PLoS Pathogens最新文献

Enteroviruses comprise a large group of mammalian pathogens that often utilize two open reading frames (ORFs) to encode their proteins: the upstream protein (UP) and the main polyprotein. In some enteroviruses, in addition to the canonical upstream AUG (uAUG), there is another AUG that may represent an alternative upstream initiation site. An analysis of enterovirus sequences containing additional upstream AUGs identified several clusters, including strains of pathogenic Enterovirus alphacoxsackie and E. coxsackiepol. Using ribosome profiling on coxsackievirus CVA13 (E. coxsackiepol), we demonstrate that both upstream AUG codons can be used for translation initiation in infected cells. Moreover, we confirm translation from both upstream AUGs using a reporter system. Mutating the additional upstream AUG in the context of CVA13 did not result in phenotypic changes in immortalized cell lines. However, the wild-type virus outcompeted this mutant in human intestinal organoids and differentiated neuronal systems, representing an advantage in physiologically relevant infection sites. Mutation of the stop codon of the shorter upstream ORF led to dysregulated translation of the other ORFs in the reporter system, suggesting a potential role for the additional uORF in modulating the expression level of the other ORFs. Additionally, we demonstrate regulation of uORF translation in response to stress. These findings reveal the remarkable plasticity of enterovirus IRES-mediated initiation and the competitive advantage of double-upstream-AUG-containing viruses in terminally differentiated intestinal organoids and neuronal systems.

The innate immune response to viral infection needs to be tightly regulated to ensure effective pathogen clearance while avoiding excessive immune activation. During SARS-CoV-2 infection, however, the immune system often fails to elicit appropriate responses, resulting in cytokine-release syndrome in patients with COVID-19. In this study, we show that reduced expression of Regnase-1, an RNase that negatively regulates immune cell activation, confers resistance to infection with the mouse-adapted SARS-CoV-2 MA10 strain. In Regnase-1+/- mice, altered neutrophil function contributed to the amelioration of MA10-induced pneumonia. Single-cell RNA sequencing of lung tissue during MA10 infection revealed four distinct neutrophil subsets, and among these, a subset characterized by an interferon-stimulated gene (ISG) signature was decreased in Regnase-1+/- mice. Furthermore, Regnase-1+/- neutrophils exhibited reduced ISG expression without corresponding changes in proinflammatory gene expression. Regnase-1 was found to repress the expression of Tsc22d3, a gene involved in the negative regulation of interferon responses, through its 3' untranslated region. Collectively, these findings suggest that Regnase-1 attenuates resistance to SARS-CoV-2 MA10 infection by promoting excessive interferon responses in neutrophils.

Members of the Stomatin, Prohibitin, Flotillin and HflK/C (SPFH) protein family form large membrane anchored or spanning complexes and are involved in various functions in different organelles. The human malaria causing parasite Plasmodium falciparum harbors four SPFH proteins, including prohibitin 1 and 2, prohibitin-like protein (PHBL), and stomatin-like protein (STOML), which all localize to the parasite mitochondrion. In the murine model parasite Plasmodium berghei, STOML appears essential for asexual blood-stage (ABS) development and is localized to puncta on mitochondrial branching points in oocyst stages. In this study, we show that deletion of P. falciparum STOML causes a significant growth defect and slower ABS development, while sexual-stage development remains unaffected. Parasites lacking STOML were not more sensitive to respiratory chain targeting drugs, rendering a function of STOML in respiratory chain assembly unlikely. Epitope tagging of endogenous STOML revealed a distinct punctate localization on branching points and endings of the ABS mitochondrial network. STOML resides in a large protein complex and pulldown experiments identified a zinc dependent metalloprotease, FtsH, as a likely interaction partner. The predicted AlphaFold2 structure of STOML shows high similarity with the bacterial HflK/C, which has been shown to form a large vault-like structure around bacterial FtsH hexamers. Combined, our results suggest that a similar STOML-FtsH complex localized to specific loci of P. falciparum mitochondria facilitates the parasite's ABS development.

Encephalitogenic alphaviruses are mosquito-borne viruses that can cause fatal disease in humans and equines. Currently, there are no licensed vaccines or antiviral treatments for these infections. Western equine encephalitis virus (WEEV) is a member of this group that had not produced a human infection in over a decade. However, an outbreak of WEEV encephalitis in humans and equines was reported recently in South America, indicating a need for additional countermeasures. Blind passage approaches to generation of RNA virus live attenuated vaccines (LAVs) frequently result in acquisition of positively charged amino acid mutations that confer heparan sulfate (HS) binding and that are attenuating factors in resultant LAVs. To develop an informed approach for creation of alphavirus LAVs, we have utilized the WEEV McMillan (McM) strain as an HS weak/non-binding platform into which we have placed positively charged amino acid substitution mutations at positions in the E2 glycoprotein previously shown to confer HS-dependent infection upon other alphaviruses. This approach yielded four mutants with high efficiency HS binding and avirulence in mice, which were further subjected to yield optimization by in vitro selection of second-site mutations. Interestingly, the original mutations concomitantly increased HS interactions and reduced infection promoted by VLDLR and PCDH10 protein receptors, while the second site mutations improved infectivity mediated by VLDLR. Further, we report a newly generated 4.1Å cryo-EM reconstruction of WEEV McM strain into which we have mapped the mutations to provide an E2 glycoprotein domain-based representation of receptor binding site location.

