Journal of Virology最新文献

Selective autophagy is a protein clearance mechanism mediated by evolutionarily conserved selective autophagy receptors (SARs), which specifically degrades misfolded, misassembled, or metabolically regulated proteins. SARs help the host to suppress viral infections by degrading viral proteins. However, viruses have evolved sophisticated mechanisms to counteract, evade, or co-opt autophagic processes, thereby facilitating viral replication. Therefore, this review aims to summarize the complex mechanisms of SARs involved in viral infections, specifically focusing on how viruses exploit strategies to regulate selective autophagy. We present an updated understanding of the various critical roles of SARs in viral pathogenesis. Furthermore, newly discovered evasion strategies employed by viruses are discussed and the ubiquitination-autophagy-innate immune regulatory axis is proposed to be a crucial pathway to control viral infections. This review highlights the remarkable flexibility and plasticity of SARs in viral infections.

Studying viral infections necessitates well-designed cell culture models to deepen our understanding of diseases and develop effective treatments. In this study, we present a readily available ex vivo 3D co-culture model replicating the human intestinal mucosa. The model combines fully differentiated human intestinal epithelium (HIE) with human monocyte-derived macrophages (hMDMs) and faithfully mirrors the in vivo structural and organizational properties of intestinal mucosal tissues. Specifically, it mimics the lamina propria, basement membrane, and the air-exposed epithelial layer, enabling the pioneering observation of macrophage migration through the tissue to the site of viral infection. In this study, we applied the HIE-hMDMs model for the first time in viral infection studies, infecting the model with two globally significant viruses: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human norovirus GII.4. The results demonstrate the model's capability to support the replication of both viruses and show the antiviral role of macrophages, determined by their migration to the infection site and subsequent direct contact with infected epithelial cells. In addition, we evaluated the production of cytokines and chemokines in the intestinal niche, observing an increased interleukin-8 production during infection. A parallel comparison using a classical in vitro cell line model comprising Caco-2 and THP-1 cells for SARS-CoV-2 experiments confirmed the utility of the HIE-hMDMs model in viral infection studies. Our data show that the ex vivo tissue models hold important implications for advances in virology research.IMPORTANCEThe fabrication of intricate ex vivo tissue models holds important implications for advances in virology research. The co-culture model presented here provides distinct spatial and functional attributes not found in simplified models, enabling the evaluation of macrophage dynamics under severe acute respiratory syndrome coronavirus 2 and human norovirus (HuNoV) infections in the intestine. Moreover, these models, comprised solely of primary cells, facilitate the study of difficult-to-replicate viruses such as HuNoV, which cannot be studied in cell line models, and offer the opportunity for personalized treatment evaluations using patient cells. Similar co-cultures have been established for the study of bacterial infections and different characteristics of the intestinal tissue. However, to the best of our knowledge, a similar intestinal model for the study of viral infections has not been published before.

Ubiquitin modification of viral proteins to degrade or regulate their function is one of the strategies of the host to resist viral infection. Here, we report that ubiquitin protein ligase E3C (UBE3C), an E3 ubiquitin ligase, displayed inhibitory effects on EV-A71 replication. UBE3C knockdown resulted in increased viral protein levels and virus titers, whereas overexpression of UBE3C reduced EV-A71 replication. To explore the mechanism by which UBE3C affected EV-A71 infection, we found that the C-terminal of UBE3C bound to 2C protein and facilitated K33/K48-linked ubiquitination degradation of 2C K268. Moreover, UBE3C lost its ability to degrade 2C K268R and had a diminished inhibitory impact against the replication of recombinant EV-A71-FY-2C K268R. In addition, UBE3C also promoted ubiquitination degradation of the 2C protein of CVB3 and CVA16 and inhibited viral replication. Thus, our findings reveal a novel mechanism that UBE3C acts as an enterovirus host restriction factor, including EV-A71, by targeting the 2C protein.

Importance: The highly conserved 2C protein of EV-A71 is a multifunctional protein and plays a key role in the replication cycle. In this study, we demonstrated for the first time that UBE3C promoted the degradation of 2C K268 via K33/K48-linked ubiquitination, thereby inhibiting viral proliferation. Our findings advance the knowledge related to the roles of 2C in EV-A71 virulence and the ubiquitination pathway in the host restriction of EV-A71 infection.

