N-terminal acetylation separately promotes nuclear localization and host shutoff activity of the influenza A virus ribonuclease PA-X.

To counteract host antiviral responses, influenza A virus triggers a global reduction of cellular gene expression, a process termed "host shutoff." A key effector of influenza A virus host shutoff is the viral endoribonuclease PA-X, which degrades host mRNAs. While many of the molecular determinants of PA-X activity remain unknown, a previous study found that N-terminal acetylation of PA-X is required for its host shutoff activity. However, it remains unclear how this co-translational modification promotes PA-X activity. Here, we report that PA-X N-terminal acetylation has two functions that can be separated based on the position of the acetylation, i.e. on the first amino acid, the initiator methionine, or the second amino acid following initiator methionine excision. Modification at either site is sufficient to ensure PA-X localization to the nucleus. However, modification of the second amino acid is not sufficient for host shutoff activity of ectopically expressed PA-X, which specifically requires N-terminal acetylation of the initiator methionine. Interestingly, during infection N-terminal acetylation of PA-X at any position results in host shutoff activity, which is in part due to a functional interaction with the influenza protein NS1. This result reveals an unexpected role for another viral protein in PA-X activity. Our studies uncover a multifaceted role for PA-X N-terminal acetylation in regulation of this important immunomodulatory factor.

Caspase-Mediated Regulation and Cellular Heterogeneity of the cGAS/STING Pathway in Kaposi's Sarcoma-Associated Herpesvirus Infection.

As a result of the ongoing virus-host arms race, viruses have evolved numerous immune subversion strategies, many of which are aimed at suppressing the production of type I interferons (IFNs). Apoptotic caspases have recently emerged as important regulators of type I IFN signaling both in noninfectious contexts and during viral infection. Despite being widely considered antiviral factors since they can trigger cell death, several apoptotic caspases promote viral replication by suppressing innate immune response. Indeed, we previously discovered that the AIDS-associated oncogenic gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) exploits caspase activity to suppress the antiviral type I IFN response and promote viral replication. However, the mechanism of this novel viral immune evasion strategy is poorly understood, particularly with regard to how caspases antagonize IFN signaling during KSHV infection. Here, we show that caspase activity inhibits the DNA sensor cGAS during KSHV lytic replication to block type I IFN induction. Furthermore, we used single-cell RNA sequencing to reveal that the potent antiviral state conferred by caspase inhibition is mediated by an exceptionally small percentage of IFN-ß-producing cells, thus uncovering further complexity of IFN regulation during viral infection. Collectively, these results provide insight into multiple levels of cellular type I IFN regulation that viruses co-opt for immune evasion. Unraveling these mechanisms can inform targeted therapeutic strategies for viral infections and reveal cellular mechanisms of regulating interferon signaling in the context of cancer and chronic inflammatory diseases. IMPORTANCE Type I interferons are key factors that dictate the outcome of infectious and inflammatory diseases. Thus, intricate cellular regulatory mechanisms are in place to control IFN responses. While viruses encode their own immune-regulatory proteins, they can also usurp existing cellular interferon regulatory functions. We found that caspase activity during lytic infection with the AIDS-associated oncogenic gammaherpesvirus Kaposi's sarcoma-associated herpesvirus inhibits the DNA sensor cGAS to block the antiviral type I IFN response. Moreover, single-cell RNA sequencing analyses unexpectedly revealed that an exceptionally small subset of infected cells (<5%) produce IFN, yet this is sufficient to confer a potent antiviral state. These findings reveal new aspects of type I IFN regulation and highlight caspases as a druggable target to modulate cGAS activity.

The Role of Viral RNA Degrading Factors in Shutoff of Host Gene Expression.

Many viruses induce shutoff of host gene expression (host shutoff) as a strategy to take over cellular machinery and evade host immunity. Without host shutoff activity, these viruses generally replicate poorly in vivo, attesting to the importance of this antiviral strategy. In this review, we discuss one particularly advantageous way for viruses to induce host shutoff: triggering widespread host messenger RNA (mRNA) decay. Viruses can trigger increased mRNA destruction either directly, by encoding RNA cleaving or decapping enzymes, or indirectly, by activating cellular RNA degradation pathways. We review what is known about the mechanism of action of several viral RNA degradation factors. We then discuss the consequences of widespread RNA degradation on host gene expression and on the mechanisms of immune evasion, highlighting open questions. Answering these questions is critical to understanding how viral RNA degradation factors regulate host gene expression and how this process helps viruses evade host responses and replicate.

Anti-viral and pro-inflammatory functions of Toll-like receptors during gamma-herpesvirus infections.

Toll-like receptors (TLRs) control anti-viral responses both directly in infected cells and in responding cells of the immune systems. Therefore, they are crucial for responses against the oncogenic γ-herpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus and the related murine virus MHV68, which directly infect immune system cells. However, since these viruses also cause lifelong persistent infections, TLRs may also be involved in modulation of inflammation during latent infection and contribute to virus-driven tumorigenesis. This review summarizes work on both of these aspects of TLR/γ-herpesvirus interactions, as well as results showing that TLR activity can drive these viruses' re-entry into the replicative lytic cycle.

