Structural and virological studies of the stages of virus replication that are affected by antirhinovirus compounds

Pleconaril is a broad-spectrum antirhinovirus and antienterovirus compound that binds into a hydrophobic pocket within viral protein 1, stabilizing the capsid and resulting in the inhibition of cell attachment and RNA uncoating. When crystals of human rhinovirus 16 (HRV16) and HRV14 are incubated with pleconaril, drug occupancy in the binding pocket is lower than when pleconaril is introduced during assembly prior to crystallization. This effect is far more marked in HRV16 than in HRV14 and is more marked with pleconaril than with other compounds. These observations are consistent with virus yield inhibition studies and radiolabeled drug binding studies showing that the antiviral effect of pleconaril against HRV16 is greater on the infectivity of progeny virions than the parent input viruses. These data suggest that drug integration into the binding pocket during assembly, or at some other late stage in virus replication, may contribute to the antiviral activity of capsid binding compounds.     

Influence of nonuniform geometry on nanoindentation of viral capsids

A series of recent nanoindentation experiments on the protein shells (capsids) of viruses has established atomic force microscopy (AFM) as a useful framework for probing the mechanics of large protein assemblies. Specifically these experiments provide an opportunity to study the coupling of the global assembly response to local conformational changes. AFM experiments on cowpea chlorotic mottle virus, known to undergo a pH-controlled swelling conformational change, have revealed a pH-dependent mechanical response. Previous theoretical studies have shown that homogeneous changes in shell geometry can play a significant role in the mechanical response. This article develops a method for accurately capturing the heterogeneous geometry of a viral capsid and explores its effect on mechanical response with a nonlinear continuum elasticity model. Models of both native and swollen cowpea chlorotic mottle virus capsids are generated from x-ray crystal structures, and are used in finite element simulations of AFM indentation along two-, three-, and fivefold icosahedral symmetry orientations. The force response of the swollen capsid model is observed to be softer by roughly a factor of two, significantly more nonlinear, and more orientation-dependent than that of a native capsid with equivalent elastic moduli, demonstrating that capsid geometric heterogeneity can have significant effects on the global structural response.     

Distinct effects of two HIV-1 capsid assembly inhibitor families that bind the same site within the N-terminal domain of the viral CA protein

The emergence of resistance to existing classes of antiretroviral drugs necessitates finding new HIV-1 targets for drug discovery. The viral capsid (CA) protein represents one such potential new target. CA is sufficient to form mature HIV-1 capsids in vitro, and extensive structure-function and mutational analyses of CA have shown that the proper assembly, morphology, and stability of the mature capsid core are essential for the infectivity of HIV-1 virions. Here we describe the development of an in vitro capsid assembly assay based on the association of CA-NC subunits on immobilized oligonucleotides. This assay was used to screen a compound library, yielding several different families of compounds that inhibited capsid assembly. Optimization of two chemical series, termed the benzodiazepines (BD) and the benzimidazoles (BM), resulted in compounds with potent antiviral activity against wild-type and drug-resistant HIV-1. Nuclear magnetic resonance (NMR) spectroscopic and X-ray crystallographic analyses showed that both series of inhibitors bound to the N-terminal domain of CA. These inhibitors induce the formation of a pocket that overlaps with the binding site for the previously reported CAP inhibitors but is expanded significantly by these new, more potent CA inhibitors. Virus release and electron microscopic (EM) studies showed that the BD compounds prevented virion release, whereas the BM compounds inhibited the formation of the mature capsid. Passage of virus in the presence of the inhibitors selected for resistance mutations that mapped to highly conserved residues surrounding the inhibitor binding pocket, but also to the C-terminal domain of CA. The resistance mutations selected by the two series differed, consistent with differences in their interactions within the pocket, and most also impaired virus replicative capacity. Resistance mutations had two modes of action, either directly impacting inhibitor binding affinity or apparently increasing the overall stability of the viral capsid without affecting inhibitor binding. These studies demonstrate that CA is a viable antiviral target and demonstrate that inhibitors that bind within the same site on CA can have distinct binding modes and mechanisms of action.     