Bacteria exposed to bactericidal treatment, such as antibiotics or bacteriophages (phages), often develop resistance. While phage therapy is proposed as a solution to the antibiotic resistance crisis, the bacterial resistance emerging during phage therapy remains poorly characterized. In this study, we examined a large population of phage-resistant extra-intestinal pathogenic Escherichia coli 536 clones that emerged from both in vitro (non-limited liquid medium) and in vivo (murine pneumonia) conditions. Genome sequencing uncovered a convergent mutational pattern in phage resistance mechanisms under both conditions, particularly targeting two cell-wall components, the K15 capsule and the lipopolysaccharide (LPS). This suggests that their identification in vivo could be predicted from in vitro assays. Phage-resistant clones exhibited a wide range of fitness according to in vitro tests, growth rate, and resistance to amoeba grazing, which could not distinguish between the K15 capsule and LPS mutants. In contrast, K15 capsule mutants retained virulence comparable to the wild-type strain, whereas LPS mutants showed significant attenuation in the murine pneumonia model. Additionally, we observed that resistance to the therapeutic phage through a nonspecific mechanism, such as capsule overproduction, did not systematically lead to co-resistance to other phages that were initially capable or incapable of infecting the wild-type strain. Our findings highlight the importance of incorporating a diverse range of phages in the design of therapeutic cocktails to target potential future phage-resistant clones effectively.

Importance: This study isolated more than 50 phage-resistant mutants from both in vitro and in vivo conditions, exposing an extra-intestinal pathogenic Escherichia coli strain to a single virulent phage. The characterization of these clones revealed several key findings: (1) mutations occurring during phage treatment affect the same pathways as those identified in vitro; (2) the resistance mechanisms are associated with the modification of two cell-wall components, with one involving receptor deletion (phage-specific mechanism) and the other, less frequent, involving receptor masking (phage-nonspecific mechanism); (3) an in vivo virulence assay demonstrated that the absence of the receptor abolishes virulence while masking the receptor preserves it; and (4) clones with a resistance mechanism nonspecific to a particular phage can remain susceptible to other phages. This supports the idea of incorporating diverse phages into therapeutic cocktails designed to collectively target both wild-type and phage-resistant strains, including those with resistance mechanisms nonspecific to a phage.

Pseudorabies virus (PRV) utilizes multiple strategies to inhibit type I interferon (IFN-I) production and signaling to achieve innate immune evasion. Among several other functions, mitochondria serve as a crucial immune hub in the initiation of innate antiviral responses. It is currently unknown whether PRV inhibits innate immune responses by manipulating mitochondria. In this study, we found that PRV infection damages mitochondrial structure and function, as shown by mitochondrial membrane potential depolarization, reduction in mitochondrial numbers, and an imbalance in mitochondrial dynamics. In addition, PRV infection triggered PINK1-Parkin-mediated mitophagy to eliminate the impaired mitochondria, which resulted in a suppression of IFN-I production, thereby promoting viral replication. Furthermore, we found that mitophagy resulted in the degradation of the mitochondrial antiviral signaling protein, which is located on the mitochondrial outer membrane. In conclusion, the data of the current study indicate that PRV-induced mitophagy represents a previously uncharacterized PRV evasion mechanism of the IFN-I response, thereby promoting virus replication.IMPORTANCEPseudorabies virus (PRV), a pathogen that induces different disease symptoms and is often fatal in domestic animals and wildlife, has caused great economic losses to the swine industry. Since 2011, different PRV variant strains have emerged in Asia, against which current commercial vaccines may not always provide optimal protection in pigs. In addition, there are indications that some of these PRV variant strains may sporadically infect people. In the current study, we found that PRV infection causes mitochondria injury. This is associated with the induction of mitophagy to eliminate the damaged mitochondria, which results in suppressed antiviral interferon production and signaling. Hence, our study reveals a novel mechanism that is used by PRV to antagonize the antiviral host immune response, providing a theoretical basis that may contribute to the research toward and development of new vaccines and antiviral drugs.

Viruses deploy sophisticated strategies to hijack the host's translation machinery to favor viral protein synthesis and counteract innate cellular defenses. However, little is known about the mechanisms by which Senecavirus A (SVA) controls the host's translation. Using a series of sophisticated molecular cell manipulation techniques, heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) was identified as an essential host factor involved in translation control in SVA-infected cells. It was also determined that the SVA structural protein, VP3, binds to and relocalizes hnRNPA2B1, which interferes with the host's protein synthesis machinery to establish a cellular environment that facilitates viral propagation via a two-pronged strategy: first, hnRNPA2B1 serves as a potent internal ribosome entry site (IRES) trans-acting factor, which is selectively co-opted to promote viral IRES-driven translation by supporting the assembly of translation initiation complexes. Second, a strong repression of host cell translation occurs in the context of the VP3-hnRNPA2B1 interaction, resulting in attenuation of the interferons response. This is the first study to demonstrate the interaction between SVA VP3 and hnRNPA2B1, and to characterize their key roles in manipulating translation. This novel dual mechanism, which regulates selective mRNA translation and immune evasion of virus-infected cells, highlights the VP3-hnRNPA2B1 complex as a potential target for the development of modified antiviral or oncolytic reagents.

