Journal of Virology最新文献

Bacteriophages are viruses infecting bacteria. The vast majority of them bear a tail, allowing host recognition, cell wall perforation, and DNA injection into the host cytoplasm. Using electron cryo-microscopy (cryo-EM) and single particle analysis, we determined the organization of the tail proximal extremity of siphophage T5 that possesses a long flexible tail and solved the structure of its tail terminator protein p142 (TrP₁₄₂). It allowed us to confirm the common evolutionary origin between T5 TrP_p142 and other known or putative TrPs from siphophages, myophages, and bacterial tail-like machines, despite very poor sequence conservation. By also determining the structure of the T5 tail proximal extremity after interaction with T5 bacterial receptor FhuA, we showed that no conformational changes occur in TrP_p142 and confirmed that the infection signal transduction is not carried by the tube itself. We also investigated the location of T5 Neck1 or tail completion protein p143 (TCP_p143) and showed, thanks to a combination of cryo-EM and structure prediction using Alphafold2, that it is not located at the capsid-to-tail interface as suggested by its position in the genome, but instead, very unexpectedly, on the side of T5 tail tip, and that it appears to be monomeric. Based on structure comparison with other putative TCPs predicted structures, this feature could not be shared by other TCPs and questions the affiliation of p143 to this family of protein.IMPORTANCEBacteriophages, viruses infecting bacteria, are the most abundant living entities on Earth. They are present in all ecosystems where bacteria develop and are instrumental in the regulation, diversity, evolution, and pathogeny of microbial populations. Moreover, with the increasing number of pathogenic strains resistant to antibiotics, virulent phages are considered a serious alternative or complement to classical treatments. 96% of all phages present a tail that allows host recognition and safe channeling of the DNA to the host cytoplasm. We present the atomic model of the proximal extremity of the siphophage T5 tail, confirming structural similarities with other phages. This structure, combined with results previously published and further explored, also allowed a review and a discussion on the role and localization of a mysterious tail protein, the tail completion protein, which is known to be present in the phage tails, but that was never identified in a phage structure.

Following reactivation of a latent alphaherpesvirus infection, viral particles are assembled in neuronal cell bodies, trafficked anterogradely within axons to nerve termini, and spread to adjacent epithelial cells. The virally encoded membrane proteins US9p and the glycoprotein heterodimer gE/gI of pseudorabies virus (PRV) and herpes simplex virus type 1 (HSV-1) play critical roles in anterograde spread, likely as a tripartite gE/gI-US9p complex. Two kinesin motors, kinesin-1 and kinesin-3, are implicated in the egress of these viruses, but how gE/gI-US9p coordinates their activities is poorly understood. Here, we report that PRV, in addition to associating with the kinesin-3 motor KIF1A, recruits the neuronal kinesin-1 isoforms KIF5A and KIF5C, but not the broadly expressed isoform KIF5B, during egress from differentiated CAD neurons. Similarly, in the axons of dorsal root ganglia (DRG)-derived sensory neurons, PRV colocalized with KIF5C but not KIF5B. In differentiated CAD cells, the association of KIF1A with egressing PRV was dependent upon US9p, whereas the recruitment of KIF5 isoforms required gE/gI. Consistent with these findings, the number of PRV particles trafficking within CAD neurites and the axons of DRG neurons increased when kinesin-1 motor activity was upregulated by hyperacetylating microtubules using trichostatin A (TSA) or tubacin, and this enhanced trafficking depended upon the presence of gE/gI. We propose that, following its recruitment by US9p, KIF1A delivers PRV particles to a location where KIF5 motors are subsequently added by a gE/gI-dependent mechanism. KIF5A/C isoforms then serve to traffic viral particles along axons, resulting in characteristic recrudescent infection.

Importance: Alphaherpesviruses include important human and veterinary pathogens that share a unique propensity to establish life-long latent infections in the peripheral nervous system. Upon reactivation, these viruses navigate back to body surfaces and transmit to new hosts. In this study, we demonstrate that the virus gE/gI-US9p membrane complex routes virus particles down this complex neuronal egress pathway by coordinating their association with multiple kinesin microtubule motors.

The hypoxia signaling pathway controls hypoxia adaptation and tolerance of organisms, which is regulated by multiple mechanisms. Viral infection elicits various pathophysiological responses in the host. However, whether viral infection can affect the hypoxia response is not yet fully understood. In this study, we found that Spring viremia of carp virus (SVCV) infection in zebrafish caused symptoms similar to those in zebrafish under hypoxic conditions. Further assays indicated that SVCV infection activated the hypoxia signaling pathway in zebrafish. In addition, SVCV infection caused increased glycolysis and reactive oxygen species (ROS) levels in cells. Mechanistically, SVCV-G protein interacted with hif1α-a/b and attenuated their K48-linked polyubiquitination, leading to their stabilization and subsequent enhancement of target gene expression. Moreover, treatment with the HIF1α-specific inhibitor PX478 enhanced the antiviral ability against SVCV infection in zebrafish and zebrafish cells. This study reveals a relationship between SVCV infection and the hypoxia signaling pathway in fish and provides a strategy for reducing the damage of viral disease in the aquaculture industry.

