Journal of Virology最新文献

Porcine epidemic diarrhea virus (PEDV), a highly pathogenic enteric coronavirus, has caused significant economic losses worldwide in recent years. The PEDV spike (S) protein has been reported to undergo extensive N-glycosylation, suggesting that glycosylation plays a crucial role in PEDV replication. In this study, we demonstrated that the N-glycosylation pathway promotes PEDV replication by facilitating the glycosylation of the S protein. First, we observed that pharmacological inhibition of host N-glycosylation using specific inhibitors significantly reduces viral replication. Furthermore, genetic ablation of STT3A or STT3B, the catalytically active subunits of the oligosaccharyltransferase (OST) complex, revealed that the STT3B-OST complex, but not STT3A, is preferentially required for PEDV replication. Notably, we showed that the N-glycosylation of the PEDV S protein depends on the oligosaccharyltransferase activity of STT3B. Together, the study demonstrated the critical role of the N-glycosylation pathway in PEDV replication by elucidating the relationship between the N-glycosylation of the PEDV S protein and STT3B, thereby presenting a potential new target for the prevention and control of PEDV.IMPORTANCEThe highly N-glycosylated spike protein of porcine epidemic diarrhea virus (PEDV) is a multifunctional protein that plays a crucial role in the viral replication cycle. In this study, using pharmacological inhibitors, we demonstrated the importance of the N-glycosylation pathway in PEDV replication. Genetic analysis revealed that STT3B, one of the catalytically active subunits of the oligosaccharyltransferase complex, promotes viral proliferation by regulating the N-glycosylation of the PEDV spike protein. Our findings enhance the understanding of the role of the N-glycosylation pathway in viral infection and identify STT3B as a potential therapeutic target for controlling PEDV infection.

Porcine epidemic diarrhea virus (PEDV) is a pathogenic coronavirus that targets the swine intestinal tract, leading to acute diarrhea and high mortality in neonatal piglets. PEDV is categorized into different genotypes based on genetic variations, especially in the spike (S) gene. The S protein is crucial for viral entry and a major immune target. Significant differences in virulence have been observed among PEDV genotypes, particularly between classical strains and newly emerging strains. In this study, we explored the impact of spike gene variability on PEDV pathogenicity. Using targeted RNA recombination, we generated recombinant PEDV (rPEDV) variants carrying spike genes from contemporary strains (moderately virulent strain UU and highly virulent strain GDU), all within the genetic background of the avirulent DR13 vaccine strain. Pathogenicity was assessed in 3-day-old piglets. The rPEDV carrying the DR13 spike gene was nonpathogenic, with no detectable viral RNA in feces. The rPEDV with the UU spike gene induced mild to severe diarrhea, with moderate viral shedding but no mortality. Conversely, the rPEDV with the GDU spike gene caused severe diarrhea, high viral titers, and high mortality. These findings highlight the critical role of the spike protein in PEDV virulence, informing future development of effective control strategies, including the design of live-attenuated vaccines.IMPORTANCEThis study significantly advances our understanding of how genetic variations in the spike (S) protein of porcine epidemic diarrhea virus (PEDV) influence its ability to cause disease. By engineering viruses with spike genes from different PEDV strains, variations in this protein could be directly linked to differences in disease severity. We found that the spike protein from highly virulent strains caused severe diarrhea and high mortality in piglets, while that from less virulent strains led to milder symptoms. These findings emphasize the central role of the spike protein in determining PEDV virulence, which may enable the design of more effective vaccines to combat PEDV and reduce its impact on the swine industry.

