Pyroptosis, a type of programmed necrosis associated with inflammatory, is a host defense mechanism against microbial infections. Although Chlamydia has been shown to induce pyroptosis, whether pyroptosis directly impacts the growth of Chlamydia has not been demonstrated. In this study, we found that C. trachomatis L2 infection of the mouse macrophage RAW 264.7 cells induced pyroptosis by monitoring the ultrastructural changes under transmission electron microscopy and the release of LDH and IL-1β. More importantly, this C. trachomatis-triggered pyroptosis with activation of caspase-1 and caspase-11 was also accompanied by gasdermin D (GSDMD) activation. Suppression of these two inflammatory caspases inhibited GSDMD activation. Interestingly, the C. trachomatis-triggered pyroptosis significantly inhibited the intracellular growth of C. trachomatis since inactivation of either GSDMD or caspase-1/11 significantly rescued infectious C. trachomatis yields, which suggests pyroptosis response can be utilized as an intrinsic mechanism to restrict C. trachomatis intracellular infection in addition to the well- documented extrinsic mechanisms by recruiting and enhancing inflammatory responses. This study may reveal novel targets for attenuating C. trachomatis infectivity and/or pathogenicity.

Background To contain the pandemics caused by SARS-CoV-2, early detection approaches with high accuracy and accessibility are critical. Generating an antigen-capture based detection system would be an ideal strategy complementing the current methods based on nucleic acids and antibody detection. The spike protein is found on the outside of virus particles and appropriate for antigen detection. Methods In this study, we utilized bioinformatics approaches to explore the immunodominant fragments on spike protein of SARS-CoV-2. Results The S1 subunit of spike protein was identified with higher sequence specificity. Three immunodominant fragments, Spike56-94, Spike199-264, and Spike577-612, located at the S1 subunit were finally selected via bioinformatics analysis. The glycosylation sites and high-frequency mutation sites on spike protein were circumvented in the antigen design. All the identified fragments present qualified antigenicity, hydrophilicity, and surface accessibility. A recombinant antigen with a length of 194 amino acids (aa) consisting of the selected immunodominant fragments as well as a universal Th epitope was finally constructed. Conclusion The recombinant peptide encoded by the construct contains multiple immunodominant epitopes, which is expected to stimulate a strong immune response in mice and generate qualified antibodies for SARS-CoV-2 detection.

Significance We constructed viable poliovirus (PV) variants carrying up to 2,104 synonymous mutations in the ORF. We studied proliferation phenotypes, particularly the specific infectivity ( si ) that defines the probability of a single virion to initiate a single cell infection. A recent hypothesis proposes that dozens of loosely conserved hairpins, formed within the viral genome, determine assembly specificity of +ssRNA viruses. Our analysis of recoded PVs does not support this hypothesis. We propose that the progression of +ssRNA virus assembly follows two steps. ( i ) Specificity: recognition between either ( a ) replication protein and capsid precursor or ( b ) single RNA packaging signal and cognate capsid precursor. ( ii ) Cocondensation, occurring between single genome/single capsid precursor complex and multiple cognate capsids aided by multiple genome-specific hairpins.

