Neurotropic RNA Virus Modulation of Immune Responses within the Central Nervous System.

The central nervous system (CNS) necessitates intricately coordinated immune responses to prevent neurological disease. However, the emergence of viruses capable of entering the CNS and infecting neurons threatens this delicate balance. Our CNS is protected from foreign invaders and excess solutes by a semipermeable barrier of endothelial cells called the blood-brain barrier. Thereby, viruses have implemented several strategies to bypass this protective layer and modulate immune responses within the CNS. In this review, we outline these immune regulatory mechanisms and provide perspectives on future questions in this rapidly expanding field.

SARS-CoV-2 viral proteins NSP1 and NSP13 inhibit interferon activation through distinct mechanisms.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a devastating global pandemic, infecting over 43 million people and claiming over 1 million lives, with these numbers increasing daily. Therefore, there is urgent need to understand the molecular mechanisms governing SARS-CoV-2 pathogenesis, immune evasion, and disease progression. Here, we show that SARS-CoV-2 can block IRF3 and NF-κB activation early during virus infection. We also identify that the SARS-CoV-2 viral proteins NSP1 and NSP13 can block interferon activation via distinct mechanisms. NSP1 antagonizes interferon signaling by suppressing host mRNA translation, while NSP13 downregulates interferon and NF-κB promoter signaling by limiting TBK1 and IRF3 activation, as phospho-TBK1 and phospho-IRF3 protein levels are reduced with increasing levels of NSP13 protein expression. NSP13 can also reduce NF-κB activation by both limiting NF-κB phosphorylation and nuclear translocation. Last, we also show that NSP13 binds to TBK1 and downregulates IFIT1 protein expression. Collectively, these data illustrate that SARS-CoV-2 bypasses multiple innate immune activation pathways through distinct mechanisms.

Playing Favorites: Integrin αvß5 Mediates Preferential Zika Infection of Neural Stem Cells.

The molecular basis dictating specificity of Zika virus infection in neural stem cells (NSCs) remains elusive. Two recent papers in Cell Stem Cell (Zhu et al., 2020) and Cell Reports (Wang et al., 2020) identify integrin αvß5 as an internalization factor that increases susceptibility in NSCs and glioblastoma stem cells.

Hepatitis C Virus Infection Is Inhibited by a Noncanonical Antiviral Signaling Pathway Targeted by NS3-NS4A.

The hepatitis C virus (HCV) NS3-NS4A protease complex is required for viral replication and is the major viral innate immune evasion factor. NS3-NS4A evades antiviral innate immunity by inactivating several proteins, including MAVS, the signaling adaptor for RIG-I and MDA5, and Riplet, an E3 ubiquitin ligase that activates RIG-I. Here, we identified a Tyr-16-Phe (Y16F) change in the NS4A transmembrane domain that prevents NS3-NS4A targeting of Riplet but not MAVS. This Y16F substitution reduces HCV replication in Huh7 cells, but not in Huh-7.5 cells, known to lack RIG-I signaling. Surprisingly, deletion of RIG-I in Huh7 cells did not restore Y16F viral replication. Rather, we found that Huh-7.5 cells lack Riplet expression and that the addition of Riplet to these cells reduced HCV Y16F replication, whereas the addition of Riplet lacking the RING domain restored HCV Y16F replication. In addition, TBK1 inhibition or IRF3 deletion in Huh7 cells was sufficient to restore HCV Y16F replication, and the Y16F protease lacked the ability to prevent IRF3 activation or interferon induction. Taken together, these data reveal that the NS4A Y16 residue regulates a noncanonical Riplet-TBK1-IRF3-dependent, but RIG-I-MAVS-independent, signaling pathway that limits HCV infection.IMPORTANCE The HCV NS3-NS4A protease complex facilitates viral replication by cleaving and inactivating the antiviral innate immune signaling proteins MAVS and Riplet, which are essential for RIG-I activation. NS3-NS4A therefore prevents IRF3 activation and interferon induction during HCV infection. Here, we uncover an amino acid residue within the NS4A transmembrane domain that is essential for inactivation of Riplet but does not affect MAVS cleavage by NS3-NS4A. Our study reveals that Riplet is involved in a RIG-I- and MAVS-independent signaling pathway that activates IRF3 and that this pathway is normally inactivated by NS3-NS4A during HCV infection. Our study selectively uncouples these distinct regulatory mechanisms within NS3-NS4A and defines a new role for Riplet in the antiviral response to HCV. Since Riplet is known to be inhibited by other RNA viruses, such as such influenza A virus, this innate immune signaling pathway may also be important in controlling other RNA virus infections.

The acidic domain of the hepatitis C virus NS4A protein is required for viral assembly and envelopment through interactions with the viral E1 glycoprotein.

