RESUMO
Transfer RNAs (tRNAs) are at the heart of the molecular biology central dogma, functioning to decode messenger RNAs into proteins. As obligate intracellular parasites, viruses depend on the host translation machinery, including host tRNAs. Thus, the ability of a virus to fine-tune tRNA expression elicits the power to impact the outcome of infection. DNA viruses commonly upregulate the output of RNA polymerase III (Pol III)-dependent transcripts, including tRNAs. Decades after these initial discoveries we know very little about how mature tRNA pools change during viral infection, as tRNA sequencing methodology has only recently reached proficiency. Here, we review perturbation of tRNA biogenesis by DNA virus infection, including an emerging player called tRNA-derived fragments (tRFs). We discuss how tRNA dysregulation shifts the power landscape between the host and virus, highlighting the potential for tRNA-based antivirals as a future therapeutic.
Assuntos
Infecções por Vírus de DNA , RNA de Transferência , Humanos , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA Mensageiro , BiologiaRESUMO
T-cell immunoglobin and mucin domain protein-1 (TIM-1) mediates entry of chikungunya virus (CHIKV) into some mammalian cells through the interaction with envelope phospholipids. While this interaction enhances entry, TIM-1 has been shown to tether newly formed HIV and Ebola virus particles, limiting their efficient release. In this study, we investigate the ability of surface receptors such as TIM-1 to sequester newly budded virions on the surface of infected cells. We established a luminescence reporter system to produce chikungunya viral particles that integrate nano-luciferase and easily quantify viral particles. We found that TIM-1 on the surface of host cells significantly reduced CHIKV release efficiency in comparison to other entry factors. Removal of cell surface TIM-1 through direct cellular knock-out or altering the cellular lipid distribution enhanced CHIKV release. Over the course of infection, CHIKV was able to counteract the tethering effect by gradually decreasing the surface levels of TIM-1 in a process mediated by the nonstructural protein 2. This study highlights the importance of phosphatidylserine receptors in mediating not only the entry of CHIKV but also its release and could aid in developing cell lines capable of enhanced vaccine production. IMPORTANCE: Chikungunya virus (CHIKV) is an enveloped alphavirus transmitted by the bites of infectious mosquitoes. Infection with CHIKV results in the development of fever, joint pain, and arthralgia that can become chronic and last for months after infection. Prevention of this disease is still highly focused on vector control strategies. In December 2023, a new live attenuated vaccine against CHIKV was approved by the FDA. We aimed to study the cellular factors involved in CHIKV release, to better understand CHIKV's ability to efficiently infect and spread among a wide variety of cell lines. We found that TIM-1 receptors can significantly abrogate CHIKV's ability to efficiently exit infected cells. This information can be beneficial for maximizing viral particle production in laboratory settings and during vaccine manufacturing.
Assuntos
Febre de Chikungunya , Vírus Chikungunya , Receptor Celular 1 do Vírus da Hepatite A , Fosfatidilserinas , Liberação de Vírus , Vírus Chikungunya/fisiologia , Vírus Chikungunya/metabolismo , Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Humanos , Fosfatidilserinas/metabolismo , Febre de Chikungunya/virologia , Febre de Chikungunya/metabolismo , Células HEK293 , Internalização do Vírus , Animais , Envelope Viral/metabolismo , Linhagem Celular , Vírion/metabolismo , Receptores Virais/metabolismoRESUMO
Transfer RNAs (tRNAs) are fundamental for both cellular and viral gene expression during viral infection. Moreover, mounting evidence supports a noncanonical role for tRNA cleavage products in the control of gene expression during diverse conditions of stress and infection. We previously reported that infection with the model murine gammaherpesvirus, MHV68, leads to altered tRNA transcription, suggesting that tRNA regulation may play an important role in mediating viral replication or the host response. To better understand how viral infection alters tRNA expression, we combined Ordered Two Template Relay (OTTR) with tRNA-specific bioinformatic software called tRAX to profile full-length tRNAs and fragmented tRNA-derived RNAs (tDRs) during infection with MHV68. We find that OTTR-tRAX is a powerful sequencing strategy for combined tRNA/tDR profiling and reveals that MHV68 infection triggers pre-tRNA and mature tRNA cleavage, resulting in the accumulation of specific tDRs. Fragments of virally-encoded tRNAs (virtRNAs), as well as virtRNA base modification signatures are also detectable during infection. We present evidence that tRNA splicing factors are involved in the biogenesis of MHV68-induced cleavage products from pre-tRNAs and, in the case of CLP1 kinase, impact infectious virus production. Our data offers new insights into the importance of tRNA processing during gammaherpesvirus infection.
