Journal of Virology最新文献

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.

Sputnik virophages are small double-stranded DNA (dsDNA) viruses that replicate only inside host amoebae infected with giant dsDNA viruses, mimiviruses. Sputnik infection affects mimivirus replication, but their molecular interaction remains poorly understood. Here, we performed a time-course transcriptome analysis of Acanthamoeba castellanii cells infected with Acanthamoeba polyphaga mimivirus (APMV; hereafter referred to as Sputnik^- cells) and those infected with both APMV and Sputnik 3 virophage (Sputnik⁺ cells). The gene expression patterns of the amoeba were similar between these two conditions, whereas the expression of APMV genes was drastically affected by Sputnik, depending on the timing of their expression. Early-expressed APMV genes showed similar expression patterns in both conditions at the early stage of infection. However, at later stages, their expression levels remained higher in Sputnik⁺ cells than in Sputnik^- cells, suggesting a prolongation of early gene expression by Sputnik. Late-expressed APMV genes showed lower expression at earlier stages in Sputnik⁺ cells, but their expression levels reached or exceeded those in Sputnik^- cells at later stages, indicating a delay in gene expression. Overall, our results demonstrated that Sputnik infection drastically alters the transcriptome of APMV rather than amoeba, likely by disturbing the transition from early to late stages of APMV infection.IMPORTANCEVirophages are small double-stranded DNA (dsDNA) viruses parasitizing other dsDNA viruses, such as giant viruses. Virophages inhibit the replication of giant viruses, and some protists use virophages as a defense system against giant viruses. However, molecular interactions among host cellular organisms, giant viruses, and virophages are largely unknown. Here, we performed a time-course transcriptome analysis of Acanthamoeba castellanii cells infected with Acanthamoeba polyphaga mimivirus (APMV) and those infected with both APMV and Sputnik 3 virophage. We demonstrated that the virophage has little effect on the amoeba transcriptome and primarily hijacks the transcriptional machinery of the giant virus. Furthermore, virophage infection alters giant virus gene expression, depending on their expression timing. The expression of early genes was prolonged, while that of late genes was delayed, suggesting that virophage infection disrupts the transition from the early to late stages of giant virus infection. This study provides molecular insights into the interactions within this unique tripartite system.

The rabies virus large (L) protein interacts with its cofactor phosphoprotein (P protein) to function as an RNA-dependent RNA polymerase (RdRp). The C-terminal domain (CTD) of the L protein plays a critical role in P protein binding. We previously reported that the highly conserved NPYNE sequence in the hydrophilic region of the CTD (positions 1929-1933 of the L protein [L1929-1933]) is important for both P protein binding and RdRp function. To elucidate the functional role of the CTD in detail, we examined the importance of each of the hydrophilic residues in the NPYNE sequence (underlined). A rabies virus mutant with Ala substitutions in these hydrophilic residues showed low replication capacity. Comprehensive analyses of a revertant of the mutant virus and a series of L protein mutants revealed that Asn at L1929 is crucial for both P protein binding and RdRp function. Analyses of the L protein mutants also showed that Asn at L1932 and Glu at L1933 have roles in RdRp function and P protein binding, respectively. Furthermore, we demonstrated that the NPYNE sequence is essential for stabilizing the L protein through the L-P interaction. In a previously determined L protein structure, all of the hydrophilic residues in the NPYNE sequence form the first α-helix in the CTD. Therefore, our findings indicate that this helix is important for P protein-binding ability, RdRp function, and stabilization of the L protein, thereby contributing to efficient viral replication.

Importance: Although RNA-dependent RNA polymerase of rhabdoviruses, which is composed of the large (L) protein and its cofactor phosphoprotein (P protein), has a high potential as a target for therapeutics against the viruses, the relationship between the structure and molecular functions is poorly understood. In this study, we functionally examined the C-terminal domain (CTD) of the rabies virus L protein as a model for the rhabdovirus L protein. We showed that the first α-helix in the CTD is important for the P protein-binding ability, RdRp function, and stability of the L protein. Since in the L-P complex structure, this helix does not form an interface with the P protein, we provide here the first evidence of an indirect contribution of the L protein CTD to the L-P interaction in rhabdoviruses. The findings in this study will be useful for developing therapeutics targeting the L-P interaction.