ABSTRACT The specificity of encapsidation of C-cluster enteroviruses depends on an interaction between capsid proteins and nonstructural protein 2C ATPase . In particular, residue N 252 of poliovirus 2C ATPase interacts with VP3 of coxsackievirus A20, in the context of a chimeric virus. Poliovirus 2C ATPase has important roles both in RNA replication and encapsidation. In this study, we searched for additional sites in 2C ATPase , near N 252 , that are required for encapsidation. Accordingly, segments adjacent to N 252 were analyzed by combining triple and single alanine mutations to identify residues required for function. Two triple alanine mutants exhibited defects in RNA replication. The remaining two mutations, located in secondary structures in a predicted three-dimensional model of 2C ATPase , caused lethal growth phenotypes. Most single alanine mutants, derived from the lethal variants, were either quasi-infectious and yielded variants with wild-type (wt) or temperature-sensitive ( ts ) growth phenotypes or had a lethal growth phenotype due to defective RNA replication. The K 259 A mutation, mapping to an α helix in the predicted structure of 2C ATPase , resulted in a cold-sensitive virus. In vivo protein synthesis and virus production were strikingly delayed at 33°C relative to the wt, suggesting a defect in uncoating. Studies with a reporter virus indicated that this mutant is also defective in encapsidation at 33°C. Cell imaging confirmed a much-reduced production of K 259 A mature virus at 33°C relative to the wt. In conclusion, we have for the first time linked a cold-sensitive encapsidation defect in 2C ATPase (K 259 A) to a subsequent delay in uncoating of the virus particle at 33°C during the next cycle of infection. IMPORTANCE Enterovirus morphogenesis, which involves the encapsidation of newly made virion RNA, is a process still poorly understood. Elucidation of this process is important for future drug development for a large variety of diseases caused by these agents. We have previously shown that the specificity of encapsidation of poliovirus and of C-cluster coxsackieviruses, which are prototypes of enteroviruses, is dependent on an interaction of capsid proteins with the multifunctional nonstructural protein 2C ATPase . In this study, we have searched for residues in poliovirus 2C ATPase , near a presumed capsid-interacting site, important for encapsidation. An unusual cold-sensitive mutant of 2C ATPase possessed a defect in encapsidation at 37°C and subsequently in uncoating during the next cycle of infection at 33°C. These studies not only reveal a new site in 2C ATPase that is involved in encapsidation but also identify a link between encapsidation and uncoating.

A central paradigm within virology is that each viral particle largely behaves as an independent infectious unit. Here, we demonstrate that clusters of enteroviral particles are packaged within phosphatidylserine (PS) lipid-enriched vesicles that are non-lytically released from cells and provide greater infection efficiency than free single viral particles. We show that vesicular PS lipids are co-factors to the relevant enterovirus receptors in mediating subsequent infectivity and transmission, in particular to primary human macrophages. We demonstrate that clustered packaging of viral particles within vesicles enables multiple viral RNA genomes to be collectively transferred into single cells. This study reveals a novel mode of viral transmission, where enteroviral genomes are transmitted from cell-to-cell en bloc in membrane-bound PS vesicles instead of as single independent genomes. This has implications for facilitating genetic cooperativity among viral quasispecies as well as enhancing viral replication.

The morphogenesis of viruses belonging to the genus Enterovirus in the family Picornaviridae is still poorly understood despite decades-long investigations. However, we recently provided evidence that 2C(ATPase) gives specificity to poliovirus encapsidation through an interaction with capsid protein VP3. The polypeptide 2C(ATPase) is a highly conserved non-structural protein of enteroviruses with important roles in RNA replication, encapsidation and uncoating. We have identified a site (K279/R280) near the C terminus of the polypeptide that is required for morphogenesis. The aim of the current project was to search for additional functional sites near the C terminus of the 2C(ATPase) polypeptide, with particular interest in those that are required for encapsidation. We selected for analysis a cysteine-rich site of the polypeptide and constructed four mutants in which cysteines or a histidine was changed to an alanine. The RNA transcripts were transfected into HeLa cells yielding two lethal, one temperature-sensitive and one quasi-infectious mutants. All four mutants exhibited normal protein translation in vitro and three of them possessed severe RNA replication defects. The quasi-infectious mutant (C286A) yielded variants with a pseudo-reversion at the original site (A286D), but some also contained one additional mutation: A138V or M293V. The temperature-sensitive mutant (C272A/H273A) exhibited an encapsidation and possibly also an uncoating defect at 37 °C. Variants of this mutant revealed suppressor mutations at three different sites in the 2C(ATPase) polypeptide: A138V, M293V and K295R. We concluded that the cysteine-rich site near the C terminus of 2C(ATPase) is involved in encapsidation, possibly through an interaction with an upstream segment located between boxes A and B of the nucleotide-binding domain.

