism of CVB infection and contributes to an in-depth understanding of enterovirus attachment receptor usage.

HIV-1 capsids mimic a microtubule regulator to coordinate early stages of infection.

While the microtubule end-binding protein, EB1 facilitates early stages of HIV-1 infection, how it does so remains unclear. Here, we show that beyond its effects on microtubule acetylation, EB1 also indirectly contributes to infection by delivering the plus-end tracking protein (+TIP), cytoplasmic linker protein 170 (CLIP170) to the cell periphery. CLIP170 bound to intact HIV-1 cores or in vitro assembled capsid-nucleocapsid complexes, while EB1 did not. Moreover, unlike EB1 and several other +TIPs, CLIP170 enhanced infection independently of effects on microtubule acetylation. Capsid mutants and imaging revealed that CLIP170 bound HIV-1 cores in a manner distinct from currently known capsid cofactors, influenced by pentamer composition or curvature. Structural analyses revealed an EB-like +TIP-binding motif within the capsid major homology region (MHR) that binds SxIP motifs found in several +TIPs, and variability across this MHR sequence correlated with the extent to which different retroviruses engage CLIP170 to facilitate infection. Our findings provide mechanistic insights into the complex roles of +TIPs in mediating early stages of retroviral infection, and reveal divergent capsid-based EB1 mimicry across retroviral species.

The M2 proteins of bat influenza A viruses reveal atypical features compared to conventional M2 proteins.

The influenza A virus (IAV) M2 protein has proton channel activity, which plays a role in virus uncoating and may help to preserve the metastable conformation of the IAV hemagglutinin (HA). In contrast to the highly conserved M2 proteins of conventional IAV, the primary sequences of bat IAV H17N10 and H18N11 M2 proteins show remarkable divergence, suggesting that these proteins may differ in their biological function. We, therefore, assessed the proton channel activity of bat IAV M2 proteins and investigated its role in virus replication. Here, we show that the M2 proteins of bat IAV did not fully protect acid-sensitive HA of classical IAV from low pH-induced conformational change, indicating low proton channel activity. Interestingly, the N31S substitution not only rendered bat IAV M2 proteins sensitive to inhibition by amantadine but also preserved the metastable conformation of acid-sensitive HA to a greater extent. In contrast, the acid-stable HA of H18N11 did not rely on such support by M2 protein. When mutant M2(N31S) protein was expressed in the context of chimeric H18N11/H5N1(6:2) encoding HA and NA of avian IAV H5N1, amantadine significantly inhibited virus entry, suggesting that ion channel activity supported virus uncoating. Finally, the cytoplasmic domain of the H18N11 M2 protein mediated rapid internalization of the protein from the plasma membrane leading to low-level expression at the cell surface. However, cell surface levels of H18N11 M2 protein were significantly enhanced in cells infected with the chimeric H18N11/H5N1(6:2) virus. The potential role of the N1 sialidase in arresting M2 internalization is discussed. IMPORTANCE Bat IAV M2 proteins not only differ from the homologous proteins of classical IAV by their divergent primary sequence but are also unable to preserve the metastable conformation of acid-sensitive HA, indicating low proton channel activity. This unusual feature may help to avoid M2-mediated cytotoxic effects and inflammation in bats infected with H17N10 or H18N11. Unlike classical M2 proteins, bat IAV M2 proteins with the N31S substitution mediated increased protection of HA from acid-induced conformational change. This remarkable gain of function may help to understand how single point mutations can modulate proton channel activity. In addition, the cytoplasmic domain was found to be responsible for the low cell surface expression level of bat IAV M2 proteins. Given that the M2 cytoplasmic domain of conventional IAV is well known to participate in virus assembly at the plasma membrane, this atypical feature might have consequences for bat IAV budding and egress.

Influenza A Virus: Cellular Entry.