Design, synthesis, and evaluation of dioxane-based antiviral agents targeted against the Sindbis virus capsid protein

Dioxane-based antiviral agents targeted to the hydrophobic binding pocket of Sindbis virus capsid protein were designed by computer graphics molecular modeling and synthesized. Virus production using SIN-IRES-Luc and capsid assembly were monitored to evaluate antiviral activity. A compound with a three-carbon linker chain connecting two dioxane moieties inhibited virus production by 50% at a concentration of 40 microM, while (R)-hydroxymethyldioxane inhibited virus production by 50% at a concentration of 1 microM. Both compounds were not cytotoxic in uninfected BHK cells at concentrations of 1mM.     

Imaging of the Alphavirus Capsid Protein during Virus Replication

Alphaviruses are enveloped viruses with highly organized structures. The nucleocapsid (NC) core contains a capsid protein lattice enclosing the plus-sense RNA genome, and it is surrounded by a lipid bilayer containing a lattice of the E1 and E2 envelope glycoproteins. Capsid protein is synthesized in the cytoplasm and particle budding occurs at the plasma membrane (PM), but the traffic and assembly of viral components and the exit of virions from host cells are not well understood. To visualize the dynamics of capsid protein during infection, we developed a Sindbis virus infectious clone tagged with a tetracysteine motif. Tagged capsid protein could be fluorescently labeled with biarsenical dyes in living cells without effects on virus growth, morphology, or protein distribution. Live cell imaging and colocalization experiments defined distinct groups of capsid foci in infected cells. We observed highly motile internal puncta that colocalized with E2 protein, which may represent the transport machinery that capsid protein uses to reach the PM. Capsid was also found in larger nonmotile internal structures that colocalized with cellular G3BP and viral nsP3. Thus, capsid may play an unforeseen role in these previously observed G3BP-positive foci, such as regulation of cellular stress granules. Capsid puncta were also observed at the PM. These puncta colocalized with E2 and recruited newly synthesized capsid protein; thus, they may be sites of virus assembly and egress. Together, our studies provide the first dynamic views of the alphavirus capsid protein in living cells and a system to define detailed mechanisms during alphavirus infection.     

A small molecule inhibits and misdirects assembly of hepatitis B virus capsids

Hepatitis B virus (HBV) capsids play an important role in viral nucleic acid metabolism and other elements of the virus life cycle. Misdirection of capsid assembly (leading to formation of aberrant particles) may be a powerful approach to interfere with virus production. HBV capsids can be assembled in vitro from the dimeric capsid protein. We show that a small molecule, bis-ANS, binds to capsid protein, inhibiting assembly of normal capsids and promoting assembly of noncapsid polymers. Using equilibrium dialysis to investigate binding of bis-ANS to free capsid protein, we found that only one bis-ANS molecule binds per capsid protein dimer, with an association energy of -28.0 +/- 2.0 kJ/mol (-6.7 +/- 0.5 kcal/mol). Bis-ANS inhibited in vitro capsid assembly induced by ionic strength as observed by light scattering and size exclusion chromatography. The binding energy of bis-ANS for capsid protein calculated from assembly inhibition data was -24.5 +/- 0.9 kJ/mol (-5.9 +/- 0.2 kcal/mol), essentially the same binding energy observed in studies of unassembled protein. These data indicate that capsid protein bound to bis-ANS did not participate in assembly; this mechanism of assembly inhibition is analogous to competitive or noncompetitive inhibition of enzymes. While assembly of normal capsids is inhibited, our data suggest that bis-ANS leads to formation of noncapsid polymers. Evidence of aberrant polymers was identified by light scattering and electron microscopy. We propose that bis-ANS acts as a molecular "wedge" that interferes with normal capsid protein geometry and capsid formation; such wedges may represent a new class of antiviral agent.     

衣壳蛋白靶向灭活策略应用于抗登革病毒感染的研究

衣壳蛋白靶向病毒灭活是近年来新兴的抗病毒策略。为探索该策略在抗登革病毒感染中的应用 ,首先建立了稳定表达登革 2型病毒衣壳蛋白 (D2C)与葡萄球菌核酸酶 (SN)融合蛋白D2C_SN的哺乳动物细胞系 ,然后以登革病毒攻击上述细胞系 ,研究表达的融合蛋白D2C_SN对产生的子代病毒颗粒感染性的影响。结果表明融合蛋白D2C_SN能够在病毒装配过程中与野生型衣壳蛋白共组装入子代病毒颗粒内部 ,并导致病毒基因组的降解。与正常BHK细胞相比较 ,融合蛋白D2C_SN可导致产生的子代病毒感染性滴度降低 10 3～ 10 4 ,显示出很强的抗病毒效果     

Rhodanine resistance and dependence of echovirus 12: a possible consequence of capsid flexibility.

