Transcriptomic analysis of sorted lung cells revealed a proviral activity of the NF-κB pathway toward SARS-CoV-2.

Investigations of cellular responses to viral infection are commonly performed on mixed populations of infected and uninfected cells or using single-cell RNA sequencing, leading to inaccurate and low-resolution gene expression interpretations. Here, we performed deep polyA+ transcriptome analyses and novel RNA profiling of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected lung epithelial cells, sorted based on the expression of the viral spike (S) protein. Infection caused a massive reduction in mRNAs and long non-coding RNAs (lncRNAs), including transcripts coding for antiviral factors, such as interferons (IFNs). This absence of IFN signaling probably explained the poor transcriptomic response of bystander cells co-cultured with S+ ones. NF-κB pathway and the inflammatory response escaped the global shutoff in S+ cells. Functional investigations revealed the proviral function of the NF-κB pathway and the antiviral activity of CYLD, a negative regulator of the pathway. Thus, our transcriptomic analysis on sorted cells revealed additional genes that modulate SARS-CoV-2 replication in lung cells.

Y RNAs are conserved endogenous RIG-I ligands across RNA virus infection and are targeted by HIV-1.

Pattern recognition receptors (PRRs) protect against microbial invasion by detecting specific molecular patterns found in pathogens and initiating an immune response. Although microbial-derived PRR ligands have been extensively characterized, the contribution and relevance of endogenous ligands to PRR activation remains overlooked. Here, we characterize the landscape of endogenous ligands that engage RIG-I-like receptors (RLRs) upon infection by different RNA viruses. In each infection, several RNAs transcribed by RNA polymerase III (Pol3) specifically engaged RLRs, particularly the family of Y RNAs. Sensing of Y RNAs was dependent on their mimicking of viral secondary structure and their 5'-triphosphate extremity. Further, we found that HIV-1 triggered a VPR-dependent downregulation of RNA triphosphatase DUSP11 in vitro and in vivo, inducing a transcriptome-wide change of cellular RNA 5'-triphosphorylation that licenses Y RNA immunogenicity. Overall, our work uncovers the contribution of endogenous RNAs to antiviral immunity and demonstrates the importance of this pathway in HIV-1 infection.

Essential role of hyperacetylated microtubules in innate immunity escape orchestrated by the EBV-encoded BHRF1 protein.

Innate immunity constitutes the first line of defense against viruses, in which mitochondria play an important role in the induction of the interferon (IFN) response. BHRF1, a multifunctional viral protein expressed during Epstein-Barr virus reactivation, modulates mitochondrial dynamics and disrupts the IFN signaling pathway. Mitochondria are mobile organelles that move through the cytoplasm thanks to the cytoskeleton and in particular the microtubule (MT) network. MTs undergo various post-translational modifications, among them tubulin acetylation. In this study, we demonstrated that BHRF1 induces MT hyperacetylation to escape innate immunity. Indeed, the expression of BHRF1 induces the clustering of shortened mitochondria next to the nucleus. This "mito-aggresome" is organized around the centrosome and its formation is MT-dependent. We also observed that the α-tubulin acetyltransferase ATAT1 interacts with BHRF1. Using ATAT1 knockdown or a non-acetylatable α-tubulin mutant, we demonstrated that this hyperacetylation is necessary for the mito-aggresome formation. Similar results were observed during EBV reactivation. We investigated the mechanism leading to the clustering of mitochondria, and we identified dyneins as motors that are required for mitochondrial clustering. Finally, we demonstrated that BHRF1 needs MT hyperacetylation to block the induction of the IFN response. Moreover, the loss of MT hyperacetylation blocks the localization of autophagosomes close to the mito-aggresome, impeding BHRF1 to initiate mitophagy, which is essential to inhibiting the signaling pathway. Therefore, our results reveal the role of the MT network, and its acetylation level, in the induction of a pro-viral mitophagy.

APOBEC3C S188I Polymorphism Enhances Context-Specific Editing of Hepatitis B Virus Genome.

Single-nucleotide polymorphism in APOBEC3C (resulting in a serine to isoleucine in position 188) is present in approximately 10% of African populations and greatly enhances restriction against human immunodeficiency virus-1 and simian immunodeficiency virus by improving dimerization and DNA processivity of the enzyme. In this study, we demonstrated in culture and in infected patients that hepatitis B virus (HBV) could be edited by APOBEC3CS188I. Using next-generation sequencing, we demonstrated that APOBEC3CS188I led to enhanced editing activity in 5'TpCpA→5'TpTpA context. This constitutes a new hallmark of this enzyme, which could be used to determine its impact on HBV or nuclear DNA.

Discovery of Genes that Modulate Flavivirus Replication in an Interferon-Dependent Manner.