Kaposi's sarcoma-associated herpesvirus at 27.

Kaposi's sarcoma-associated herpesvirus (KSHV) was discovered 27 years ago and its link to several pathologies - Kaposi's sarcoma, primary effusion lymphoma, and the B cell variant of Multicentric Castleman disease - is now well established. However, many questions remain about how KSHV causes tumors. Here, I will review studies from the last few years (primarily 2019-2021) that report new information about KSHV biology and tumorigenesis, including new results about KSHV proteins implicated in tumorigenesis, genetic and environmental variability in KSHV-related tumor development, and potential vulnerabilities of KSHV-caused tumors that could be novel therapeutic targets.

The influenza A virus host shutoff factor PA-X is rapidly turned over in a strain-specific manner.

The influenza A endoribonuclease PA-X regulates virulence and transmission of the virus by reducing host gene expression and thus regulating immune responses to influenza A virus. Despite this key function in viral biology, the levels of PA-X protein remain markedly low during infection, and previous results suggest that these low levels are not solely the result of regulation of the level of translation and RNA stability. How PA-X is regulated post-translationally remains unknown. We now report that the PA-X protein is rapidly turned over. PA-X from multiple viral strains are short-lived, although the half-life of PA-X ranges from ∼30 minutes to ∼3.5 hours depending on the strain. Moreover, sequences in the variable PA-X C-terminal domain are primarily responsible for regulating PA-X half-life, although the N-terminal domain also accounts for some differences among strains. Interestingly, we find that the PA-X from the 2009 pandemic H1N1 strain has a longer half-life compared to the other variants we tested. This PA-X isoform has been reported to have a higher host shutoff activity, suggesting a role for protein turnover in regulating PA-X activity. Collectively, this study reveals a novel regulatory mechanism of PA-X protein levels that may impact host shutoff activity during influenza A virus infection.IMPORTANCE The PA-X protein from influenza A virus reduces host immune responses to infection through suppressing host gene expression, including genes encoding the antiviral response. Thus, it plays a central role in influenza A virus biology. Despite its key function, PA-X was only discovered in 2012 and much remains to be learned including how PA-X activity is regulated to promote optimal levels of viral infection. In this study, we reveal that PA-X protein levels are very low likely because of rapid turnover. We show that instability is a conserved property among PA-X variants from different strains of influenza A virus, but that the half-lives of PA-X variants differ. Moreover, the longer half-life of PA-X from the 2009 pandemic H1N1 strain correlates with its reported higher activity. Therefore, PA-X stability may be a way to regulate its activity and may contribute to the differential virulence of influenza A virus strains.

The Kaposi's Sarcoma-Associated Herpesvirus Protein ORF42 Is Required for Efficient Virion Production and Expression of Viral Proteins.

Kaposi's sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi's sarcoma and other aggressive AIDS-associated malignancies, encodes over 90 genes, most of which are expressed only during the lytic replication cycle. The role of many of the KSHV lytic proteins in the KSHV replication cycle remains unknown, and many proteins are annotated based on known functions of homologs in other herpesviruses. Here we investigate the role of the previously uncharacterized KSHV lytic protein ORF42, a presumed tegument protein. We find that ORF42 is dispensable for reactivation from latency but is required for efficient production of viral particles. Like its alpha- and beta-herpesviral homologs, ORF42 is a late protein that accumulates in the viral particles. However, unlike its homologs, ORF42 appears to be required for efficient expression of at least some viral proteins and may potentiate post-transcriptional stages of gene expression. These results demonstrate that ORF42 has an important role in KSHV replication and may contribute to shaping viral gene expression.

Host Shutoff in Influenza A Virus: Many Means to an End.

Influenza A virus carries few of its own proteins, but uses them effectively to take control of the infected cells and avoid immune responses. Over the years, host shutoff, the widespread down-regulation of host gene expression, has emerged as a key process that contributes to cellular takeover in infected cells. Interestingly, multiple mechanisms of host shutoff have been described in influenza A virus, involving changes in translation, RNA synthesis and stability. Several viral proteins, notably the non-structural protein NS1, the RNA-dependent RNA polymerase and the endoribonuclease PA-X have been implicated in host shutoff. This multitude of host shutoff mechanisms indicates that host shutoff is an important component of the influenza A virus replication cycle. Here we review the various mechanisms of host shutoff in influenza A virus and the evidence that they contribute to immune evasion and/or viral replication. We also discuss what the purpose of having multiple mechanisms may be.

Transcriptome-Wide Cleavage Site Mapping on Cellular mRNAs Reveals Features Underlying Sequence-Specific Cleavage by the Viral Ribonuclease SOX.