Recombinant viruses of echovirus 12 carrying mutations of a rhodanine-resistant or -dependent variant, were investigated, and five single mutations each inducing a rhodanine-resistant or -dependent phenotype were defined. Four mutations are localized in the capsid protein VP1, and the fifth exchange is in VP4. All original and recombinant viruses were shown to be stabilized by the antiviral drug rhodanine against heat inactivation. Hence, resistant and dependent variants still seem able to bind rhodanine, and apparently none of the exchanges affects the putative drug binding site. We hypothesize that drug resistance and dependence are consequences of an increased flexibility of the virus capsid.     

VP1 sequencing of all human rhinovirus serotypes: insights into genus phylogeny and susceptibility to antiviral capsid-binding compounds

Rhinoviruses are the most common infectious agents of humans. They are the principal etiologic agents of afebrile viral upper-respiratory-tract infections (the common cold). Human rhinoviruses (HRVs) comprise a genus within the family Picornaviridae. There are >100 serotypically distinct members of this genus. In order to better understand their phylogenetic relationship, the nucleotide sequence for the major surface protein of the virus capsid, VP1, was determined for all known HRV serotypes and one untyped isolate (HRV-Hanks). Phylogenetic analysis of deduced amino acid sequence data support previous studies subdividing the genus into two species containing all but one HRV serotype (HRV-87). Seventy-five HRV serotypes and HRV-Hanks belong to species HRV-A, and twenty-five HRV serotypes belong to species HRV-B. Located within VP1 is a hydrophobic pocket into which small-molecule antiviral compounds such as pleconaril bind and inhibit functions associated with the virus capsid. Analyses of the amino acids that constitute this pocket indicate that the sequence correlates strongly with virus susceptibility to pleconaril inhibition. Further, amino acid changes observed in reduced susceptibility variant viruses recovered from patients enrolled in clinical trials with pleconaril were distinct from those that confer natural phenotypic resistance to the drug. These observations suggest that it is possible to differentiate rhinoviruses naturally resistant to capsid function inhibitors from those that emerge from susceptible virus populations as a result of antiviral drug selection pressure based on sequence analysis of the drug-binding pocket.     

Effects of hydrostatic pressure on a membrane-enveloped virus: high immunogenicity of the pressure-inactivated virus.

A new approach to the preparation of antiviral vaccines relying on the inactivation of the virus particle by hydrostatic pressure is described. The enveloped virus vesicular stomatitis virus was utilized as a model; a pressure of 260 MPa applied for 12 h reduced infectivity by a factor of 10(4), and the antibodies against pressurized material were as effective as those against the intact virus when measured by their neutralization titer. Fluorescence measurements indicate that application of pressure results in perturbations of the particle interactions that permit binding of specific molecular probes. Electron microscopy showed that the membrane of the pressurized virus was partially preserved, presenting the spike pattern of the membrane G protein. Unlike the icosahedral viruses, dissociation into smaller particles was not observed, but a constant change in the morphology was the presence of a bulge in the surface of the pressurized virus, indicating a displacement of the capsid subunits, retained under the lipid and protein membrane.     

Sulfamoylbenzamide Derivatives Inhibit the Assembly of Hepatitis B Virus Nucleocapsids

Chronic hepatitis B virus (HBV) infection, a serious public health problem leading to cirrhosis and hepatocellular carcinoma, is currently treated with either pegylated alpha interferon (pegIFN-α) or one of the five nucleos(t)ide analogue viral DNA polymerase inhibitors. However, neither pegIFN-α nor nucleos(t)ide analogues are capable of reliably curing the viral infection. In order to develop novel antiviral drugs against HBV, we established a cell-based screening assay by using an immortalized mouse hepatocyte-derived stable cell line supporting a high level of HBV replication in a tetracycline-inducible manner. Screening of a library consisting of 26,900 small molecules led to the discovery of a series of sulfamoylbenzamide (SBA) derivatives that significantly reduced the amount of cytoplasmic HBV DNA. Structure-activity relationship studies have thus far identified a group of fluorine-substituted SBAs with submicromolar antiviral activity against HBV in human hepatoma cells. Mechanistic analyses reveal that the compounds dose dependently inhibit the formation of pregenomic RNA (pgRNA)-containing nucleocapsids of HBV but not other animal hepadnaviruses, such as woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV). Moreover, heterologous genetic complementation studies of capsid protein, DNA polymerase, and pgRNA between HBV and WHV suggest that HBV capsid protein confers sensitivity to the SBAs. In summary, SBAs represent a novel chemical entity with superior activity and a unique antiviral mechanism and are thus warranted for further development as novel antiviral therapeutics for the treatment of chronic hepatitis B.     