HIV persists in people living with HIV (PLHIV) on antiretroviral therapy (ART) in long-lived and proliferating latently infected CD4+ T cells that selectively express pro-survival proteins, including the zinc finger proteins, Ikaros and Aiolos. In this study, we investigated whether pomalidomide, an immunomodulatory agent that induces degradation of Ikaros and Aiolos, could increase the death of HIV-infected cells and/or reverse HIV latency. Using an in vitro model of CD4+ T cells infected with a green fluorescent protein (GFP) reporter virus, pomalidomide increased the expression of the pro-survival protein B cell lymphoma (Bcl)-2 and did not increase apoptosis of GFP+ HIV productively infected CD4+ T cells. Pomalidomide also increased the expression of CD155 and UL16-binding protein (ULBP) stress proteins on GFP+ HIV productively infected CD4+ T cells, but this did not translate to enhanced clearance following co-culture with a natural killer (NK) cell line. Using CD4+ T cells from PLHIV on ART, pomalidomide ex vivo activated memory CD4+ T cells resulting in elevated HLA-DR expression and induced CD4+ T cell proliferation but only in the presence of T cell receptor stimulation with anti-CD3 and anti-CD28. There was no effect on cell-associated HIV RNA or the frequency of intact HIV DNA. In conclusion, despite an increase in stress protein expression, promoting Ikaros and Aiolos degradation in CD4+ T cells using pomalidomide did not directly induce apoptosis of HIV-infected cells or induce HIV latency reversal.IMPORTANCEPeople living with HIV (PLHIV) require lifelong antiretroviral therapy (ART) due to the persistence of latently infected cells. The zinc finger proteins, Ikaros and Aiolos, have recently been implicated in promoting the persistence of latently infected cells. In this study, we investigated the effects of pomalidomide, an immunomodulatory imide drug that induces the degradation of Ikaros and Aiolos, on HIV latency reversal and death of infected cells. Using CD4+ T cells from people living with HIV on suppressive antiretroviral therapy, as well as an in vitro model of productive HIV infection, we found that pomalidomide induced T cell activation and expression of stress proteins but no evidence of latency reversal or selective death of infected cells.

Like other RNA viruses, foot-and-mouth disease virus (FMDV) has a high mutation rate. After the acute phase of infection, about half of infected cattle develop a persistent FMDV infection that can last for weeks or months. During this persistent phase, the virus continues to replicate, resulting in the emergence of genomic heterogeneity. We have documented the pattern of mutations in the persistent phase by obtaining consensus-level sequences directly from oropharyngeal fluid (OPF) without prior virus isolation in culture. OPF samples were repeatedly collected from 22 experimentally infected cattle, 20 of which were virus positive in the OPF on day 21 after infection or later. We observed that during the persistent phase, the amount of non-synonymous mutations causing an amino acid change increased over time. Two amino acid changes that showed a striking increase during the persistent phase, VP3 A75T and VP2 Y79H, were present neither in the inoculum nor during the acute phase. Another amino acid change in VP3, R56C, which was previously implicated in FMDV pathogenicity, was already present in the inoculum and dominated toward the end of the trial in most samples. Several other amino acid changes occurred, particularly on the surface of VP2 around residue VP2 79. By functional analysis, we show that the persistent isolates evolve distinctly compared with cell culture adaptation but do not show signs of antigenic escape from neutralizing antibodies. In agreement with previous observations, we conclude that these amino acid changes are indeed associated with persistent infection of cattle with FMDV serotype O.

Importance: Our research article describes the genetic changes that occur during the acute and persistent foot-and-mouth disease (FMDV) infection. This is of particular interest to understand viral dynamics within an infected population from which new viral strains could emerge. Especially FMDV, with its high antigenic diversity and very limited cross-reactivity between strains and serotypes, has already demonstrated in the past that new variants can quickly emerge and evade vaccine responses. In our study, we have observed that this dynamic evolution continues during the persistent phase. Persistently infected animals, which are clinically indistinguishable from healthy animals, also pose a reservoir for recombination. A better understanding of viral dynamics is essential for improved vaccines to prevent the emergence of antigenic variants.