The frequent emergence of pathogenic viruses with pandemic potential has posed a significant threat to human health and economy, despite enormous advances in our understanding of infection mechanisms and devising countermeasures through developing various prophylactic and therapeutic strategies. The recent coronavirus disease (COVID-19) pandemic has re-emphasised the importance of rigorous research on virus infection mechanisms and highlighted the need for our preparedness for potential pandemics. Although viruses cannot self-replicate, they tap into host cell factors and processes for their entry, propagation and dissemination. Upon entering the host cells, viruses ingeniously utilise the innate biological functions of the host cell to replicate themselves and maintain their existence in the hosts. Influenza A virus (IAV), which has a negative-sense, single-stranded RNA as its genome, is no exception. IAVs are enveloped viruses with a lipid bilayer derived from the host cell membrane and have a surface covered with the spike glycoprotein haemagglutinin (HA) and neuraminidase (NA). Viral genome is surrounded by an M1 shell, forming a "capsid" in the virus particle. IAV particles use HA to recognise sialic acids on the cell surface of lung epithelial cells for their attachment. After attachment to the cell surface, IAV particles are endocytosed and sorted into the early endosomes. Subsequently, as the early endosomes mature into late endosomes, the endosomal lumen becomes acidified, and the low pH of the late endosomes induces conformational reaggangements in the HA to initiate fusion between the endosomal and viral membranes. Upon fusion, the viral capsid disintegrates and the viral ribonucleoprotein (vRNP) complexes containing the viral genome are released into the cytosol. The process of viral capsid disintegration is called "uncoating". After successful uncoating, the vRNPs are imported into the nucleus by importin α/ß (IMP α/ß), where viral replication and transcription take place and the new vRNPs are assembled. Recently, we have biochemically elucidated the molecular mechanisms of the processes of viral capsid uncoating subsequent viral genome dissociation. In this chapter, we present the molecular details of the viral uncoating process.

The Art of Viral Membrane Fusion and Penetration.

As obligate pathogens, viruses have developed diverse mechanisms to deliver their genome across host cell membranes to sites of virus replication. While enveloped viruses utilize viral fusion proteins to accomplish fusion of their envelope with the cellular membrane, non-enveloped viruses rely on machinery that causes local membrane ruptures and creates an opening through which the capsid or viral genome is released. Both membrane fusion and membrane penetration take place at the plasma membrane or in intracellular compartments, often involving the engagement of the cellular machinery and antagonism of host restriction factors. Enveloped and non-enveloped viruses have evolved intricate mechanisms to enable virus uncoating and modulation of membrane fusion in a spatiotemporally controlled manner. This chapter summarizes and discusses the current state of understanding of the mechanisms of viral membrane fusion and penetration. The focus is on the role of lipids, viral scaffold uncoating, viral membrane fusion inhibitors, and host restriction factors as physicochemical modulators. In addition, recent advances in visualizing and detecting viral membrane fusion and penetration using cryo-electron microscopy methods are presented.

Dynactin 1 negatively regulates HIV-1 infection by sequestering the host cofactor CLIP170.

Many viruses directly engage and require the dynein-dynactin motor-adaptor complex in order to transport along microtubules (MTs) to the nucleus and initiate infection. HIV type 1 (HIV-1) exploits dynein, the dynein adaptor BICD2, and core dynactin subunits but unlike several other viruses, does not require dynactin-1 (DCTN1). The underlying reason for HIV-1's variant dynein engagement strategy and independence from DCTN1 remains unknown. Here, we reveal that DCTN1 actually inhibits early HIV-1 infection by interfering with the ability of viral cores to interact with critical host cofactors. Specifically, DCTN1 competes for binding to HIV-1 particles with cytoplasmic linker protein 170 (CLIP170), one of several MT plus-end tracking proteins (+TIPs) that regulate the stability of viral cores after entry into the cell. Outside of its function as a dynactin subunit, DCTN1 also functions as a +TIP that we find sequesters CLIP170 from incoming particles. Deletion of the Zinc knuckle (Zn) domain in CLIP170 that mediates its interactions with several proteins, including DCTN1, increased CLIP170 binding to virus particles but failed to promote infection, further suggesting that DCTN1 blocks a critical proviral function of CLIP170 mediated by its Zn domain. Our findings suggest that the unique manner in which HIV-1 binds and exploits +TIPs to regulate particle stability leaves them vulnerable to the negative effects of DCTN1 on +TIP availability and function, which may in turn have driven HIV-1 to evolve away from DCTN1 in favor of BICD2-based engagement of dynein during early infection.