Establishment of the interferon (IFN)-mediated antiviral state provides a crucial initial line of defense against viral infection. Numerous genes that contribute to this antiviral state remain to be identified. Using a loss-of-function strategy, we screened an original library of 1156 siRNAs targeting 386 individual curated human genes in stimulated microglial cells infected with Zika virus (ZIKV), an emerging RNA virus that belongs to the flavivirus genus. The screen recovered twenty-one potential host proteins that modulate ZIKV replication in an IFN-dependent manner, including the previously known IFITM3 and LY6E. Further characterization contributed to delineate the spectrum of action of these genes towards other pathogenic RNA viruses, including Hepatitis C virus and SARS-CoV-2. Our data revealed that APOL3 acts as a proviral factor for ZIKV and several other related and unrelated RNA viruses. In addition, we showed that MTA2, a chromatin remodeling factor, possesses potent flavivirus-specific antiviral functions induced by IFN. Our work identified previously unrecognized genes that modulate the replication of RNA viruses in an IFN-dependent manner, opening new perspectives to target weakness points in the life cycle of these viruses.

Herpes Simplex Virus Type 1 Infection Disturbs the Mitochondrial Network, Leading to Type I Interferon Production through the RNA Polymerase III/RIG-I Pathway.

Viruses have evolved a plethora of mechanisms to impair host innate immune responses. Herpes simplex virus type 1 (HSV-1), a double-stranded linear DNA virus, impairs the mitochondrial network and dynamics predominantly through the UL12.5 gene. We demonstrated that HSV-1 infection induced a remodeling of mitochondrial shape, resulting in a fragmentation of the mitochondria associated with a decrease in their volume and an increase in their sphericity. This damage leads to the release of mitochondrial DNA (mtDNA) to the cytosol. By generating a stable THP-1 cell line expressing the DNase I-mCherry fusion protein and a THP-1 cell line specifically depleted of mtDNA upon ethidium bromide treatment, we showed that cytosolic mtDNA contributes to type I interferon and APOBEC3A upregulation. This was confirmed by using an HSV-1 strain (KOS37 UL98-SPA) with a deletion of the UL12.5 gene that impaired its ability to induce mtDNA stress. Furthermore, by using an inhibitor of RNA polymerase III, we demonstrated that upon HSV-1 infection, cytosolic mtDNA enhanced type I interferon induction through the RNA polymerase III/RIG-I pathway. APOBEC3A was in turn induced by interferon. Deep sequencing analyses of cytosolic mtDNA mutations revealed an APOBEC3A signature predominantly in the 5'TpCpG context. These data demonstrate that upon HSV-1 infection, the mitochondrial network is disrupted, leading to the release of mtDNA and ultimately to its catabolism through APOBEC3-induced mutations. IMPORTANCE Herpes simplex virus 1 (HSV-1) impairs the mitochondrial network through the viral protein UL12.5. This leads to the fusion of mitochondria and simultaneous release of mitochondrial DNA (mtDNA) in a mouse model. We have shown that released mtDNA is recognized as a danger signal, capable of stimulating signaling pathways and inducing the production of proinflammatory cytokines. The expression of the human cytidine deaminase APOBEC3A is highly upregulated by interferon responses. This enzyme catalyzes the deamination of cytidine to uridine in single-stranded DNA substrates, resulting in the catabolism of edited DNA. Using human cell lines deprived of mtDNA and viral strains deficient in UL12, we demonstrated the implication of mtDNA in the production of interferon and APOBEC3A expression during viral infection. We have shown that HSV-1 induces mitochondrial network fragmentation in a human model and confirmed the implication of RNA polymerase III/RIG-I signaling in the capture of cytosolic mtDNA.

Genomic diversity contributes to the neuroinvasiveness of the Yellow fever French neurotropic vaccine.

Mass vaccination with the live attenuated vaccine YF-17D is the current way to prevent infection with Yellow fever virus (YFV). However, 0.000012-0.00002% of vaccinated patients develop post-vaccination neurological syndrome (YEL-AND). Understanding the factors responsible for neuroinvasion, neurotropism, and neurovirulence of the vaccine is critical for improving its biosafety. The YF-FNV vaccine strain, known to be associated with a higher frequency of YEL-AND (0.3-0.4%) than YF-17D, is an excellent model to study vaccine neuroinvasiveness. We determined that neuroinvasiveness of YF-FNV occured both via infection and passage through human brain endothelial cells. Plaque purification and next generation sequencing (NGS) identified several neuroinvasive variants. Their neuroinvasiveness was not higher than that of YF-FNV. However, rebuilding the YF-FNV population diversity from a set of isolated YF-FNV-N variants restored the original neuroinvasive phenotype of YF-FNV. Therefore, we conclude that viral population diversity is a critical factor for YFV vaccine neuroinvasiveness.

Retinoic Acid Inducible Gene I and Protein Kinase R, but Not Stress Granules, Mediate the Proinflammatory Response to Yellow Fever Virus.