Glutathione (GSH) is the most abundant cellular thiol playing an essential role in preserving a reduced cellular environment. Cellular GSH levels can be efficiently reduced by the GSH biosynthesis inhibitor, L-buthionine sulfoximine (BSO). The aim of our study was to determine the role of GSH in the growth of two C-cluster enteroviruses, poliovirus type 1 (PV1) and coxsackievirus A20 (CAV20). Our results show that the growth of both PV1 and CAV20 is strongly inhibited by BSO and can be partially reversed by the addition of GSH. BSO has no effect on viral protein synthesis or RNA replication but it strikingly reduces the accumulation of 14S pentamers in infected cells. GSH-pull down assays show that GSH directly interacts with capsid precursors and mature virus made in the absence of BSO whereas capsid precursors produced under GSH-depletion do not bind to GSH. In particular, the loss of binding of GSH may debilitate the stability of 14S pentamers, resulting in their failure to assemble into mature virus. Immunofluorescence cell imaging demonstrated that GSH-depletion did not affect the localization of viral capsid proteins to the replication complex. PV1 BSO resistant (BSOr) mutants evolved readily during passaging of the virus in the presence of BSO. Structural analyses revealed that the BSOr mutations, mapping to VP1 and VP3 capsid proteins, are primarily located at protomer/protomer interfaces. BSOr mutations might, in place of GSH, aid the stability of 14S particles that is required for virion maturation. Our observation that BSOr mutants are more heat resistant and need less GSH than wt virus to be protected from heat inactivation suggests that they possess a more stable capsid. We propose that the role of GSH during enterovirus morphogenesis is to stabilize capsid structures by direct interaction with capsid proteins both during and after the formation of mature virus particles.

Enteroviruses (family of the Picornaviridae) cover a large group of medically important human pathogens for which no antiviral treatment is approved. Although these viruses have been extensively studied, some aspects of the viral life cycle, in particular morphogenesis, are yet poorly understood. We report the discovery of TP219 as a novel inhibitor of the replication of several enteroviruses, including coxsackievirus and poliovirus. We show that TP219 binds directly glutathione (GSH), thereby rapidly depleting intracellular GSH levels and that this interferes with virus morphogenesis without affecting viral RNA replication. The inhibitory effect on assembly was shown not to depend on an altered reducing environment. Using TP219, we show that GSH is an essential stabilizing cofactor during the transition of protomeric particles into pentameric particles. Sequential passaging of coxsackievirus B3 in the presence of low GSH-levels selected for GSH-independent mutants that harbored a surface-exposed methionine in VP1 at the interface between two protomers. In line with this observation, enteroviruses that already contained this surface-exposed methionine, such as EV71, did not rely on GSH for virus morphogenesis. Biochemical and microscopical analysis provided strong evidence for a direct interaction between GSH and wildtype VP1 and a role for this interaction in localizing assembly intermediates to replication sites. Consistently, the interaction between GSH and mutant VP1 was abolished resulting in a relocalization of the assembly intermediates to replication sites independent from GSH. This study thus reveals GSH as a novel stabilizing host factor essential for the production of infectious enterovirus progeny and provides new insights into the poorly understood process of morphogenesis.

The Picornaviridae represent a large family of small plus-strand RNA viruses that cause a bewildering array of important human and animal diseases. Morphogenesis is the least-understood step in the life cycle of these viruses, and this process is difficult to study because encapsidation is tightly coupled to genome translation and RNA replication. Although the basic steps of assembly have been known for some time, very few details are available about the mechanism and factors that regulate this process. Most of the information available has been derived from studies of enteroviruses, in particular poliovirus, where recent evidence has shown that, surprisingly, the specificity of encapsidation is governed by a viral protein-protein interaction that does not involve an RNA packaging signal. In this review, we make an attempt to summarize what is currently known about the following topics: (i) encapsidation intermediates, (ii) the specificity of encapsidation (iii), viral and cellular factors that are required for encapsidation, (iv) inhibitors of encapsidation, and (v) a model of enterovirus encapsidation. Finally, we compare some features of picornavirus morphogenesis with those of other plus-strand RNA viruses.