RESUMO
Transfer RNAs (tRNAs) are fundamental for both cellular and viral gene expression during viral infection. In addition, mounting evidence supports biological function for tRNA cleavage products, including in the control of gene expression during conditions of stress and infection. We previously reported that infection with the model murine gammaherpesvirus, MHV68, leads to enhanced tRNA transcription. However, whether this has any influence on tRNA transcript processing, viral replication, or the host response is not known. Here, we combined two new approaches, sequencing library preparation by Ordered Two Template Relay (OTTR) and tRNA bioinformatic analysis by tRAX, to quantitatively profile full-length tRNAs and tRNA fragment (tRF) identities during MHV68 infection. We find that MHV68 infection triggers both pre-tRNA and mature tRNA cleavage, resulting in the accumulation of specific tRFs. OTTR-tRAX revealed not only host tRNAome changes, but also the expression patterns of virally-encoded tRNAs (virtRNAs) and virtRFs made from the MHV68 genome, including their base modification signatures. Because the transcript ends of several host tRFs matched tRNA splice junctions, we tested and confirmed the role of tRNA splicing factors TSEN2 and CLP1 in MHV68-induced tRF biogenesis. Further, we show that CLP1 kinase, and by extension tRNA splicing, is required for productive MHV68 infection. Our findings provide new insight into how gammaherpesvirus infection both impacts and relies on tRNA transcription and processing.
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The menstrual cycle influences the risk of acquiring sexually transmitted infections (STIs), including Chlamydia trachomatis (C. trachomatis), although the underlying immune contributions are poorly defined. A mouse model simulating the immune-mediated process of menstruation could provide valuable insights into tissue-specific determinants of protection against chlamydial infection within the cervicovaginal and uterine mucosae comprising the female reproductive tract (FRT). Here, we used the pseudopregnancy approach in naïve C57Bl/6 mice and performed vaginal challenge with Chlamydia muridarum (C. muridarum) at decidualization, endometrial tissue remodeling, or uterine repair. This strategy identified that the time frame comprising uterine repair correlated with robust infection and greater bacterial burden as compared with mice on hormonal contraception, while challenges during endometrial remodeling were least likely to result in a productive infection. By comparing the infection site at early time points following chlamydial challenge, we found that a greater abundance of innate effector populations and proinflammatory signaling, including IFNγ correlated with protection. FRT immune profiling in uninfected mice over pseudopregnancy or in pig-tailed macaques over the menstrual cycle identified NK cell infiltration into the cervicovaginal tissues and lumen over the course of endometrial remodeling. Notably, NK cell depletion over this time frame reversed protection, with mice now productively infected with C. muridarum following challenge. This study shows that the pseudopregnancy murine menstruation model recapitulates immune changes in the FRT as a result of endometrial remodeling and identifies NK cell localization at the FRT as essential for immune protection against primary C. muridarum infection.
RESUMO
T-cell immunoglobin and mucin domain protein-1 (TIM-1) mediates entry of Chikungunya virus (CHIKV) into some mammalian cells through the interaction with envelope phospholipids. While this interaction enhances entry, TIM has been shown to tether newly formed HIV and Ebola virus particles, limiting their efficient release. In this study, we investigate the ability of surface receptors such as TIM-1 to sequester newly budded virions on the surface of infected cells. We established a luminescence reporter system to produce Chikungunya viral particles that integrate nano-luciferase and easily quantify viral particles. We found that TIM-1 on the surface of host cells significantly reduced CHIKV release efficiency in comparison to other entry factors. Removal of cell surface TIM-1 through direct cellular knock-out or altering the cellular lipid distribution enhanced CHIKV release. Over the course of infection, CHIKV was able to counteract the tethering effect by gradually decreasing the surface levels of TIM-1 in a process that appears to be mediated by the nonstructural protein 2. This study highlights the importance of phosphatidylserine receptors in mediating not only the entry of CHIKV but also its release and could aid in developing cell lines capable of enhanced vaccine production.