Importance: Viral infection triggers various pathophysiological responses in the host. The hypoxia signaling pathway controls hypoxia adaptation and tolerance of organisms. However, whether viral infection can affect the hypoxia response is not yet fully understood. This study showed that Spring viremia of carp virus (SVCV) infection activated the hypoxia signaling pathway and induced a hypoxia response. The SVCV-G protein interacted with hif1α-a/b and reduced their K48-linked polyubiquitination, leading to their stabilization and subsequent enhancement of target gene expression. Additionally, treatment with the HIF1α-specific inhibitor PX478 enhanced the antiviral ability against SVCV infection in zebrafish and zebrafish cells. Our findings not only reveal a relationship between SVCV infection and the hypoxia signaling pathway in fish but also provide a strategy for reducing the damage of viral disease in the aquaculture industry.

Porcine reproductive and respiratory syndrome viruses (PRRSVs) are significant pathogens that affect the global swine industry. Its virions consist of a central core composed of nucleocapsid (N) protein, surrounded by multiple distinct viral envelope proteins. However, the mechanisms underlying the recognition and packaging of N protein by viral envelope proteins remain elusive. In this study, we elucidated the role of nonstructural protein 2 (nsp2) from highly pathogenic PRRSV-2 (HP-PRRSV-2) in viral assembly. Firstly, among all the tested envelope proteins, only glycoprotein 5 (GP5) exhibits limited interaction with N protein. Interestingly, we demonstrated that full-length nsp2 co-immunoprecipitates (Co-IPs) with the N protein and all tested viral envelope proteins. In the presence of full-length nsp2, the N protein interacts with distinct viral envelope proteins. Moreover, upon viral infection, Co-IP experiments using nsp2-specific antibodies or N-specific antibodies revealed the formation of a complex between N and nsp2 with the M protein, GP2a, and GP5. However, neither of the two short forms of nsp2-namely nsp2TF nor nsp2N-participates in this process as they fail to interact with the N protein. Finally, our results demonstrate that this process occurs in the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment (ERGIC). Overall, our findings unveil a novel functional role for full-length nsp2 of HP-PRRSV-2 in facilitating the assembly of the N protein with viral envelope proteins.IMPORTANCEThe virus assembly process of arteriviruses remains largely elusive, including the direct interaction between N protein and viral envelope proteins or the potential requirement for additional proteins in facilitating assembly. Moreover, where the N protein assembles with viral envelope proteins during the virus lifecycle remains unclear. This study reveals a novel role for nonstructural protein 2 (nsp2) in highly pathogenic porcine reproductive and respiratory syndrome virus type 2 (HP-PRRSV-2), highlighting its involvement in HP-PRRSV-2 assembly. These findings provide crucial insights into HP-PRRSV-2 assembly and enhance our understanding of their lifecycle. Overall, this study offers an alternative approach to developing a new antiviral strategy targeting PRRSV-2 assembly.

Adeno-associated viruses (AAVs) are the most extensively researched viral vectors for gene therapy globally. The AAV viral protein 1 (VP1) N-terminus controls the capsid's ability to translocate into the cell nucleus; however, the exact mechanism of this process is largely unknown. In this study, we sought to elucidate the precise interactions between AAV serotype 6 (AAV6), a promising vector for immune disorders, and host transport receptors responsible for vector nuclear localization. Focusing on the positively charged basic areas within the N-terminus of AAV6 VP1, we identified a 53-amino acid region that interacts with nuclear import receptors. We measured the binding affinities between this region and various nuclear import receptors, discovering a notably strong interaction with IMPα5 and IMPα7 in the low nanomolar range. We also elucidated the X-ray crystal structure of this region in complex with an importin alpha (IMPα) isoform, uncovering its binding as a bipartite nuclear localization signal (NLS). Furthermore, we show that using this bipartite NLS, AAV6 VP1 capsid protein can localize to the nucleus of mammalian cells in a manner dependent on the IMPα/IMPβ nuclear import pathway. This study provides detailed insights into the interaction between the AAV6 VP1 capsid protein and nuclear import receptors, deepening our knowledge of AAV nuclear import mechanisms and establishing a basis for the improvement of AAV6-based gene therapy vectors.IMPORTANCEAAVs, recognized as the most extensively researched viral vectors for gene therapy globally, offer significant advantages over alternatives due to their small size, non-pathogenic nature, and innate ability for tissue-specific targeting. AAVs are required to localize to the nucleus to perform their role as a gene therapy vector; however, the precise mechanisms that facilitate this process remain unknown. Despite sharing overt genomic similarities with AAV1 and AAV2, AAV6 is a unique serotype. It is currently recognized for its ability to effectively transduce hematopoietic cell lineages and, consequently, is considered promising for the treatment of immune disorders. Identifying the exact mechanisms that permit AAV6 to access the nucleus can open up new avenues for gene therapy vector engineering, which can ultimately lead to increased therapeutic benefits.