Weak protein-protein interactions are sufficient to drive assembly of hepatitis B virus capsids

Hepatitis B virus (HBV) is an enveloped DNA virus with a spherical capsid (or core). The capsid is constructed from 120 copies of the homodimeric capsid protein arranged with T = 4 icosahedral symmetry. We examined in vitro assembly of purified E. coli expressed HBV capsid protein. After equilibration, concentrations of capsid and dimer were evaluated by size exclusion chromatography. The extent of assembly increased as temperature and ionic strength increased. The concentration dependence of capsid assembly conformed to the equilibrium expression: K(capsid) = [capsid]/[dimer](120). Given the known geometry for HBV capsids and dimers, the per capsid assembly energy was partitioned into energy per subunit-subunit contact. We were able to make three major conclusions. (i) Weak interactions (from -2.9 kcal/mol at 21 degrees C in low salt to -4.4 kcal/mol at 37 degrees C in high salt) at each intersubunit contact result in a globally stable capsid; weak intersubunit interactions may be the basis for the phenomenon of capsid breathing. (ii) HBV assembly is characterized by positive enthalpy and entropy. The reaction is entropy-driven, consistent with the largely hydrophobic contacts found in the crystal structure. (iii) Increasing NaCl concentration increases the magnitude of free energy, enthalpy, and entropy, as if ionic strength were increasing the amount of hydrophobic surface buried by assembly. This last point leads us to suggest that salt acts by inducing a conformational change in the dimer from an assembly-inactive form to an assembly-active form. This model of conformational change linked to assembly is consistent with immunological differences between dimer and capsid.     

An in vitro fluorescence screen to identify antivirals that disrupt hepatitis B virus capsid assembly

Virus assembly has not been routinely targeted in the development of antiviral drugs, in part because of the lack of tractable methods for screening in vitro. We have developed an in vitro assay of hepatitis B virus (HBV) capsid assembly, based on fluorescence quenching of dye-labeled capsid protein, for testing potential inhibitors. This assay is adaptable to high-throughput screening and can identify small-molecule inhibitors of virus assembly that prevent, inappropriately accelerate and/or misdirect capsid formation to yield aberrant particles. An in vitro primary screen has the advantage of identifying promising lead compounds affecting assembly without the requirement that they be taken up by cells in culture and be nontoxic. Our approach may facilitate the identification of antivirals targeting viruses other than HBV, such as avian influenza and HIV.     

Effect of macromolecular crowding agents on human immunodeficiency virus type 1 capsid protein assembly in vitro

Previous studies on the self-assembly of capsid protein CA of human immunodeficiency virus type 1 (HIV-1) in vitro have provided important insights on the structure and assembly of the mature HIV-1 capsid. However, CA polymerization in vitro was previously observed to occur only at very high ionic strength. Here, we have analyzed the effects on CA assembly in vitro of adding unrelated, inert macromolecules (crowding agents), aimed at mimicking the crowded (very high macromolecular effective concentration) environment within the HIV-1 virion. Crowding agents induced fast and efficient polymerization of CA even at low (close to physiological) ionic strength. The hollow cylinders thus assembled were indistinguishable in shape and dimensions from those formed in dilute protein solutions at high ionic strength. However, two important differences were noted: (i) disassembly by dilution of the capsid-like particles was undetectable at very high ionic strength, but occurred rapidly at low ionic strength in the presence of a crowding agent, and (ii) a variant CA from a presumed infectious HIV-1 with mutations at the CA dimerization interface was unable to assemble at any ionic strength in the absence of a crowding agent; in contrast, this mutation allowed efficient assembly, even at low ionic strength, when a crowding agent was used. The use of a low ionic strength and inert macromolecules to mimic the crowded environment inside the HIV-1 virion may lead to a better in vitro evaluation of the effects of conditions, mutations or/and other molecules, including potential antiviral compounds, on HIV-1 capsid assembly, stability and disassembly.     

