Biology subjects

Gaglia, M. M.

Publications and source records attributed to Gaglia, M. M..

8 recordsLinked to original sources

bioRxiv2026

Dual roles for influenza A protein PA-X: limiting inflammatory response and disrupting MHC I antigen presentation in human respiratory epithelium

The mechanisms of immune evasion of influenza A virus are key to its success as an infectious agent. To replicate effectively, influenza A virus must suppress both early innate immune responses, like antiviral type I and III interferons, and adaptive immune responses. The latter is particularly important during infections of animals and humans with pre-existing immunity and explains the continued circulation of influenza A viruses. Here we report how influenza A virus employes a single immunomodulatory protein, the endoribonuclease PA-X, to modulate both arms of the immune responses. To define how influenza A virus uses PA-X to evade immune responses, we characterized its impact on the host response to infection in the infected and bystander cells of the airway epithelium using a 3D ex vivo model and primary cells from multiple human donors. PA- X significantly decreases secretion of multiple cytokines from the airway epithelium, including IFN- {lambda}, inflammatory cytokines, and growth factors related to lung damage, which likely plays a role in its ability to modulate inflammation and lung pathology in vivo. In addition, we discovered that PA-X also decreases surface levels of major histocompatibility complex I (MHC I) on infected cells. This reduction may impact antigen-specific T cells and reduce adaptive immune detection. These dual functions for PA-X highlight how influenza A virus employs active mechanisms to block not only innate immunity but also adaptive immune detection, in addition to tolerating high levels of antigen mutations to escape it. Author SummaryInfluenza viruses must evade both innate and adaptive immune responses to infect, replicate, and transmit to new hosts. Through innate immunity, infected cells activate proteins to disrupt viral replication and alert surrounding cells. Through adaptive immunity, antigen-specific T cells recognize specific viral peptides and kill infected cells. We studied how influenza overcomes immune responses. Using a model of human airway tissue, we found a single influenza protein PA-X, can impinge on both innate and adaptive responses. PA-X decreases innate immune signaling between infected and surrounding cells and interferes with the major histocompatibility class I antigen presentation pathway. These dual functions allow PA-X to help the virus continue to circulate and infect new hosts, even in populations with immunity to the flu.

microbiology↗

bioRxiv2024

Interferon-β induction heterogeneity during KSHV infection is determined by interferon-β enhanceosome transcription factors other than IRF3

Careful regulation of type I interferons (IFN) is vital for balancing tissue damage and protection against infections. We previously found that during Kaposis sarcoma-associated herpesvirus infection, type I IFN induction was limited to a small percentage of infected cells. This heterogeneity was not explained by viral gene expression. Here, we used a fluorescent reporter and fluorescence activated cell sorting to investigate the source of this heterogeneity. Surprisingly, the canonical IFN induction pathway culminating in the activation of the IRF3 transcription factor was similarly activated between cells that made high vs. low/no IFN-{beta}. In contrast, the activation or expression of the two other IFN transcription factors, the NF-{kappa}B subunit RelA and the AP-1 subunit ATF2, correlated with IFN-{beta} induction. Our results suggest that during viral infection, activation of IRF3 does not automatically result in IFN responses at the level of individual cells, but that other factors, such as NF-{kappa}B and AP-1, are limiting for type I IFN induction. ImportanceThe ability of mammalian cells to react to viral infections is a crucial step in the induction of immune responses. The first course of action for the cell is to express and release type I interferons like interferon-{beta} (IFN-{beta}), secreted molecules that warn surrounding cells. Single-cell level examination of gene expression has revealed that surprisingly, during many viral infections, only a small fraction of infected cells makes IFN-{beta}. This is likely a mechanism to prevent immune system overreactions. However, it remains unclear why only some cells respond. Here, we find that during infection with Kaposis sarcoma-associated herpesvirus, an oncogenic virus that affects immunocompromised individuals, the transcription factors AP-1 and NF-{kappa}B, rather than the more commonly studied IRF3, may decide which cells go on to make IFN-{beta}. Our findings contribute to a better understanding of complex gene regulation and shed light on a process that fights an oncogenic virus.