Hepatitis C virus (HCV) assembly and envelopment are coordinated by a complex protein interaction network that includes most of the viral structural and nonstructural proteins. While the nonstructural protein 4A (NS4A) is known to be important for viral particle production, the specific function of NS4A in this process is not well understood. We performed mutagenesis of the C-terminal acidic domain of NS4A and found that mutation of several of these amino acids prevented the formation of the viral envelope, and therefore the production of infectious virions, without affecting viral RNA replication. In an overexpression system, we found that NS4A interacted with several viral proteins known to coordinate envelopment, including the viral E1 glycoprotein. One of the NS4A C-terminal mutations, Y45F, disrupted the interaction of NS4A with E1. Specifically, NS4A interacted with the first hydrophobic region of E1, a region previously described as regulating viral particle production. Indeed, we found that an E1 mutation in this region, D72A, also disrupted the interaction of NS4A with E1. Supernatants from HCV NS4A Y45F transfected cells had significantly reduced levels of HCV RNA, however they contained equivalent levels of Core protein. Interestingly, the Core protein secreted from these cells formed high order oligomers with a density matching the infectious virus secreted from wild-type cells. These results suggest that this Y45F mutation in NS4A causes secretion of low-density Core particles lacking genomic HCV RNA. These results corroborate previous findings showing that the E1 D72A mutation also causes secretion of Core complexes lacking genomic HCV RNA, and therefore suggest that the interaction between NS4A and E1 is involved in the incorporation of viral RNA into infectious HCV particles. Our findings define a new role for NS4A in the HCV lifecycle and help elucidate the protein interactions necessary for production of infectious virus.

A Fluorescent Cell-Based System for Imaging Zika Virus Infection in Real-Time.

Zika virus (ZIKV) is a re-emerging flavivirus that is transmitted to humans through the bite of an infected mosquito or through sexual contact with an infected partner. ZIKV infection during pregnancy has been associated with numerous fetal abnormalities, including prenatal lethality and microcephaly. However, until recent outbreaks in the Americas, ZIKV has been relatively understudied, and therefore the biology and pathogenesis of ZIKV infection remain incompletely understood. Better methods to study ZIKV infection in live cells could enhance our understanding of the biology of ZIKV and the mechanisms by which ZIKV contributes to fetal abnormalities. To this end, we developed a fluorescent cell-based reporter system allowing for live imaging of ZIKV-infected cells. This system utilizes the protease activity of the ZIKV non-structural proteins 2B and 3 (NS2B-NS3) to specifically mark virus-infected cells. Here, we demonstrate the utility of this fluorescent reporter for identifying cells infected by ZIKV strains of two lineages. Further, we use this system to determine that apoptosis is induced in cells directly infected with ZIKV in a cell-autonomous manner. Ultimately, approaches that can directly track ZIKV-infected cells at the single cell-level have the potential to yield new insights into the host-pathogen interactions that regulate ZIKV infection and pathogenesis.

Methods to Visualize MAVS Subcellular Localization.

The mitochondrial antiviral signaling (MAVS) protein is a central adaptor protein required for antiviral innate immune signaling. To facilitate its roles in innate immunity, MAVS localizes to multiple intracellular membranous compartments, including the mitochondria, the mitochondrial-associated ER membrane (MAM), and peroxisomes. Studies of MAVS function therefore often require an analysis of MAVS localization. To detect MAVS protein on intracellular membranes, biochemical fractionation to isolate MAMs, mitochondria, or peroxisomes can be used. Further, immunofluorescence with antibodies against specific membrane markers can be used to visualize MAVS distribution throughout the cell. Here, we describe the biochemical fractionation and immunofluorescence protocols used to detect MAVS subcellular localization.

N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection.

The RNA modification N6-methyladenosine (m6A) post-transcriptionally regulates RNA function. The cellular machinery that controls m6A includes methyltransferases and demethylases that add or remove this modification, as well as m6A-binding YTHDF proteins that promote the translation or degradation of m6A-modified mRNA. We demonstrate that m6A modulates infection by hepatitis C virus (HCV). Depletion of m6A methyltransferases or an m6A demethylase, respectively, increases or decreases infectious HCV particle production. During HCV infection, YTHDF proteins relocalize to lipid droplets, sites of viral assembly, and their depletion increases infectious viral particles. We further mapped m6A sites across the HCV genome and determined that inactivating m6A in one viral genomic region increases viral titer without affecting RNA replication. Additional mapping of m6A on the RNA genomes of other Flaviviridae, including dengue, Zika, yellow fever, and West Nile virus, identifies conserved regions modified by m6A. Altogether, this work identifies m6A as a conserved regulatory mark across Flaviviridae genomes.

MAVS Coordination of Antiviral Innate Immunity.

RNA virus infection is sensed in the cytoplasm by the retinoic acid-inducible gene I (RIG-I)-like receptors. These proteins signal through the host adaptor protein MAVS to trigger the antiviral innate immune response. Here, we describe how MAVS subcellular localization impacts its function and the regulation underlying MAVS signaling. We propose a model to describe how the coordination of MAVS functions at the interface between the mitochondria and the mitochondrion-associated endoplasmic reticulum (ER) membrane programs antiviral signaling.