HIV-1 cores retain their integrity until minutes before uncoating in the nucleus.

We recently reported that HIV-1 cores that retained >94% of their capsid (CA) protein entered the nucleus and disassembled (uncoated) near their integration site <1.5 h before integration. However, whether the nuclear capsids lost their integrity by rupturing or a small loss of CA before capsid disassembly was unclear. Here, we utilized a previously reported vector in which green fluorescent protein is inserted in HIV-1 Gag (iGFP); proteolytic processing efficiently releases GFP, some of which remains trapped inside capsids and serves as a fluid phase content marker that is released when the capsids lose their integrity. We found that nuclear capsids retained their integrity until shortly before integration and lost their GFP content marker ∼1 to 3 min before loss of capsid-associated mRuby-tagged cleavage and polyadenylation specificity factor 6 (mRuby-CPSF6). In contrast, loss of GFP fused to CA and mRuby-CPSF6 occurred simultaneously, indicating that viral cores retain their integrity until just minutes before uncoating. Our results indicate that HIV-1 evolved to retain its capsid integrity and maintain a separation between macromolecules in the viral core and the nuclear environment until uncoating occurs just before integration. These observations imply that intact HIV-1 capsids are imported through nuclear pores; that reverse transcription occurs in an intact capsid; and that interactions between the preintegration complex and LEDGF/p75, and possibly other host factors that facilitate integration, must occur during the short time period between loss of capsid integrity and integration.

Morpholinodiazenyl chalcone blocks influenza A virus capsid uncoating by perturbing the clathrin-mediated vesicular trafficking pathway.

Influenza A virus (IAV) is a highly contagious respiratory pathogen that significantly threatens global health by causing seasonal epidemics and occasional, unpredictable pandemics. To identify new compounds with therapeutic potential against IAV, we designed and synthesized a series of 4'-morpholinodiazenyl chalcones using the molecular hybridization method, performed a high-content screen against IAV, and found that (E)-1-{4-[(E)-morpholinodiazenyl]phenyl}-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (MC-22) completely neutralized IAV infection. While MC-22 allowed IAV to successfully internalize into the cell and fuse at the acidic late endosomes, it prevented viral capsid uncoating and genome release. Since IAV majorly utilizes clathrin-mediated endocytosis (CME) for cellular entry, we examined whether MC-22 had any effect on CME, using nonviral cargoes that enter cells via clathrin-dependent or -independent pathways. Although MC-22 showed no effect on the uptake of choleratoxin B, a cargo that enters cells majorly via the clathrin-independent pathway, it significantly attenuated the clathrin-dependent internalization of both epidermal growth factor and transferrin. Cell biological analyses revealed a marked increase in the size of early endosomes upon MC-22 treatment, indicating an endosomal trafficking/maturation defect. This study reports the identification of MC-22 as a novel CME-targeting, highly potent IAV entry inhibitor, which is expected to neutralize a broad spectrum of viruses that enter the host cells via CME.

May I Help You with Your Coat? HIV-1 Capsid Uncoating and Reverse Transcription.

The human immunodeficiency virus type 1 (HIV-1) capsid is a protein core formed by multiple copies of the viral capsid (CA) protein. Inside the capsid, HIV-1 harbours all the viral components required for replication, including the genomic RNA and viral enzymes reverse transcriptase (RT) and integrase (IN). Upon infection, the RT transforms the genomic RNA into a double-stranded DNA molecule that is subsequently integrated into the host chromosome by IN. For this to happen, the viral capsid must open and release the viral DNA, in a process known as uncoating. Capsid plays a key role during the initial stages of HIV-1 replication; therefore, its stability is intimately related to infection efficiency, and untimely uncoating results in reverse transcription defects. How and where uncoating takes place and its relationship with reverse transcription is not fully understood, but the recent development of novel biochemical and cellular approaches has provided unprecedented detail on these processes. In this review, we present the latest findings on the intricate link between capsid stability, reverse transcription and uncoating, the different models proposed over the years for capsid uncoating, and the role played by other cellular factors on these processes.

