Gammaherpesvirus infection triggers the formation of tRNA fragments from premature tRNAs.

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.

Gammaherpesvirus infection alters transfer RNA splicing and triggers tRNA cleavage.

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.

"Transfer" of power: The intersection of DNA virus infection and tRNA biology.

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.

Manipulation of RNA polymerase III by Herpes Simplex Virus-1.

RNA polymerase III (Pol III) transcribes noncoding RNA, including transfer RNA (tRNA), and is commonly targeted during cancer and viral infection. We find that Herpes Simplex Virus-1 (HSV-1) stimulates tRNA expression 10-fold. Perturbation of host tRNA synthesis requires nuclear viral entry, but not synthesis of specific viral transcripts. tRNA with a specific codon bias were not targeted-rather increased transcription was observed from euchromatic, actively transcribed loci. tRNA upregulation is linked to unique crosstalk between the Pol II and III transcriptional machinery. While viral infection results in depletion of Pol II on host mRNA promoters, we find that Pol II binding to tRNA loci increases. Finally, we report Pol III and associated factors bind the viral genome, which suggests a previously unrecognized role in HSV-1 gene expression. These findings provide insight into mechanisms by which HSV-1 alters the host nuclear environment, shifting key processes in favor of the pathogen.

Alteration of the Premature tRNA Landscape by Gammaherpesvirus Infection.

Transfer RNAs (tRNAs) are transcribed by RNA polymerase III (RNAPIII) and play a central role in decoding our genome, yet their expression and noncanonical function remain understudied. Many DNA tumor viruses enhance the activity of RNAPIII, yet whether infection alters tRNA expression is largely unknown. Here, we present the first genome-wide analysis of how viral infection alters the tRNAome. Using a tRNA-specific sequencing method (DM-tRNA-seq), we find that the murine gammaherpesvirus MHV68 induces global changes in premature tRNA (pre-tRNA) expression, with 14% of tRNA genes upregulated more than 3-fold, indicating that differential tRNA gene induction is a characteristic of DNA virus infection. Elevated pre-tRNA expression corresponds to increased RNAPIII occupancy for the subset of tRNA genes tested; additionally, posttranscriptional mechanisms contribute to the accumulation of pre-tRNA species. We find increased abundance of tRNA fragments derived from pre-tRNAs upregulated by viral infection, suggesting that noncanonical tRNA cleavage is also affected. Furthermore, pre-tRNA accumulation, but not RNAPIII recruitment, requires gammaherpesvirus-induced degradation of host mRNAs by the virally encoded mRNA endonuclease muSOX. We hypothesize that depletion of pre-tRNA maturation or turnover machinery contributes to robust accumulation of full-length pre-tRNAs in infected cells. Collectively, these findings reveal pervasive changes to tRNA expression during DNA virus infection and highlight the potential of using viruses to explore tRNA biology.IMPORTANCE Viral infection can dramatically change the gene expression landscape of the host cell, yet little is known regarding changes in noncoding gene transcription by RNA polymerase III (RNAPIII). Among these are transfer RNAs (tRNAs), which are fundamental in protein translation, yet whose gene regulatory features remain largely undefined in mammalian cells. Here, we perform the first genome-wide analysis of tRNA expression changes during viral infection. We show that premature tRNAs accumulate during infection with the model gammaherpesvirus MHV68 as a consequence of increased transcription, but that transcripts do not undergo canonical maturation into mature tRNAs. These findings underscore how tRNA expression is a highly regulated process, especially during conditions of elevated RNAPIII activity.

The molecular virology of coronaviruses.

Few human pathogens have been the focus of as much concentrated worldwide attention as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of COVID-19. Its emergence into the human population and ensuing pandemic came on the heels of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), two other highly pathogenic coronavirus spillovers, which collectively have reshaped our view of a virus family previously associated primarily with the common cold. It has placed intense pressure on the collective scientific community to develop therapeutics and vaccines, whose engineering relies on a detailed understanding of coronavirus biology. Here, we present the molecular virology of coronavirus infection, including its entry into cells, its remarkably sophisticated gene expression and replication mechanisms, its extensive remodeling of the intracellular environment, and its multifaceted immune evasion strategies. We highlight aspects of the viral life cycle that may be amenable to antiviral targeting as well as key features of its biology that await discovery.

Ribosome Biogenesis Modulates Ty1 Copy Number Control in Saccharomyces cerevisiae.