microbiology↗

bioRxiv2023

N-terminal acetylation of the influenza ribonuclease PA-X promotes nuclear localization and host shutoff activity in a multifaceted manner

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 whether acetylation occurs 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, suggesting that additional factors during infection can augment the host shutoff activity of PA-X. Our studies thus uncover a multifaceted role for PA-X N-terminal acetylation in regulation of this important immunomodulatory factor. ImportanceInfluenza A viruses pose a significant threat to human health in the form of seasonal epidemics and recurrent pandemics, leading to large burdens on the healthcare system. During infection, host immune and inflammatory responses have a key role in disease outcome. They are needed to clear the virus, but when excessive they can cause lung damage. Influenza A viruses encode several factors that modulate the host immune response, influencing viral replication and pathogenesis. In this study, we focused on one of these influenza factors, PA-X, which destroys cellular mRNAs and reduces cellular gene expression (a phenomenon called "host shutoff") to control immune responses. This protein is modified with an acetylation at its N-terminus, and this modification is needed for its activity, but it has remained unclear why. Our results demonstrate that N-terminal acetylation of PA-X is needed for multiple aspects of its function. It ensures that PA-X goes to the nucleus, where it accesses its RNA targets, but it also separately contributes to host shutoff activity. However, for host shutoff activity, the specific location of the modification matters, whereas for entry in the nucleus it does not. For full activity, the modification must be placed on the very first amino acid of the protein, and this amino acid must be methionine. These findings uncover how influenza A viruses exploit a widespread protein modification to support the host shutoff activity of one of its important immunomodulatory proteins.

microbiology↗

bioRxiv2022

The cut site specificity of the influenza A virus endoribonuclease PA-X allows it to discriminate between host and viral mRNAs

Widespread shutoff of host gene expression through RNA degradation is an advantageous way for many viruses to block antiviral responses. However, viruses still need to maintain expression of their own genes and host genes necessary for replication. The influenza A virus host shutoff endoribonuclease PA-X solves this problem by sparing viral mRNAs and some host RNAs. To understand how PA-X distinguishes between RNAs, we characterized PA-X cut sites transcriptome-wide. This analysis shows that PA-Xs from multiple influenza strains cleave RNAs at GCUG tetramers in hairpin loops. Importantly, GCUG tetramers are enriched in the human but not the influenza transcriptome. Moreover, optimal PA-X cut sites inserted in the influenza A virus genome are quickly selected against during viral replication. This finding suggests that PA-X evolved these cleavage characteristics to target host but not viral mRNAs, in a manner reminiscent of cellular self vs. non-self discrimination.

microbiology↗

bioRxiv2021

Analysis of caspase-mediated regulation of the cGAS/STING pathway in Kaposi's sarcoma associated herpesvirus lytic infection reveals a dramatic cellular heterogeneity in type I interferon responses

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 in both non-infectious contexts and during viral infection. Despite being widely considered anti-viral 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 Kaposis 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 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 use single-cell RNA-sequencing to reveal that the potent antiviral state conferred by caspase inhibition is mediated by an exceptionally small percentage of IFN-{beta}-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. ImportanceType 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 Kaposis 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.

microbiology↗

bioRxiv2020

A novel role for the C-terminal domain of the influenza A virus host shutoff endoribonuclease PA-X in mediating rapid turnover of the PA-X protein

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. IMPORTANCEThe 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.

microbiology↗

bioRxiv2020

Defective influenza A virus RNA products mediate MAVS-dependent upregulation of human leukocyte antigen class I proteins