[This corrects the article DOI: 10.1371/journal.ppat.1013754.].

Harnessing antimicrobial peptides as bactericidal agents affords an attractive approach to developing new anti-infective therapies. We found that abolishing disulfide bonding in mouse cryptdin 1 (Crp1), a weakly bactericidal α-defensin of 35 residues, turned it into a potent antimicrobial peptide against Gram-negative bacteria. Here we report that Crp1 in its natively folded β-sheet structure forms high-ordered nanonets to cloak, but not kill, Escherichia coli, whereas its disulfide-devoid linear counterpart (L-Crp1) readily disintegrates the bacterial membrane as monomers. L-Crp1 adopts a helix-loop-helix conformation in molecular dynamics simulations, likely conducive to productive peptide-membrane interactions detrimental to bacteria. A truncated peptide spanning the helix-loop-helix, L-Crp11-25, maintains the same conformation as and similar membranolytic and bactericidal activities to L-Crp1. Remarkably, intraperitoneally administered L-Crp11-25 rescues E. coli-challenged mice from lethality in a sepsis model by effectively reducing bacterial burden, inflammation and tissue damage. Our studies cultivate additional mechanistic insights into the mode of action of defensins and shed new light on how to harness these host factors for potential therapeutic use.

Plant-parasitic nematodes secrete molecules to manipulate their hosts, but little is known about their mode of delivery and packaging. Here, we describe microRNA-containing exosomes that are secreted by root-knot nematodes and systemically increase host susceptibility. By revealing a novel mode of nematode-plant communication, our findings outline a mechanism for the delivery of nematode patho-molecules, offering a new target for disrupting parasitism at the level of vesicle-mediated delivery.

The polymerase acidic (PA) protein is a subunit of the trimeric influenza A virus (IAV) RNA-dependent RNA polymerase and the target of the anti-influenza drug baloxavir marboxil (BXM). As with other direct-acting antivirals, treatment with BXM can lead to selection of viruses carrying resistance mutations. If these mutations have negligible fitness costs, resistant viruses can spread widely and render existing treatments obsolete. Multiple BXM resistance mutations in the nuclease domain of PA have been identified, with I38T and I38M amino acid substitutions occurring frequently. These mutations have minimal to no effects on viral polymerase activity, virus replication, or transmission. However, for reasons that are not well understood, viruses with BXM resistance substitutions have not been able to compete with parental wild-type strains. The IAV genome segment encoding PA also encodes the host shutoff nuclease PA-X, which shares the endonuclease domain with PA but has a unique C-terminal domain generated by ribosomal frameshifting during translation. Unlike their effects on PA activity, the effects of BXM or the I38T/M substitutions on PA-X function remain uncharacterized. In our work, for the first time, we directly examine the effects of baloxavir and the I38T/M substitutions on PA-X activity and show that baloxavir inhibits PA-X activity in a dose dependent manner. Most importantly, we also demonstrate that the I38T/M mutations significantly impair the host shutoff activity of PA-X proteins from different IAV strains of H1N1, H3N2, and H5N1 subtypes. Our work reveals that the deleterious effects of I38T/M on PA-X function may represent an important barrier to the spread of BXM-resistant viruses.

Viruses are recognized by host cell innate immunity through viral RNA/DNA sensing by cyclic GMP-AMP synthase (cGAS) stimulator of interferon genes (STING). However, many viruses evade cGAS-STING signaling and antiviral IFN-β response. Here, we show that natural killer (NK) cells counteract immune evasion of type I interferon response upon human cytomegalovirus (HCMV) infection. NK cells enhance IFN-β response in virus-infected cells more efficiently than perforin-knockout and GrM-knockout NK cells. Mechanistically, GrM cleaves viral pp71 into two fragments, the first, like full-length pp71, still inhibits cGAS-STING-IFN-β response but is rapidly degraded by the proteasome, and the second fragment that rather augments IFN-β and outperforms full-length pp71 inhibition of STING. NK cells cannot enhance IFN-β response in cells infected with HCMV that harbors a pp71 with a mutated GrM cleavage site. We conclude that NK cells use GrM to counteract cytomegaloviral innate immune evasion through pp71-mediated inhibition of cGAS-STING-IFN-β innate immune response.