Atomic Structures of Coxsackievirus B5 Provide Key Information on Viral Evolution and Survival.

Coxsackie virus B5 (CVB5), a main serotype in human Enterovirus B (EVB), can cause severe viral encephalitis and aseptic meningitis among infants and children. Currently, there is no approved vaccine or antiviral therapy available against CVB5 infection. Here, we determined the atomic structures of CVB5 in three forms: mature full (F) particle (2.73 Å), intermediate altered (A) particle (2.81 Å), and procapsid empty (E) particle (2.95 Å). Structural analysis of F particle of CVB5 unveiled similar structures of "canyon," "puff," and "knob" as those other EV-Bs. We observed structural rearrangements that are alike during the transition from F to A particle, indicative of similar antigenicity, cell entry, and uncoating mechanisms shared by all EV-Bs. Further comparison of structures and sequences among all structure-known EV-Bs revealed that while the residues targeted by neutralizing MAbs are diversified and drive the evolution of EV-Bs, the relative conserved residues recognized by uncoating receptors could serve as the basis for the development of antiviral vaccines and therapeutics. IMPORTANCE As one of the main serotypes in Enterovirus B, CVB5 has been commonly reported in recent years. The atomic structures of CVB5 shown here revealed classical features found in EV-Bs and the structural rearrangement occurring during particle expansion and uncoating. Also, structure- and sequence-based comparison between CVB5 and other structure-known EV-Bs screened out key domains important for viral evolution and survival. All these provide insights into the development of vaccine and therapeutics for EV-Bs.

Membrane Interactions and Uncoating of Aichi Virus, a Picornavirus That Lacks a VP4.

Kobuviruses are an unusual and poorly characterized genus within the picornavirus family and can cause gastrointestinal enteric disease in humans, livestock, and pets. The human kobuvirus Aichi virus (AiV) can cause severe gastroenteritis and deaths in children below the age of 5 years; however, this is a very rare occurrence. During the assembly of most picornaviruses (e.g., poliovirus, rhinovirus, and foot-and-mouth disease virus), the capsid precursor protein VP0 is cleaved into VP4 and VP2. However, kobuviruses retain an uncleaved VP0. From studies with other picornaviruses, it is known that VP4 performs the essential function of pore formation in membranes, which facilitates transfer of the viral genome across the endosomal membrane and into the cytoplasm for replication. Here, we employ genome exposure and membrane interaction assays to demonstrate that pH plays a critical role in AiV uncoating and membrane interactions. We demonstrate that incubation at low pH alters the exposure of hydrophobic residues within the capsid, enhances genome exposure, and enhances permeabilization of model membranes. Furthermore, using peptides we demonstrate that the N terminus of VP0 mediates membrane pore formation in model membranes, indicating that this plays an analogous function to VP4. IMPORTANCE To initiate infection, viruses must enter a host cell and deliver their genome into the appropriate location. The picornavirus family of small nonenveloped RNA viruses includes significant human and animal pathogens and is also a model to understand the process of cell entry. Most picornavirus capsids contain the internal protein VP4, generated from cleavage of a VP0 precursor. During entry, VP4 is released from the capsid. In enteroviruses this forms a membrane pore, which facilitates genome release into the cytoplasm. Due to high levels of sequence similarity, it is expected to play the same role for other picornaviruses. Some picornaviruses, such as Aichi virus, retain an intact VP0, and it is unknown how these viruses rearrange their capsids and induce membrane permeability in the absence of VP4. Here, we have used Aichi virus as a model VP0 virus to test for conservation of function between VP0 and VP4. This could enhance understanding of pore function and lead to development of novel therapeutic agents that block entry.

Region-Specific Hepatitis B Virus Genome Exposure from Nucleocapsid Modulated by Capsid Linker Sequence and Inhibitor: Implications for Uncoating.