Transposons can impact the host genome by altering gene expression and participating in chromosome rearrangements. Therefore, organisms evolved different ways to minimize the level of transposition. In Saccharomyces cerevisiae and its close relative S. paradoxus, Ty1 copy number control (CNC) is mediated by the self-encoded restriction factor p22, which is derived from the GAG capsid gene and inhibits virus-like particle (VLP) assembly and function. Based on secondary screens of Ty1 cofactors, we identified LOC1, a RNA localization/ribosome biogenesis gene that affects Ty1 mobility predominantly in strains harboring Ty1 elements. Ribosomal protein mutants rps0bΔ and rpl7aΔ displayed similar CNC-specific phenotypes as loc1Δ, suggesting that ribosome biogenesis is critical for CNC. The level of Ty1 mRNA and Ty1 internal (Ty1i) transcripts encoding p22 was altered in these mutants, and displayed a trend where the level of Ty1i RNA increased relative to full-length Ty1 mRNA. The level of p22 increased in these mutants, and the half-life of p22 also increased in a loc1Δ mutant. Transcriptomic analyses revealed small changes in the level of Ty1 transcripts or efficiency of translation initiation in a loc1Δ mutant. Importantly, a loc1Δ mutant had defects in assembly of Gag complexes and packaging Ty1 RNA. Our results indicate that defective ribosome biogenesis enhances CNC by increasing the level of p22, and raise the possibility for versatile links between VLP assembly, its cytoplasmic environment, and a novel stress response.

Host Noncoding Retrotransposons Induced by DNA Viruses: a SINE of Infection?

Our genomes are dominated by repetitive elements. The majority of these elements derive from retrotransposons, which expand throughout the genome through a process of reverse transcription and integration. Short interspersed nuclear elements, or SINEs, are an abundant class of retrotransposons that are transcribed by RNA polymerase III, thus generating exclusively noncoding RNA (ncRNA) that must hijack the machinery required for their transposition. SINE loci are generally transcriptionally repressed in somatic cells but can be robustly induced upon infection with multiple DNA viruses. Recent research has focused on the gene expression and signaling events that are modulated by SINE ncRNAs, particularly during gammaherpesvirus infection. Here, we review the biology of these SINE ncRNAs, explore how DNA virus infection may lead to their induction, and describe how novel gene regulatory and immune-related functions of these ncRNAs may impact the viral life cycle.

Ty1 escapes restriction by the self-encoded factor p22 through mutations in capsid.

Ty1 is a long terminal repeat (LTR) retrotransposon belonging to the Ty1/copia family and is present in up to 32 full-length copies in Saccharomyces. Like retroviruses, Ty1 contains GAG and POL genes, LTRs, and replicates via an RNA intermediate within a virus-like particle (VLP). Although Ty1 retrotransposition is not infectious, uncontrolled replication can lead to detrimental effects on the host genome, including insertional mutagenesis and chromosomal rearrangements. Ty1 copy number control (CNC) limits replication and is mediated through a self-encoded protein called p22. p22 is translated from a subgenomic Ty1 RNA and encodes an amino-truncated version of the Gag protein. We highlight a recent study identifying Ty1 Gag, which comprises the VLP capsid and provides nucleic acid chaperone functions, as a direct target of p22-mediated inhibition. CNC-resistant (CNCR) mutations map within predicted helical domains of Gag, including those in the Ty1/copia pfam domain Retrotran_gag_2 (formerly UBN2) and a central region we refer to as the CNCR domain. CNCR Gag forms VLPs that exclude p22, thus restoring Ty1 replication. We discuss possible mechanisms for p22 inclusion in Ty1 VLPs and compare Ty1 CNC with retroviral restriction factors targeting capsid (CA).

A self-encoded capsid derivative restricts Ty1 retrotransposition in Saccharomyces.

Retrotransposons and retroviral insertions have molded the genomes of many eukaryotes. Since retroelements transpose via an RNA intermediate, the additive nature of the replication cycle can result in massive increases in copy number if left unchecked. Host organisms have countered with several defense systems, including domestication of retroelement genes that now act as restriction factors to minimize propagation. We discovered a novel truncated form of the Saccharomyces Ty1 retrotransposon capsid protein, dubbed p22 that inhibits virus-like particle (VLP) assembly and function. The p22 restriction factor expands the repertoire of defense proteins targeting the capsid and highlights a novel host-parasite strategy. Instead of inhibiting all transposition by domesticating the restriction gene as a distinct locus, Ty1 and budding yeast may have coevolved a relationship that allows high levels of transposition when Ty1 copy numbers are low and progressively less transposition as copy numbers rise. Here, we offer a perspective on p22 restriction, including its mode of expression, effect on VLP functions, interactions with its target, properties as a nucleic acid chaperone, similarities to other restriction factors, and future directions.

The Ty1 Retrotransposon Restriction Factor p22 Targets Gag.