Importance: Viral reproduction is contingent on viral protein synthesis, which relies entirely on the host's translation machinery. As such, viruses often need to control the cellular translational apparatus to favor viral protein production and avoid host innate defenses. Senecavirus A (SVA) is an important virus, both as an emerging pathogen in the pork industry and as a potential oncolytic virus for neuroendocrine cancers. Here, heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) was identified as a critical regulator of the translational landscape during SVA infection. This study supports a model whereby the VP3 protein of SVA efficiently subverts the host's protein synthesis machinery through its ability to bind to and relocalize hnRNPA2B1, not only selectively promoting viral internal ribosome entry site-driven translation but also resulting in global translation shutdown and immune evasion. Together, these data provide new insights into how the complex interactions between translation machinery, SVA, and innate immunity contribute to the pathogenicity of the SVA.

Cardiovascular manifestations of coronavirus disease 2019 (COVID-19) include myocardial injury, heart failure, and myocarditis and are associated with long-term disability and mortality. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and antigens are found in the myocardium of COVID-19 patients, and human cardiomyocytes are susceptible to infection in cell or organoid cultures. While these observations raise the possibility that cardiomyocyte infection may contribute to the cardiac sequelae of COVID-19, a causal relationship between cardiomyocyte infection and myocardial dysfunction and pathology has not been established. Here, we generated a mouse model of cardiomyocyte-restricted infection by selectively expressing human angiotensin-converting enzyme 2 (hACE2), the SARS-CoV-2 receptor, in cardiomyocytes. Inoculation of Myh6-Cre Rosa26^{loxP-STOP-loxP-hACE2} mice with an ancestral, non-mouse-adapted strain of SARS-CoV-2 resulted in viral replication within the heart, accumulation of macrophages, and moderate left ventricular (LV) systolic dysfunction. Cardiac pathology in this model was transient and resolved with viral clearance. Blockade of monocyte trafficking reduced macrophage accumulation, suppressed the development of LV systolic dysfunction, and promoted viral clearance in the heart. These findings establish a mouse model of SARS-CoV-2 cardiomyocyte infection that recapitulates features of cardiac dysfunctions of COVID-19 and suggests that both viral replication and resultant innate immune responses contribute to cardiac pathology.IMPORTANCEHeart involvement after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection occurs in multiple ways and is associated with worse outcomes in coronavirus disease 2019 (COVID-19) patients. It remains unclear if cardiac disease is driven by primary infection of the heart or immune response to the virus. SARS-CoV-2 is capable of entering contractile cells of the heart in a culture dish. However, it remains unclear how such infection affects the function of the heart in the body. Here, we designed a mouse in which only heart muscle cells can be infected with a SARS-CoV-2 strain to study cardiac infection in isolation from other organ systems. In our model, infected mice show viral infection, worse function, and accumulation of immune cells in the heart. A subset of immune cells facilitates such worsening heart function. As this model shows features similar to those observed in patients, it may be useful for understanding the heart disease that occurs as a part of COVID-19.

While investigating methods to target gene delivery vectors to specific cell types, we examined the potential of using a nanobody against the SARS-CoV-2 Spike protein receptor-binding domain to direct lentivirus infection of Spike-expressing cells. Using four different approaches, we found that lentiviruses with surface-exposed nanobody domains selectively infect Spike-expressing cells. Targeting is dependent on the fusion function of the Spike protein, and conforms to a model in which nanobody binding to the Spike protein triggers the Spike fusion machinery. The nanobody-Spike interaction also is capable of directing cell-cell fusion and the selective infection of nanobody-expressing cells by Spike-pseudotyped lentivirus vectors. Significantly, cells infected with SARS-CoV-2 are efficiently and selectively infected by lentivirus vectors pseudotyped with a chimeric nanobody protein. Our results suggest that cells infected by any virus that forms syncytia may be targeted for gene delivery by using an appropriate nanobody or virus receptor mimic. Vectors modified in this fashion may prove useful in the delivery of immunomodulators to infected foci to mitigate the effects of viral infections.IMPORTANCEWe have discovered that lentiviruses decorated on their surfaces with a nanobody against the SARS-CoV-2 Spike protein selectively infect Spike-expressing cells. Infection is dependent on the specificity of the nanobody and the fusion function of the Spike protein and conforms to a reverse fusion model, in which nanobody binding to Spike triggers the Spike fusion machinery. The nanobody-Spike interaction also can drive cell-cell fusion and infection of nanobody-expressing cells with viruses carrying the Spike protein. Importantly, cells infected with SARS-CoV-2 are selectively infected with nanobody-decorated lentiviruses. These results suggest that cells infected by any virus that expresses an active receptor-binding fusion protein may be targeted by vectors for delivery of cargoes to mitigate infections.