Hepatitis B virus (HBV) contains a partially double-stranded, relaxed circular (RC) DNA genome synthesized within a nucleocapsid (NC) in the host cell cytoplasm. The release of RC DNA from the NC, in an ill-defined process called uncoating, to the nucleus is required for its conversion to the covalently closed circular (CCC) DNA, the viral episome serving as the transcriptional template for all viral RNAs necessary for replication and, thus, essential for establishing and sustaining viral infection. In efforts to better understand uncoating, we analyzed HBV core (HBc) mutants that show various levels of nuclear CCC DNA but little to no cytoplasmic RC DNA. We found that RC DNA could be synthesized by these mutants outside the cell, but in contrast to the wild type (wt), the mutant NCs were unable to protect RC DNA from digestion by the endogenous nuclease(s) in cellular lysates or exogenous DNase. Subcellular fractionation suggested that the major RC DNA-degrading activity was membrane associated. Digestion with sequence-specific and nonspecific DNases revealed the exposure of specific regions of RC DNA from the mutant NC. Similarly, treatment of wt NCs with a core inhibitor known to increase CCC DNA by affecting uncoating also led to region-specific exposure of RC DNA. Furthermore, a subpopulation of untreated wild type (wt) mature NCs showed site-specific exposure of RC DNA as well. Competition between RC DNA degradation and its conversion to CCC DNA during NC uncoating thus likely plays an important role in the establishment and persistence of HBV infection and has implications for the development of capsid-targeted antivirals. IMPORTANCE Disassembly of the hepatitis B virus (HBV) nucleocapsid (NC) to release its genomic DNA, in an ill-understood process called uncoating, is required to form the viral nuclear episome in the host cell nucleus, a viral DNA essential for establishing and sustaining HBV infection. The elimination of the HBV nuclear episome remains the holy grail for the development of an HBV cure. We report here that the HBV genomic DNA is exposed in a region-specific manner during uncoating, which is enhanced by mutations of the capsid protein and a capsid-targeted antiviral compound. The exposure of the viral genome can result in its rapid degradation or, alternatively, can enhance the formation of the nuclear episome, thus having a major impact on HBV infection and persistence. These results are thus important for understanding fundamental mechanisms of HBV replication and persistence and for the ongoing pursuit of an HBV cure.

Molecular Determinants of Human Rhinovirus Infection, Assembly, and Conformational Stability at Capsid Protein Interfaces.

Human rhinovirus (HRV), one of the most frequent human pathogens, is the major causative agent of common colds. HRVs also cause or exacerbate severe respiratory diseases, such as asthma or chronic obstructive pulmonary disease. Despite the biomedical and socioeconomic importance of this virus, no anti-HRV vaccines or drugs are available yet. Protein-protein interfaces in virus capsids have increasingly been recognized as promising virus-specific targets for the development of antiviral drugs. However, the specific structural elements and residues responsible for the biological functions of these extended capsid regions are largely unknown. In this study, we performed a thorough mutational analysis to determine which particular residues along the capsid interpentamer interfaces are relevant to HRV infection as well as the stage(s) in the viral cycle in which they are involved. The effect on the virion infectivity of the individual mutation to alanine of 32 interfacial residues that, together, removed most of the interpentamer interactions was analyzed. Then, a representative sample that included many of those 32 single mutants were tested for capsid and virion assembly as well as virion conformational stability. The results indicate that most of the interfacial residues, and the interactions they establish, are biologically relevant, largely because of their important roles in virion assembly and/or stability. The HRV interpentamer interface is revealed as an atypical protein-protein interface, in which infectivity-determining residues are distributed at a high density along the entire interface. Implications for a better understanding of the relationship between the molecular structure and function of HRV and the development of novel capsid interface-binding anti-HRV agents are discussed. IMPORTANCE The rising concern about the serious medical and socioeconomic consequences of respiratory infections by HRV has elicited a renewed interest in the development of anti-HRV drugs. The conversion into effective drugs of compounds identified via screening, as well as antiviral drug design, rely on the acquisition of fundamental knowledge about the targeted viral elements and their roles during specific steps of the infectious cycle. The results of this study provide a detailed view on structure-function relationships in a viral capsid protein-protein interface, a promising specific target for antiviral intervention. The high density and scattering of the interfacial residues found to be involved in HRV assembly and/or stability support the possibility that any compound designed to bind any particular site at the interface will inhibit infection by interfering with virion morphogenesis or stabilization of the functional virion conformation.