As a key regulator of human immunodeficiency virus type 1 (HIV-1) transcription, Tat plays an essential role in viral replication and latency, making it a promising target for designing viral control strategies. Identifying host factors that modulate Tat and exploring the underlying mechanisms will benefit our understanding of HIV-1 transcriptional regulation and provide valuable insights into Tat-based therapeutic strategies. Here, by employing the TurboID approach, we discovered high-affinity binding between FBXO45 and Tat. Our findings demonstrate that FBXO45 negatively regulates Tat by promoting Tat ubiquitination and directing it to autophagic degradation. Autophagic degradation of Tat has been reported, but the specific underlying mechanisms remain unidentified. We elucidated this issue by providing evidence that FBXO45-mediated Tat polyubiquitination is an essential prerequisite for this process. Silencing of FBXO45 leads to a deficiency of autophagy receptor SQSTM1/p62 to bind and facilitate the autophagic degradation of Tat. Our results further underscore the crosstalk between post-translational modifications of Tat by demonstrating that the phosphorylation site of the Tat S62 residue is required for ubiquitination induced by FBXO45. Furthermore, in the context of the regulation of HIV-1, FBXO45 inhibits viral replication and maintains the latency of HIV-1 by suppressing viral transcription. Importantly, FBXO45 overexpression significantly attenuated viral rebound after antiretroviral therapy withdrawal. In summary, our findings suggest a novel role for FBXO45 in regulating HIV-1 replication by inducing the ubiquitination and SQSTM1/p62-dependent autophagic degradation of Tat. Considering the indispensable role of Tat in the regulation of HIV-1 replication and reactivation, FBXO45 may be a potential target for therapeutic intervention against HIV-1.IMPORTANCEHIV-1 Tat plays an indispensable role in regulating viral transcription and is a promising target for achieving a functional cure for AIDS. Identifying the host factors that modulate Tat expression could benefit the development of anti-HIV-1 strategies targeting Tat. Using TurboID assay, we identified a significant interaction between FBXO45 and Tat. Functionally, FBXO45 ubiquitinates and directs Tat for SQSTM1/p62-mediated autophagic degradation, thereby effectively restricting HIV-1 replication and maintaining HIV-1 latency by suppressing Tat-dependent viral transcription. These findings uncover a novel role for FBXO45 in regulating Tat and broaden our understanding of the host mechanisms involved in Tat processing.

The porcine epidemic diarrhea virus (PEDV), a highly pathogenic coronavirus, poses significant challenges to global swine agriculture with severe economic consequences. Our research reveals that in addition to known transmission routes, PEDV can be airborne, initially invading the nasal mucosa and subsequently being transported by dendritic cells and peripheral blood T cells, ultimately leading to intestinal disease in piglets. This study elucidates the cellular mechanisms behind the process, demonstrating how PEDV is internalized by CD4⁺ T cells after being transferred by dendritic cells, where it establishes a latent infection. Crucially, PEDV induces the upregulation of the integrin α4β7 homing receptor, facilitating the migration of these infected CD4⁺ T cells to the small intestine. Furthermore, our findings reveal that the activation of the α4β7-Rho-GTPases-Cofilin signaling pathway by PEDV reorganizes the actin cytoskeleton, enabling CD4⁺ T-cell transmigration through high endothelial venules into the intestinal mucosa, resulting in the infection of intestinal epithelial cells. These insights not only illuminate the molecular mechanisms PEDV employs to hijack CD4⁺ T cells for transmission from the respiratory tract to the intestine but also identify novel targets for therapeutic intervention, providing new perspectives for effectively preventing and managing PEDV infection with broader implications for controlling similar pathogens in diverse hosts.IMPORTANCEPorcine epidemic diarrhea virus (PEDV), characterized by rapid transmission and widespread prevalence, poses a significant long-term threat to the global pig farming industry. Our previous research revealed that, in addition to the classic fecal-oral infection route, PEDV can invade through the nasal mucosa, leading to intestinal infection. This study further investigated the molecular mechanisms by which the virus is transported by T lymphocytes from the respiratory tract to the intestines. We found that PEDV establishes a latent infection in CD4⁺ T cells and promotes their intestinal homing by upregulating the homing receptor integrin α4β7. Additionally, we elucidated the activation of the integrin α4β7-mediated Rho-GTPase-Cofilin signaling axis by PEDV, which regulates pseudopod formation and facilitates CD4⁺ T-cell migration to the intestinal mucosal lamina propria post-homing. This study elucidates the mechanism underlying the lymphocyte-dependent dissemination of PEDV following nasal infection, providing new insights into strategies for preventing PEDV invasion.