Influenza A virus (IAV) increases presentation of class I human leukocyte antigen (HLA) proteins that limit antiviral responses mediated by natural killer (NK) cells, but molecular mechanisms have not yet been fully elucidated. We observed that infection with A/Fort Monmouth/1/1947 (H1N1) IAV significantly increased presentation of HLA-B, -C and -E on lung epithelial cells. Virus entry was not sufficient to induce HLA upregulation, because UV-inactivated virus had no effect. We found that HLA upregulation was elicited by aberrant internally-deleted viral RNAs (vRNAs) known as mini viral RNAs (mvRNAs) and defective interfering RNAs (DI RNAs), which bind to retinoic acid-inducible gene-I (RIG-I) and initiate mitochondrial antiviral signaling (MAVS) protein-dependent antiviral interferon (IFN) responses. Indeed, MAVS was required for HLA upregulation in response to IAV infection or ectopic mvRNA/DI RNA expression. The effect was partially due to paracrine signalling, as we observed that IAV infection or mvRNA/DI RNA-expression stimulated production of IFN-{beta} and IFN-{lambda}1, and conditioned media from these cells elicited a modest increase in HLA surface levels in naive epithelial cells. HLA upregulation in response to aberrant viral RNAs could be prevented by chemical blockade of IFN receptor signal transduction. While HLA upregulation would seem to be advantageous to the virus, it is kept in check by the viral non-structural 1 (NS1) protein; we determined that NS1 limits cell-intrinsic and paracrine mechanisms of HLA upregulation. Taken together, our findings indicate that aberrant IAV RNAs stimulate HLA presentation, which may aid viral evasion of innate immunity. IMPORTANCEHuman leukocyte antigens (HLA) are cell surface proteins that regulate innate and adaptive immune responses to viral infection by engaging with receptors on immune cells. Many viruses have evolved ways to evade host immune responses by modulating HLA expression and/or processing. Here, we provide evidence that aberrant RNA products of influenza virus genome replication can trigger RIG-I/MAVS-dependent remodeling of the cell surface, increasing surface presentation of HLA proteins known to inhibit the activation of an immune cell known as a natural killer (NK) cell. While this HLA upregulation would seem to be advantageous to the virus, it is kept in check by the viral non-structural 1 (NS1) protein, which limits RIG-I activation and interferon production by the infected cell.

microbiology↗

bioRxiv2018

A screen for gene regulators encoded by Kaposi’s sarcoma-associated herpesvirus uncovers a novel role for the ORF42 protein in regulating the production of cellular and viral proteins

The tight control of viral and host gene expression is critical to the replication of herpesviruses, including the gamma-herpesvirus Kaposis sarcoma-associated herpesvirus (KSHV). While some of the KSHV proteins that contribute to viral and host gene regulation are known, it is clear that there are additional uncharacterized contributing viral factors. Identifying these proteins and their role in gene regulation is important to determine the mechanistic underpinnings of the complex replication cycle of KSHV. Through a reporter-based screen, we have identified several new potential KSHV-encoded gene regulators, including the previously uncharacterized protein ORF42, which we find stimulates global protein production upon overexpression. We have generated an ORF42-null virus, which revealed that ORF42 is required for wild-type levels of virus production. Moreover, global protein synthesis and the accumulation of viral proteins are reduced in infected cells in the absence of ORF42, suggesting that ORF42 regulates protein synthesis during infection. A comparison of the effects of ORF42 on the levels of RNA and protein suggests that ORF42 acts post-transcriptionally to control protein levels. In addition to gene regulation, ORF42 may have other functions in virion formation, as it is found in viral particles, which is consistent with the described roles of the ORF42 homologs in alpha- and beta-herpesviruses.\n\nImportanceKaposis sarcoma-associated herpesvirus (KSHV) causes Kaposis sarcoma, an AIDS-associated malignancy that remains one of the leading causes of cancer deaths in sub-Saharan Africa. Replication of the virus is important for tumor formation and inhibition of viral replication may be used for treatment. The correct levels and temporal expression of viral and host genes during KSHV replication are key to viral replication, but the mechanisms that control this regulation remain enigmatic. Here we identify several new KSHV proteins involved in viral and cellular gene regulation and characterize the previously unstudied KSHV ORF42 protein in regulation of viral and host protein levels and efficient formation of viral progeny.

microbiology↗