A novel form of copy number control (CNC) helps maintain a low number of Ty1 retrovirus-like transposons in the Saccharomyces genome. Ty1 produces an alternative transcript that encodes p22, a trans-dominant negative inhibitor of Ty1 retrotransposition whose sequence is identical to the C-terminal half of Gag. The level of p22 increases with copy number and inhibits normal Ty1 virus-like particle (VLP) assembly and maturation through interactions with full length Gag. A forward genetic screen for CNC-resistant (CNCR) mutations in Ty1 identified missense mutations in GAG that restore retrotransposition in the presence of p22. Some of these mutations map within a predicted UBN2 domain found throughout the Ty1/copia family of long terminal repeat retrotransposons, and others cluster within a central region of Gag that is referred to as the CNCR domain. We generated multiple alignments of yeast Ty1-like Gag proteins and found that some Gag proteins, including those of the related Ty2 elements, contain non-Ty1 residues at multiple CNCR sites. Interestingly, the Ty2-917 element is resistant to p22 and does not undergo a Ty1-like form of CNC. Substitutions conferring CNCR map within predicted helices in Ty1 Gag that overlap with conserved sequence in Ty1/copia, suggesting that p22 disturbs a central function of the capsid during VLP assembly. When hydrophobic residues within predicted helices in Gag are mutated, Gag level remains unaffected in most cases yet VLP assembly and maturation is abnormal. Gag CNCR mutations do not alter binding to p22 as determined by co-immunoprecipitation analyses, but instead, exclude p22 from Ty1 VLPs. These findings suggest that the CNCR alleles enhance retrotransposition in the presence of p22 by allowing productive Gag-Gag interactions during VLP assembly. Our work also expands the strategies used by retroviruses for developing resistance to Gag-like restriction factors to now include retrotransposons.

The NIH Extracellular RNA Communication Consortium.

The Extracellular RNA (exRNA) Communication Consortium, funded as an initiative of the NIH Common Fund, represents a consortium of investigators assembled to address the critical issues in the exRNA research arena. The overarching goal is to generate a multi-component community resource for sharing fundamental scientific discoveries, protocols, and innovative tools and technologies. The key initiatives include (a) generating a reference catalogue of exRNAs present in body fluids of normal healthy individuals that would facilitate disease diagnosis and therapies, (b) defining the fundamental principles of exRNA biogenesis, distribution, uptake, and function, as well as development of molecular tools, technologies, and imaging modalities to enable these studies,

Pain catastrophizing and pain-related fear in osteoarthritis patients: relationships to pain and disability.

This study examined the degree to which pain catastrophizing and pain-related fear explain pain, psychological disability, physical disability, and walking speed in patients with osteoarthritis (OA) of the knee. Participants in this study were 106 individuals diagnosed as having OA of at least one knee, who reported knee pain persisting for six months or longer. Results suggest that pain catastrophizing explained a significant proportion (all Ps < or = 0.05) of variance in measures of pain (partial r(2) [pr(2)] = 0.10), psychological disability (pr(2) = 0.20), physical disability (pr(2) = 0.11), and gait velocity at normal (pr(2) = 0.04), fast (pr(2) = 0.04), and intermediate speeds (pr(2) = 0.04). Pain-related fear explained a significant proportion of the variance in measures of psychological disability (pr(2) = 0.07) and walking at a fast speed (pr(2) = 0.05). Pain cognitions, particularly pain catastrophizing, appear to be important variables in understanding pain, disability, and walking at normal, fast, and intermediate speeds in knee OA patients. Clinicians interested in understanding variations in pain and disability in this population may benefit by expanding the focus of their inquiries beyond traditional medical and demographic variables to include an assessment of pain catastrophizing and pain-related fear.

Arthritis self-efficacy and self-efficacy for resisting eating: relationships to pain, disability, and eating behavior in overweight and obese individuals with osteoarthritic knee pain.

This study examined arthritis self-efficacy and self-efficacy for resisting eating as predictors of pain, disability, and eating behaviors in overweight or obese patients with osteoarthritis (OA) of the knee. Patients (N=174) with a body mass index between 25 and 42 completed measures of arthritis-related self-efficacy, weight-related self-efficacy, pain, physical disability, psychological disability, overeating, and demographic and medical information. Hierarchical linear regression analyses were conducted to examine whether arthritis self-efficacy (efficacy for pain control, physical function, and other symptoms) and self-efficacy for resisting eating accounted for significant variance in pain, disability, and eating behaviors after controlling for demographic and medical characteristics. Analyses also tested whether the contributions of self-efficacy were domain specific. Results showed that self-efficacy for pain accounted for 14% (p=.01) of the variance in pain, compared to only 3% accounted for by self-efficacy for physical function and other symptoms. Self-efficacy for physical function accounted for 10% (p=.001) of the variance in physical disability, while self-efficacy for pain and other symptoms accounted for 3%. Self-efficacy for other (emotional) symptoms and resisting eating accounted for 21% (p<.05) of the variance in psychological disability, while self-efficacy for pain control and physical function were not significant predictors. Self-efficacy for resisting eating accounted for 28% (p=.001) of the variance in eating behaviors. Findings indicate that self-efficacy is important in understanding pain and behavioral adjustment in overweight or obese OA patients. Moreover, the contributions of self-efficacy were domain specific. Interventions targeting both arthritis self-efficacy and self-efficacy for resisting eating may be helpful in this population.