RESUMO
To identify proteins specific to the proximal ciliary axoneme, we used iTRAQ to compare short (~2 µm) and full-length (~11 µm) axonemes of Chlamydomonas. Known compoents of the proximal axoneme such as minor dynein heavy chains and LF5 kinase as well as the ciliary tip proteins FAP256 (CEP104) and EB1 were enriched in short axonemes whereas proteins present along the length of the axoneme were of similar abundance in both samples. The iTRAQ analysis revealed that FAP93, a protein of unknown function, and protein phosphatase 2A (PP2A) are enriched in the short axonemes. Consistently, immunoblots show enrichment of FAP93 and PP2A in short axonemes and immunofluorescence confirms the localization of FAP93 and enrichment of PP2A at the proximal axoneme. Ciliary regeneration reveals that FAP93 assembles continuously but more slowly than other axonemal structures and terminates at 1.03 µm in steady-state axonemes. The length of FAP93 assembly correlates with ciliary length, demonstrating ciliary length-dependent assembly of FAP93. Dikaryon rescue experiments show that FAP93 can assemble independently of IFT transport. In addition, FRAP analysis of GFP-tagged FAP93 demonstrates that FAP93 is stably anchored in axoneme. FAP93 may function as a scaffold for assembly of other specific proteins at the proximal axoneme.
RESUMO
Chikungunya virus (CHIKV) is the causative agent of the human disease chikungunya fever, characterized by debilitating acute and chronic arthralgia. No licensed vaccines or antivirals are currently available for CHIKV. Therefore, the prevention of attachment of viral particles to host cells is a potential intervention strategy. As an arbovirus, CHIKV infects a wide variety of cells in both its mammalian and mosquito host. This broad cell tropism might stem from CHIKV's ability to bind to a variety of entry factors in the host cell including phosphatidylserine receptors (PSRs), glycosaminoglycans (GAGs), and the proteinaceous receptor Mxra8, among others. In this study, we aimed to determine the relevance of each attachment factor during CHIKV entry into a panel of mammalian and mosquito cells. Our data suggest that the importance of particular binding factors during CHIKV infection is highly cell line dependent. Entry into mammalian Vero cells was mediated through attachment to PSRs, mainly T-cell immunoglobulin mucin domain-1 (TIM-1). Conversely, CHIKV infection into HAP1 and NIH3T3 was predominantly mediated by heparan sulfate (HS) and Mxra8, respectively. Entry into mosquito cells was independent of PSRs, HS, and Mxra8. Although entry into mosquito cells remains unclear, our data denotes the importance of careful evaluation of reagents used to identify receptor use in invertebrate cells. While PSRs, GAGs, and Mxra8 all enhance entry in a cell line dependent manner, none of these factors are necessary for CHIKV entry, suggesting additional host factors are involved.
RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the most recent global pandemic that has caused more than a million deaths around the world. The spike glycoprotein (S) drives the entry and fusion of this virus and is the main determinant of cell tropism. To explore S requirements for entry under BSL2 conditions, S has been pseudotyped onto vesicular stomatitis virus (VSV) or retroviral particles with varied success. Several alterations to S were demonstrated to improve pseudoparticle titers, but they have not been systematically compared. In this study, we produced pseudotyped VSV particles with multiple modifications to S, including truncation, mutation, and tagging strategies. The main objective of this study was to determine which modifications of the S protein optimize cell surface expression, incorporation into pseudotyped particles, and pseudoparticle entry. Removal of the last 19 residues of the cytoplasmic tail produced a hyper-fusogenic S, while removal of 21 residues increased S surface production and VSV incorporation. Additionally, we engineered a replication-competent VSV (rVSV) virus to produce the S-D614G variant with a truncated cytoplasmic tail. While the particles can be used to assess S entry requirements, the rVSV∆G/SMet1D614G∆21 virus has a poor specific infectivity (particle to infectious titer ratio).