The human cytomegalovirus (HCMV) encoded chemokine receptor US28 plays a critical role in viral pathogenesis, mediating several processes such as cellular migration, differentiation, transformation, and viral latency and reactivation. Despite significant research examining the signal transduction pathways utilized by US28, the precise mechanism by which US28 activates these pathways remains unclear. We performed a mutational analysis of US28 to identify signaling domains that are critical for functional activities. Our results indicate that specific residues within the third intracellular loop (ICL3) of US28 are major determinants of G-protein coupling and downstream signaling activity. Alanine substitutions at positions S218, K223, and R225 attenuated US28-mediated activation of MAPK and RhoA signal transduction pathways. Furthermore, we show that mutations at positions S218, K223, or R225 result in impaired coupling to multiple Gα isoforms. However, these substitutions did not affect US28 plasma membrane localization or the receptor internalization rate. Utilizing CD34⁺ HPC models, we demonstrate that attenuation of US28 signaling via mutation of residues within the ICL3 region results in an inability of the virus to efficiently reactivate from latency. These results were recapitulated in vivo, utilizing a humanized mouse model of HCMV infection. Together, our results provide new insights into the mechanism by which US28 manipulates host signaling networks to mediate viral latency and reactivation. The results reported here will guide the development of targeted therapies to prevent HCMV-associated disease.IMPORTANCEHuman cytomegalovirus (HCMV) is a β-herpesvirus that infects between 44% and 100% of the world population. Primary infection is typically asymptomatic and results in the establishment of latent infection within CD34⁺hematopoietic progenitor cells (HPCs). However, reactivation from latent infection remains a significant cause of morbidity and mortality in immunocompromised individuals. The viral chemokine receptor US28 influences various cellular processes crucial for viral latency and reactivation, yet the precise mechanism by which US28 functions remains unclear. Through mutational analysis, we identified key residues within the third intracellular loop (ICL3) of US28 that govern G-protein coupling, downstream signaling, and viral reactivation in vitro and in vivo. These findings offer novel insights into how US28 manipulates host signaling networks to regulate HCMV latency and reactivation and expand our understanding of HCMV pathogenesis.

The attack and defense of infected cells and cytotoxic CD8 T cells occur in germinal centers in lymphoid tissue in chronic persistent HIV/SIV infection. Latently infected cells, the therapeutic target of HIV infection, accumulate in follicular helper T (Tfh) cells in lymphoid tissue; the impact of HIV-specific follicular CD8 (fCD8) T cells in lymphoid tissue on the latently infected cells remains unknown. We infected 15 cynomolgus macaques with SIVmac239 and examined the contribution of SIV-Gag-specific fCD8 T cells, defined by activation-induced markers (AIMs), to SIV-infected cells. Eight out of the 15 infected macaques served as progressors; a chronic phase combination antiretroviral therapy (cART) model was established for the eight macaques (progressors) with chronic persistent infection status, wherein cART was started in the chronic phase and discontinued after 27 weeks. Seven macaques that naturally controlled the viremia served as natural controllers. The frequency of SIV-Gag-specific fCD8 T cells was inversely correlated with the amount of cell-associated SIV-gag RNA in the Tfh only under cART or in the controllers but not in untreated progressors. scRNA-seq of SIV-Gag-specific fCD8 T cells in various conditions revealed that the gene expression pattern of SIV-Gag-specific fCD8 T cells in the controllers was closer to that of those under cART than the untreated progressors. Comparing the SIV-Gag-specific fCD8 T cells of those under cART to the controllers revealed their more exhausted and immunosenescent nature under cART. Improving the HIV/SIV-specific fCD8 T cells under cART by targeting those pathways might contribute to the development of potential curative strategies.IMPORTANCEWe infected cynomolgus macaques with SIVmac239 to establish an SIV-chronically infected cART model. We performed an in-depth characterization of Tfh and fCD8 T cells in three conditions-chronic stage of untreated, cART-treated, and natural controller cynomolgus macaques-by combining tissue section analysis and single-cell analyses of sorted cells. We revealed the inverse relationship between Tfh infection and SIV-Gag-specific fCD8 T cell frequencies as observed in HIV-infected individuals, thereby establishing the cynomolgus macaque as a relevant animal model to study the determinants of HIV/SIV persistence in lymphoid tissue. Additionally, scRNA-seq analysis of SIV-Gag-specific fCD8 T cells revealed an enrichment of exhausted or senescent transcriptomic signatures under cART. These data will provide the basic insights into virus-host CD8 T cell interactions, particularly within the follicular region, during latent HIV infection under ART.

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