Chemical Evolution of Rhinovirus Identifies Capsid-Destabilizing Mutations Driving Low-pH-Independent Genome Uncoating.

Rhinoviruses (RVs) cause recurrent infections of the nasal and pulmonary tracts, life-threatening conditions in chronic respiratory illness patients, predisposition of children to asthmatic exacerbation, and large economic cost. RVs are difficult to treat. They rapidly evolve resistance and are genetically diverse. Here, we provide insight into RV drug resistance mechanisms against chemical compounds neutralizing low pH in endolysosomes. Serial passaging of RV-A16 in the presence of the vacuolar proton ATPase inhibitor bafilomycin A1 (BafA1) or the endolysosomotropic agent ammonium chloride (NH4Cl) promoted the emergence of resistant virus populations. We found two reproducible point mutations in viral proteins 1 and 3 (VP1 and VP3), A2526G (serine 66 to asparagine [S66N]), and G2274U (cysteine 220 to phenylalanine [C220F]), respectively. Both mutations conferred cross-resistance to BafA1, NH4Cl, and the protonophore niclosamide, as identified by massive parallel sequencing and reverse genetics, but not the double mutation, which we could not rescue. Both VP1-S66 and VP3-C220 locate at the interprotomeric face, and their mutations increase the sensitivity of virions to low pH, elevated temperature, and soluble intercellular adhesion molecule 1 receptor. These results indicate that the ability of RV to uncoat at low endosomal pH confers virion resistance to extracellular stress. The data endorse endosomal acidification inhibitors as a viable strategy against RVs, especially if inhibitors are directly applied to the airways. IMPORTANCE Rhinoviruses (RVs) are the predominant agents causing the common cold. Anti-RV drugs and vaccines are not available, largely due to rapid evolutionary adaptation of RVs giving rise to resistant mutants and an immense diversity of antigens in more than 160 different RV types. In this study, we obtained insight into the cell biology of RVs by harnessing the ability of RVs to evolve resistance against host-targeting small chemical compounds neutralizing endosomal pH, an important cue for uncoating of normal RVs. We show that RVs grown in cells treated with inhibitors of endolysosomal acidification evolved capsid mutations yielding reduced virion stability against elevated temperature, low pH, and incubation with recombinant soluble receptor fragments. This fitness cost makes it unlikely that RV mutants adapted to neutral pH become prevalent in nature. The data support the concept of host-directed drug development against respiratory viruses in general, notably at low risk of gain-of-function mutations.

HIV-1 uncoats in the nucleus near sites of integration.

HIV-1 capsid core disassembly (uncoating) must occur before integration of viral genomic DNA into the host chromosomes, yet remarkably, the timing and cellular location of uncoating is unknown. Previous studies have proposed that intact viral cores are too large to fit through nuclear pores and uncoating occurs in the cytoplasm in coordination with reverse transcription or at the nuclear envelope during nuclear import. The capsid protein (CA) content of the infectious viral cores is not well defined because methods for directly labeling and quantifying the CA in viral cores have been unavailable. In addition, it has been difficult to identify the infectious virions because only one of ∼50 virions in infected cells leads to productive infection. Here, we developed methods to analyze HIV-1 uncoating by direct labeling of CA with GFP and to identify infectious virions by tracking viral cores in living infected cells through viral DNA integration and proviral DNA transcription. Astonishingly, our results show that intact (or nearly intact) viral cores enter the nucleus through a mechanism involving interactions with host protein cleavage and polyadenylation specificity factor 6 (CPSF6), complete reverse transcription in the nucleus before uncoating, and uncoat <1.5 h before integration near (<1.5 µm) their genomic integration sites. These results fundamentally change our current understanding of HIV-1 postentry replication events including mechanisms of nuclear import, uncoating, reverse transcription, integration, and evasion of innate immunity.