Yellow fever virus (YFV) is an RNA virus primarily targeting the liver. Severe YF cases are responsible for hemorrhagic fever, plausibly precipitated by excessive proinflammatory cytokine response. Pathogen recognition receptors (PRRs), such as the cytoplasmic retinoic acid inducible gene I (RIG-I)-like receptors (RLRs), and the viral RNA sensor protein kinase R (PKR), are known to initiate a proinflammatory response upon recognition of viral genomes. Here, we sought to reveal the main determinants responsible for the acute cytokine expression occurring in human hepatocytes following YFV infection. Using a RIG-I-defective human hepatoma cell line, we found that RIG-I largely contributes to cytokine secretion upon YFV infection. In infected RIG-I-proficient hepatoma cells, RIG-I was localized in stress granules. These granules are large aggregates of stalled translation preinitiation complexes known to concentrate RLRs and PKR and are so far recognized as hubs orchestrating RNA virus sensing. Stable knockdown of PKR in hepatoma cells revealed that PKR contributes to both stress granule formation and cytokine induction upon YFV infection. However, stress granule disruption did not affect the cytokine response to YFV infection, as assessed by small interfering RNA (siRNA)-knockdown-mediated inhibition of stress granule assembly. Finally, no viral RNA was detected in stress granules using a fluorescence in situ hybridization approach coupled with immunofluorescence. Our findings suggest that both RIG-I and PKR mediate proinflammatory cytokine induction in YFV-infected hepatocytes, in a stress granule-independent manner. Therefore, by showing the uncoupling of the cytokine response from the stress granule formation, our model challenges the current view in which stress granules are required for the mounting of the acute antiviral response.IMPORTANCE Yellow fever is a mosquito-borne acute hemorrhagic disease caused by yellow fever virus (YFV). The mechanisms responsible for its pathogenesis remain largely unknown, although increased inflammation has been linked to worsened outcome. YFV targets the liver, where it primarily infects hepatocytes. We found that two RNA-sensing proteins, RIG-I and PKR, participate in the induction of proinflammatory mediators in human hepatocytes infected with YFV. We show that YFV infection promotes the formation of cytoplasmic structures, termed stress granules, in a PKR- but not RIG-I-dependent manner. While stress granules were previously postulated to be essential platforms for immune activation, we found that they are not required for the production of proinflammatory mediators upon YFV infection. Collectively, our work uncovered molecular events triggered by the replication of YFV, which could prove instrumental in clarifying the pathogenesis of the disease, with possible repercussions for disease management.

Targeting Kaposi's Sarcoma-Associated Herpesvirus ORF21 Tyrosine Kinase and Viral Lytic Reactivation by Tyrosine Kinase Inhibitors Approved for Clinical Use.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the cause of three human malignancies: Kaposi's sarcoma, primary effusion lymphoma, and the plasma cell variant of multicentric Castleman disease. Previous research has shown that several cellular tyrosine kinases play crucial roles during several steps in the virus replication cycle. Two KSHV proteins also have protein kinase function: open reading frame (ORF) 36 encodes a serine-threonine kinase, while ORF21 encodes a thymidine kinase (TK), which has recently been found to be an efficient tyrosine kinase. In this study, we explore the role of the ORF21 tyrosine kinase function in KSHV lytic replication. By generating a recombinant KSHV mutant with an enzymatically inactive ORF21 protein, we show that the tyrosine kinase function of ORF21/TK is not required for the progression of the lytic replication in tissue culture but that it is essential for the phosphorylation and activation to toxic moieties of the antiviral drugs zidovudine and brivudine. In addition, we identify several tyrosine kinase inhibitors, already in clinical use against human malignancies, which potently inhibit not only ORF21 TK kinase function but also viral lytic reactivation and the development of KSHV-infected endothelial tumors in mice. Since they target both cellular tyrosine kinases and a viral kinase, some of these compounds might find a use in the treatment of KSHV-associated malignancies.IMPORTANCE Our findings address the role of KSHV ORF21 as a tyrosine kinase during lytic replication and the activation of prodrugs in KSHV-infected cells. We also show the potential of selected clinically approved tyrosine kinase inhibitors to inhibit KSHV TK, KSHV lytic replication, infectious virion release, and the development of an endothelial tumor. Since they target both cellular tyrosine kinases supporting productive viral replication and a viral kinase, these drugs, which are already approved for clinical use, may be suitable for repurposing for the treatment of KSHV-related tumors in AIDS patients or transplant recipients.

LGP2 binds to PACT to regulate RIG-I- and MDA5-mediated antiviral responses.

The retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) RIG-I, MDA5, and LGP2 stimulate inflammatory and antiviral responses by sensing nonself RNA molecules produced during viral replication. Here, we investigated how LGP2 regulates the RIG-I- and MDA5-dependent induction of type I interferon (IFN) signaling and showed that LGP2 interacted with different components of the RNA-silencing machinery. We identified a direct protein-protein interaction between LGP2 and the IFN-inducible, double-stranded RNA binding protein PACT. The LGP2-PACT interaction was mediated by the regulatory C-terminal domain of LGP2 and was necessary for inhibiting RIG-I-dependent responses and for amplifying MDA5-dependent responses. We described a point mutation within LGP2 that disrupted the LGP2-PACT interaction and led to the loss of LGP2-mediated regulation of RIG-I and MDA5 signaling. These results suggest a model in which the LGP2-PACT interaction regulates the inflammatory responses mediated by RIG-I and MDA5 and enables the cellular RNA-silencing machinery to coordinate with the innate immune response.