HIV cure strategies that aim to induce viral reactivation for immune clearance leverage latency reversal agents to modulate host pathways which directly or indirectly facilitate viral reactivation. Inhibition of bromo and extra-terminal domain (BET) family member BRD4 reverses HIV latency, but enthusiasm for the use of BET inhibitors in HIV cure studies is tempered by concerns over inhibition of other BET family members and dose-limiting toxicities in oncology trials. Here, we evaluated the potential for bivalent chemical degraders targeted to the BET family as alternative latency reversal agents. We observed that despite highly potent and selective BRD4 degradation in primary CD4+ T-cells from ART-suppressed donors, BRD4 degraders failed to induce latency reversal as compared to BET inhibitors. Furthermore, BRD4 degraders failed to mimic previously observed synergistic HIV reactivation between BET inhibitors and an activator of the non-canonical NF-κB pathway. Mechanistic investigation of this discrepancy revealed that latency reversal by BET inhibitors is not related to the abatement of competition between Tat and BRD4 for P-TEFb, but rather the ability of BRD4 to disrupt 7SK and increase the levels of free P-TEFb. This activity is dependent on the shift of BRD4 from chromatin-bound to soluble and retargeting of P-TEFb to chromatin, which is dependent on intact BRD4 but independent of the bromodomains.

Importance: Multiple factors and pathways contribute to the maintenance of HIV latency, including bromo and extra-terminal domain (BET) family member BRD4. While small molecule inhibitors of the BET family result in latency reversal, enthusiasm for the use of BET inhibitors in HIV cure is limited due to toxicity concerns. We examined BRD4-selective chemical degraders as alternatives to BET inhibitors but found two robust degraders failed to induce latency reversal. We observed key differences in the ability of BET inhibitors versus BET degraders to disrupt P-TEFb, a key cellular activator of transcription and a complex required for HIV reactivation. We present a new model for the role of BRD4 in HIV latency and propose that BRD4 be reconsidered as an activator rather than a repressor of HIV transcription in the context of HIV cure strategies.

Zika virus (ZIKV) is spread by mosquito bites and is unique among known flaviviruses for being able to cause microcephaly. Entry factors for ZIKV are incompletely understood, but phosphatidylserine (PS) receptors, including the TAM (Tyro3, AXL, and Mer) and TIM (T-cell Ig mucin) families, can serve as cofactors for flavivirus entry in a cell type-specific manner. We identify AXL as the top hit in a CRISPR/Cas9 genome-wide screen in human glioblastoma cells and establish a definitive role of AXL, but not TYRO3 or MerTK, for ZIKV infection. Additionally, Spondweni virus also shows AXL dependency, while dengue virus infection is not affected by AXL knockout. Passage of ZIKV in AXL knockout (KO) cells generated a mutant virus capable of infection via AXL-independent mechanisms, and multiple independent selections identified a common mutation, H83R, in the prM coding region of the ZIKV genome. The mutant virus exhibits an increased infectivity rate in AXL KO cells as compared to wild-type ZIKV and is dependent upon the single H83R mutation. The mutant virus' ability to infect cells in an AXL-independent manner is unrelated to interferon signaling antagonism but likely pertains to a change in virus maturation that leads to a structural disturbance of the ZIKV virion. Our study provides evidence for a potential mechanism linking the viral structural proteins and host PS receptor usage during flavivirus infection.IMPORTANCEA major challenge in elucidating the mechanism of Zika virus (ZIKV) pathogenesis is the multitude of cell types it infects with distinct requirements. The role of phosphatidylserine (PS) receptors in ZIKV infection is cell type-specific, and the controversy surrounds their function in flavivirus entry. Here, we establish a definitive requirement of AXL for infection of human glioblastoma cells by both Zika and Spondweni virus. We then identified a single amino acid mutation (H83R) in the prM protein of ZIKV that allowed AXL-independent infection of these cells. The H83R-mediated escape of AXL requirement is independent of interferon (IFN) signaling suppression by AXL; instead, the mutation has the potential to disrupt the virus assembly and virion structure. This study reveals a previously unknown connection between the PS receptor usage and the flavivirus prM gene, which can guide detailed molecular mechanism studies of the interplay between virion assembly and virus entry.