Many viruses express factors that reduce host gene expression through widespread degradation of cellular mRNA. An example of this class of proteins is the mRNA-targeting endoribonuclease SOX from the gamma-herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV). Previous studies indicated that cleavage of messenger RNAs (mRNA) by SOX occurs at specific locations defined by the sequence of the target RNA, which is at odds with the down-regulation of a large portion of cellular transcripts. In this study, we address this paradox by using high-throughput sequencing of cleavage intermediates combined with a custom bioinformatics-based analysis pipeline to identify SOX cleavage sites across the mRNA transcriptome. These data, coupled with targeted mutagenesis, reveal that while cleavage sites are specific and reproducible, they are defined by a degenerate sequence motif containing a small number of conserved residues rather than a strong consensus sequence. This degenerate element is well represented in both human and KSHV mRNA, and its presence correlates with RNA destabilization by SOX. This represents a new endonuclease targeting strategy, in which use of a degenerate targeting element enables RNA cleavage at specific locations without restricting the range of targets. Furthermore, it shows that strong target selectivity can be achieved without a high degree of sequence specificity.

A common strategy for host RNA degradation by divergent viruses.

Infection with gammaherpesviruses, alphaherpesviruses, and betacoronaviruses can result in widespread mRNA degradation, in each case initiated predominantly by a single viral factor. Although not homologous, these factors exhibit significant mechanistic similarities. In cells, each targets translatable RNAs for cleavage and requires host Xrn1 to complete RNA degradation, although the mechanism of targeting and the position of the primary cleavage differ. Thus, multiple host shutoff factors have converged upon a common mRNA degradation pathway.

The somatic reproductive tissues of C. elegans promote longevity through steroid hormone signaling.

In Caenorhabditis elegans and Drosophila melanogaster, removing the germline precursor cells increases lifespan. In worms, and possibly also in flies, this lifespan extension requires the presence of somatic reproductive tissues. How the somatic gonad signals other tissues to increase lifespan is not known. The lifespan increase triggered by loss of the germ cells is known to require sterol hormone signaling, as reducing the activity of the nuclear hormone receptor DAF-12, or genes required for synthesis of the DAF-12 ligand dafachronic acid, prevents germline loss from extending lifespan. In addition to sterol signaling, the FOXO transcription factor DAF-16 is required to extend lifespan in animals that lack germ cells. DAF-12/NHR is known to assist with the nuclear accumulation of DAF-16/FOXO in these animals, yet we find that loss of DAF-12/NHR has little or no effect on the expression of at least some DAF-16/FOXO target genes. In this study, we show that the DAF-12-sterol signaling pathway has a second function to activate a distinct set of genes and extend lifespan in response to the somatic reproductive tissues. When germline-deficient animals lacking somatic reproductive tissues are given dafachronic acid, their expression of DAF-12/NHR-dependent target genes is restored and their lifespan is increased. Together, our findings indicate that in C. elegans lacking germ cells, the somatic reproductive tissues promote longevity via steroid hormone signaling to DAF-12.

Viruses and the cellular RNA decay machinery.

The ability to control cellular and viral gene expression, either globally or selectively, is central to a successful viral infection, and it is also crucial for the host to respond and eradicate pathogens. In eukaryotes, regulation of message stability contributes significantly to the control of gene expression and plays a prominent role in the normal physiology of a cell as well as in its response to environmental and pathogenic stresses. Not surprisingly, emerging evidence indicates that there are significant interactions between the eukaryotic RNA turnover machinery and a wide variety of viruses. Interestingly, in many cases viruses have evolved mechanisms not only to evade eradication by these pathways, but also to manipulate them for enhanced viral replication and gene expression. Given our incomplete understanding of how many of these pathways are normally regulated, viruses should be powerful tools to help deconstruct the complex networks and events governing eukaryotic RNA stability.

Stimulation of movement in a quiescent, hibernation-like form of Caenorhabditis elegans by dopamine signaling.

One of the characteristics of animals in hibernation is reduced behavioral activity. The Caenorhabditis elegans dauer state is a hibernation-like state of diapause that displays a dramatic reduction in spontaneous locomotion. A similar dauer-like quiescent state is produced in adults by relatively strong mutations in the insulin/IGF-1 receptor homolog daf-2. In this study, we show that mutations affecting the neurotransmitter dopamine, which regulates voluntary movement in many organisms, can stimulate movement in dauers and dauer-like quiescent adults. Surprisingly, the movement of quiescent animals is stimulated by conditions that reduce dopamine signaling and also by conditions predicted to increase dopamine signaling. Reducing dopamine signaling is likely to stimulate movement by activating a foraging response also seen in nondauers after withdrawal of food. In contrast, the stimulation of movement by increased dopamine is much more pronounced in quiescent daf-2(-) dauer and dauer-like adult animals than in nondauaer animals. This altered response to dopamine is primarily attributable to activity of the FOXO (forkhead box O) transcription factor DAF-16 in neurons. We suggest that dauers and dauer-like quiescent adults may have underlying changes in the dopamine system that enable them to respond differently to environmental stimulation.