Mechanical fatigue in repetitively stretched single molecules of titin

Relaxed striated muscle cells exhibit mechanical fatigue when exposed to repeated stretch and release cycles. To understand the molecular basis of such mechanical fatigue, single molecules of the giant filamentous protein titin, which is the main determinant of sarcomeric elasticity, were repetitively stretched and released while their force response was characterized with optical tweezers. During repeated stretch-release cycles titin becomes mechanically worn out in a process we call molecular fatigue. The process is characterized by a progressive shift of the stretch-force curve toward increasing end-to-end lengths, indicating that repeated mechanical cycles increase titin's effective contour length. Molecular fatigue occurs only in a restricted force range (0-25 pN) during the initial part of the stretch half-cycle, whereas the rest of the force response is repeated from one mechanical cycle to the other. Protein-folding models fail to explain molecular fatigue on the basis of an incomplete refolding of titin's globular domains. Rather, the process apparently derives from the formation of labile nonspecific bonds cross-linking various sites along a pre-unfolded titin segment. Because titin's molecular fatigue occurs in a physiologically relevant force range, the process may play an important role in dynamically adjusting muscle's response to the recent history of mechanical perturbations.     

Mechanisms of Antiviral Action of Plant Antimicrobials against Murine Norovirus

Numerous plant compounds have antibacterial or antiviral properties; however, limited research has been conducted with nonenveloped viruses. The efficacies of allspice oil, lemongrass oil, and citral were evaluated against the nonenveloped murine norovirus (MNV), a human norovirus surrogate. The antiviral mechanisms of action were also examined using an RNase I protection assay, a host cell binding assay, and transmission electron microscopy. All three antimicrobials produced significant reductions (P ≤ 0.05) in viral infectivity within 6 h of exposure (0.90 log₁₀ to 1.88 log₁₀). After 24 h, the reductions were 2.74, 3.00, and 3.41 log₁₀ for lemongrass oil, citral, and allspice oil, respectively. The antiviral effect of allspice oil was both time and concentration dependent; the effects of lemongrass oil and citral were time dependent. Based on the RNase I assay, allspice oil appeared to act directly upon the viral capsid and RNA. The capsids enlarged from ≤35 nm to up to 75 nm following treatment. MNV adsorption to host cells was not significantly affected. Alternatively, the capsid remained intact following exposure to lemongrass oil and citral, which appeared to coat the capsid, causing nonspecific and nonproductive binding to host cells that did not lead to successful infection. Such contrasting effects between allspice oil and both lemongrass oil and citral suggest that though different plant compounds may yield similar reductions in virus infectivity, the mechanisms of inactivation may be highly varied and specific to the antimicrobial. This study demonstrates the antiviral properties of allspice oil, lemongrass oil, and citral against MNV and thus indicates their potential as natural food and surface sanitizers to control noroviruses.     

New class of orthopoxvirus antiviral drugs that block viral maturation

By using a homology-based bioinformatics approach, a structural model of the vaccinia virus (VV) I7L proteinase was developed. A unique chemical library of approximately 51,000 compounds was computationally queried to identify potential active site inhibitors. The resulting biased subset of compounds was assayed for both toxicity and the ability to inhibit the growth of VV in tissue culture cells. A family of chemotypically related compounds was found which exhibits selective activity against orthopoxviruses, inhibiting VV with 50% inhibitory concentrations of 3 to 12 microM. These compounds exhibited no significant cytotoxicity in the four cell lines tested and did not inhibit the growth of other organisms such as Saccharomyces cerevisiae, Pseudomonas aeruginosa, adenovirus, or encephalomyocarditis virus. Phenotypic analyses of virus-infected cells were conducted in the presence of active compounds to verify that the correct biochemical step (I7L-mediated core protein processing) was being inhibited. Electron microscopy of compound-treated VV-infected cells indicated a block in morphogenesis. Compound-resistant viruses were generated and resistance was mapped to the I7L open reading frame. Transient expression with the mutant I7L gene rescued the ability of wild-type virus to replicate in the presence of compound, indicating that this is the only gene necessary for resistance. This novel class of inhibitors has potential for development as an efficient antiviral drug against pathogenic orthopoxviruses, including smallpox.