Endogenous retroviruses (ERVs) are chromosomally integrated viral copies that represent relics of past infections. Analysis of the sequenced genomes of 17 mouse strains, Mus musculus subspecies, and Mus spretus identified 29 ERVs of mouse mammary tumor viruses (MMTVs), termed Mtvs. The 15 laboratory mouse Mtvs are each present in multiple strains reflecting their common breeding history; most predate the development of inbred strains and were likely acquired by Mus musculus domesticus progenitors but have no orthologs in wild mice, whereas four, including the intact Mtv1, were likely endogenized more recently. One of the 14 Mtvs found in wild mice was distributed over a broad geographic range in southeast Asia. Most Mtvs are full-length, with multiple open reading frames, but Mtvs from many wild mice have an unusual envelope deletion corresponding to an intron of the viral rem accessory gene, suggesting its derivation from spliced MMTV cDNAs. These deleted envs have open reading frames, are found in globally distributed mice, and show subspecies-specific sequence variation consistent with their recurrent generation. The highly variable MMTV sag gene, responsible for resistance to exogenous infection, exhibits evidence of recombination as well as positive selection, consistent with its role in antiviral defense. In contrast, the spread of Mtvs in Mus musculus populations is not marked by an active arms race pitting the MMTV envelope against its cellular receptor. Thus, the acquisition of potentially disease-inducing Mtvs is a recent and ongoing process in Mus accompanied by recombination, positive selection, and a recurrent envelope deletion.

Importance: Endogenous retroviruses (ERVs) are copies of viral genomes inserted into host chromosomes, producing a fossil record of past infections and virus-host co-adaptations. ERVs of mouse mammary tumor viruses (Mtvs) were found in all common laboratory strains, all Mus musculus subspecies, and a sister species, Mus spretus. Most laboratory mouse Mtvs predate inbred strain origins and were acquired by M. musculus domesticus, but although widely shared among strains, none of these were found in wild mice. Among wild mouse Mtvs, only one showed a broad geographic distribution. All M. musculus subspecies carry Mtvs with a large envelope deletion corresponding to the processed mRNA for the viral rem gene; such Mtvs likely derive from spliced viral mRNA. The Mtv sag gene responsible for resistance to exogenous infection is under purifying selection and has been subject to recombination, whereas the Mtv envelope and its cellular receptor show no evidence of genetic conflicts.

Porcine reproductive and respiratory syndrome virus (PRRSV) isolates share a restricted cellular tropism. Marc-145 cells derived from African green monkey are one of the few cell lines supporting PRRSV propagation in vitro and are commonly used for PRRS vaccine development. However, currently prevalent PRRSV isolates display different Marc-145 cell tropism while the exact determinant is not clarified yet. In this study, we identified for the first time that the 91/97/98 amino acid (aa) substitutions in GP2a of PRRSV play critical roles in determining Marc-145 adaptation. Specifically, multiple series of chimeric viruses were constructed based on four PRRSV infectious clones including Marc-145 adaptive HP-PRRSV-2 strain and Marc-145 non-adaptive NADC34-like PRRSV-2, NADC30-like PRRSV-2, and PRRSV-1 strains. The GP2a 91/97/98 aa substitutions are a sufficient and necessary determinant in NADC34-like and NADC30-like PRRSV-2, a sufficient but not necessary determinant in HP-PRRSV-2, a necessary but not sufficient determinant in PRRSV-1, respectively. In addition, the GP2a substitutions also influenced PRRSV infectivity in PAMs and piglets. Noticeably, the GP2a substitutions did not significantly affect the levels of neutralizing antibodies, porcine T follicular helper (Tfh) cells, and PRRSV-specific IFNγ secreting cells. Overall, our results not only provide new insights into PRRSV tropism and infectivity but also will facilitate PRRS vaccine development.

Importance: Prevalent PRRSV isolates present different cell tropisms in vitro. Clarifying the exact determinant of PRRSV tropism is crucial for PRRSV isolation and vaccine development. By constructing chimeric viruses based on four representative PRRSV infectious clones, we identified for the first time that the 91/97/98 amino acid substitutions in GP2a play critical but distinct roles in determining Marc-145 cell tropism for different PRRSV strains. The GP2a 91/97/98 amino acid substitutions also affect PRRSV infectivity in PAMs and piglets but do not influence immune responses. This study not only deciphers an exact determinant of PRRSV tropism and infectivity but also has guiding significance for PRRS vaccine development.