ABSTRACT Polypeptide 2C ATPase is one of the most thoroughly studied but least understood proteins in the life cycle of poliovirus. Within the protein, multiple functional domains important for uncoating, host cell membrane alterations, and RNA replication and encapsidation have previously been identified. In this study, charged to alanine-scanning mutagenesis was used to generate conditional-lethal mutations in hitherto-uncharacterized domains of the 2C ATPase polypeptide, particularly those involved in morphogenesis. Adjacent or clustered charged amino acids (2 to 4), scattered along the 2C ATPase coding sequence, were replaced with alanines. RNA transcripts of mutant poliovirus cDNA clones were transfected into HeLa cells. Subsequently, 10 lethal, 1 severely temperature-sensitive, 2 quasi-infectious, and 3 wild type-like mutants were identified. Using a luciferase-containing reporter virus, we demonstrated RNA replication defects in all lethal and quasi-infectious mutants. Temperature-sensitive mutants were defective in RNA replication only at the restricted temperatures. Furthermore, we characterized a quasi-infectious mutant (K 6 A/K 7 A) that produced a suppressor mutation (G 1 R) and a novel 2B∧2C ATPase cleavage site (Q∧R). Surprisingly, this cleavage site mutation did not interfere with normal processing of the polyprotein. These mutants have led to the identification of several new sites within the 2C ATPase polypeptide that are required for RNA replication. In addition, analysis of the suppressor mutants has revealed a new domain near the C terminus of 2C ATPase that is involved in encapsidation, possibly achieved through interaction with an amino acid sequence between NTP binding motifs A and B of 2C ATPase . Most importantly, the identification of suppressor mutations in both 2C ATPase and the capsid domains (VP1 and VP3) of poliovirus has confirmed that an interaction between 2C ATPase and capsid proteins is involved in viral morphogenesis.

In spite of decades-long studies, the mechanism of morphogenesis of plus-stranded RNA viruses belonging to the genus Enterovirus of Picornaviridae, including poliovirus (PV), is not understood. Numerous attempts to identify an RNA encapsidation signal have failed. Genetic studies, however, have implicated a role of the non-structural protein 2C(ATPase) in the formation of poliovirus particles. Here we report a novel mechanism in which protein-protein interaction is sufficient to explain the specificity in PV encapsidation. Making use of a novel "reporter virus", we show that a quasi-infectious chimera consisting of the capsid precursor of C-cluster coxsackie virus 20 (C-CAV20) and the nonstructural proteins of the closely related PV translated and replicated its genome with wild type kinetics, whereas encapsidation was blocked. On blind passages, encapsidation of the chimera was rescued by a single mutation either in capsid protein VP3 of CAV20 or in 2C(ATPase) of PV. Whereas each of the single-mutation variants expressed severe proliferation phenotypes, engineering both mutations into the chimera yielded a virus encapsidating with wild type kinetics. Biochemical analyses provided strong evidence for a direct interaction between 2C(ATPase) and VP3 of PV and CAV20. Chimeras of other C-CAVs (CAV20/CAV21 or CAV18/CAV20) were blocked in encapsidation (no virus after blind passages) but could be rescued if the capsid and 2C(ATPase) coding regions originated from the same virus. Our novel mechanism explains the specificity of encapsidation without apparent involvement of an RNA signal by considering that (i) genome replication is known to be stringently linked to translation, (ii) morphogenesis is known to be stringently linked to genome replication, (iii) newly synthesized 2C(ATPase) is an essential component of the replication complex, and (iv) 2C(ATPase) has specific affinity to capsid protein(s). These conditions lead to morphogenesis at the site where newly synthesized genomes emerge from the replication complex.

The poliovirus (PV) eradication campaign is conducted on the premise that this virus, because of the lack of a zoonotic reservoir, will not reemerge once eradicated. This report examines the origin of PV using theoretical and experimental approaches. Our rooted phylogenetic analysis suggests a speciation of PV from a C-cluster coxsackie A virus (C-CAV) ancestor through mutation of the capsid that caused a receptor switch from intercellular adhesion molecule-1 to CD155, leading to a change of pathogenicity. This hypothesis is supported experimentally with chimeras generated from three different pairs of PV and C-CAV. Those carrying the PV capsid and the replication proteins of C-CAVs replicated well, whereas their reciprocal counterparts were either debilitated or dead. This phenomenon of asymmetry is observed also in recombinants between PV1 and C-CAV20, selected in tissue culture cells using a previously undescribed protocol. The recombinants are generated at frequencies of 10(-6) typical for PV interserotype recombination. Strikingly, they resemble genetically and phenotypically, including neurovirulence in CD155 transgenic mice, the large majority of circulating vaccine-derived PVs that have caused poliomyelitis outbreaks in different parts of the world. These data provide experimental evidence for C-CAVs being partners to PVs in generating diverse PV progeny by homologous recombination. They support speciation of a novel human pathogen (PV) from a pool of different human pathogens (C-CAVs). In a PV-free world without PV neutralizing antibodies, contemporary C-CAV, like their ancestor(s), could be fertile ground for a PV-like agent to emerge by mutation.