Porcine epidemic diarrhea virus (PEDV) is a primary cause of viral diarrhea in neonatal piglets, leading to substantial economic losses in the swine industry globally. It primarily targets epithelial cells of the small intestine, compromising intestinal function and resulting in the death of affected animals. As mitochondria are essential for maintaining gut health, this study investigates the effects of PEDV infection on mitochondrial function in small intestinal epithelial cells and its subsequent impacts. Using small RNA sequencing, fluorescence in situ hybridization, dual luciferase reporter assay, gene overexpression, and silencing experiments, we investigated the mitochondrial structural and functional impairments induced by PEDV infection in jejunum epithelial cells of piglets and characterized the regulatory pattern of miRNAs in mitochondria of jejunum epithelial cells during PEDV infection. The results indicate that PEDV infection leads to the upregulation and mitochondrial localization of the nuclear-encoded microRNA, miR-34c, which in turn suppresses COX1 expression. The activation of the miR-34c/COX1 axis diminishes mitochondrial complex III, IV, and V activities, depletes ATP, lowers mitochondrial oxygen consumption, induces mitochondrial depolarization, increases the accumulation of mitochondrial reactive oxygen species (mtROS), and stimulates mitophagy. Furthermore, we confirm that CREB3L1 acts as an upstream transcription factor regulating the miR-34c/COX1 axis during PEDV infection, modulating mitochondrial damage in the epithelial cells of the jejunum. These findings demonstrate for the first time that PEDV infection activates the miR-34c/COX1 axis via the transcription factor CREB3L1 and regulates the nuclear-mitochondrial communication and mitochondrial fate, providing a new perspective on the pathogenesis of PEDV.IMPORTANCEThis study reveals the mechanism by which the porcine epidemic diarrhea virus (PEDV) disrupts mitochondrial function in piglets, enhancing viral pathogenicity. By demonstrating how PEDV infection upregulates miR-34c, leading to COX1 suppression and subsequent mitochondrial dysfunction, the research highlights a novel aspect of viral manipulation of host cellular mechanisms. These findings provide a deeper understanding of the PEDV pathogenesis and identify potential targets for therapeutic intervention, advancing efforts to mitigate the economic impact of PEDV on the swine industry.

HIV-1 protease (PR) activation is triggered by Gag-Pol dimerization. We previously reported that reverse transcriptase (RT) amino acid substitution mutations resulted in p66/51RT heterodimer instability associated with impaired PR activation, and that treatment with efavirenz (EFV, an RT dimerization enhancer) increased PR activation, suggesting RT involvement. However, the contribution of RT to PR activation via the promotion of Gag-Pol dimerization has not been corroborated. To determine whether RT/RT interaction affects Gag-Pol dimerization, RT amino acid substitution mutations known to impair PR activation were cloned into a p6gag-containing construct, Gagp6-Pol, which assembles and releases virus-like particles (VLPs) when PR is inactivated. To map domains involved in Gag-Pol/Gag-Pol interaction, the major Gag assembly domain, with or without additional p6*, PR, or integrase (IN) deletions, was removed from Gagp6-Pol. Resulting constructs were transiently expressed in HEK293T cells. Sucrose density gradient fractionation and electron microscopy results suggest that p6gag-containing RT could form VLPs with lower densities and smaller sizes compared to wild-type particles. RT-PCR results suggest that p6-RT is capable of viral RNA packaging. RT-destabilizing amino acid mutations associated with PR-mediated virus processing deficiencies were found to be capable of reducing Gagp6-Pol VLP yields and attenuating EFV enhancement of Gagp6-Pol VLP assembly. Our results support the proposal that impaired RT stability or RT/RT interaction can disrupt Gag-Pol/Gag-Pol interaction, leading to impaired PR activation. This Gagp6-Pol VLP assembly system offers a potential assay method for probing domains involved in Gag-Pol/Gag-Pol interaction.

Importance: HIV-1 protease (PR) activation for mediating virus particle processing is essential for virus infectivity. As part of our attempt to determine whether Gag-Pol dimerization triggers PR activation, we found that RT point mutations that impair RT heterodimer stability and virus particle processing markedly reduced VLP assembly efficiencies in a p6gag-containing Gag-Pol expression vector (designated Gagp6-Pol). Further, these unstable RT point mutations markedly inhibited the facilitating effect of an RT dimerization enhancer on Gagp6-Pol VLP assembly. Our data support the proposal that RT/RT interaction contributes to PR activation by promoting Gag-Pol/Gag-Pol interaction, thus suggesting that targeting Gag-Pol dimerization may serve as an alternative HIV/AIDS treatment strategy. A Gag-Pol VLP assembly assay might be usable for probing the potential impacts of Gag-Pol dimerization on PR activation.