Family history of inflammatory bowel disease among patients with ulcerative colitis: a systematic review and meta-analysis.

BACKGROUND AND AIMS: Despite numerous shared susceptibility loci between Crohn's disease and ulcerative colitis, the prevalence of family history among ulcerative colitis patients is not well-established and considered to be less prevalent. A systemic review and meta-analysis were conducted to estimate the prevalence of family history of inflammatory bowel disease in ulcerative colitis patients, and its effect on disease outcomes. METHODS: PubMED was searched to identify studies reporting the prevalence of family history of inflammatory bowel disease among ulcerative colitis patients. Definitions of family history, study type, and subtypes of family history prevalence were abstracted, as were disease outcomes including age at ulcerative colitis diagnosis, disease location, surgery and extraintestinal manifestations. Pooled prevalence estimates were calculated using random effects models. RESULTS: Seventy-one studies (86,824 patients) were included. The prevalence of a family history of inflammatory bowel disease in ulcerative colitis patients was 12% (95% confidence interval [CI] 11 to 13%; range 0-39%). Family history of ulcerative colitis (9%; 22 studies) was more prevalent than Crohn's disease (2%; 18 studies). Patients younger than 18years of age at time of diagnosis had a greater family history of inflammatory bowel disease (prevalence 15%, 95% CI: 11-20%; 13 studies). There were no differences in disease location, need for surgery, or extraintestinal manifestations among those with a family history, although very few studies reported on these outcomes. CONCLUSIONS: Overall, 12% of ulcerative colitis patients have a family history of inflammatory bowel disease, and were more likely to have a family history of ulcerative colitis than Crohn's disease. Pediatric-onset ulcerative colitis patients were more likely to have a family history of inflammatory bowel disease.

Hepatitis C Virus. Strategies to Evade Antiviral Responses.

Hepatitis C virus (HCV) causes chronic liver disease and poses a major clinical and economic burden worldwide. HCV is an RNA virus that is sensed as non-self in the infected liver by host pattern recognition receptors, triggering downstream signaling to interferons (IFNs). The type III IFNs play an important role in immunity to HCV, and human genetic variation in their gene loci is associated with differential HCV infection outcomes. HCV evades host antiviral innate immune responses to mediate a persistent infection in the liver. This review focuses on anti-HCV innate immune sensing, innate signaling and effectors, and the processes and proteins used by HCV to evade and regulate host innate immunity.

MicroRNA-224 negatively regulates p21 expression during late neoplastic progression in inflammatory bowel disease.

BACKGROUND: The development of colon cancer represents a major complication in patients with inflammatory bowel disease (IBD). The importance of microRNAs (miRs) in carcinogenesis is becoming clearer because miRs have been implicated in the regulation of cancer-related cellular processes to include apoptosis, differentiation, cell cycle progression, and immune function. In the current study, we sought to identify miR dysregulation specific to progression along the normal-inflammation-cancer axis in colonic specimens from patients with IBD. METHODS: MiR microarrays and quantitative reverse transcription PCR were used to detect and confirm dysregulated miRs. Receiver operating characteristic curve analysis was applied to evaluate the potential use of miR-224 as a neoplastic disease marker in IBD. For miR-224 target messenger RNA (mRNA) identification, mRNA microarrays were employed in combination with bioinformatic analyses, Western blotting, and luciferase activity measurements. RESULTS: We identified 30 miRs that were differentially expressed between chronically inflamed mucosae and cancers arising from IBD tissues. MiR-224 levels increased successively at each stage of IBD progression and accurately discriminated cancers from normal or chronically inflamed IBD tissues. Moreover, mRNA arrays combined with bioinformatic analyses suggested the participation of miR-224 in cell cycle regulation. Subsequently, cell cycle experiments indicated that miR-224 regulates the G1-S checkpoint. Finally, in silico prediction analyses, confirmed by Western blotting and luciferase assays, identified p21 as a specific direct mRNA target of miR-224. CONCLUSIONS: These findings reveal miR dysregulation specific to IBD-associated colorectal carcinoma. MiR-224 is overexpressed in IBD cancers and targets p21, a key cell cycle regulator. Moreover, these results establish the participation of miR-224 in IBD carcinogenesis.

A manganese-rich environment supports superoxide dismutase activity in a Lyme disease pathogen, Borrelia burgdorferi.

The Lyme disease pathogen Borrelia burgdorferi represents a novel organism in which to study metalloprotein biology in that this spirochete has uniquely evolved with no requirement for iron. Not only is iron low, but we show here that B. burgdorferi has the capacity to accumulate remarkably high levels of manganese. This high manganese is necessary to activate the SodA superoxide dismutase (SOD) essential for virulence. Using a metalloproteomic approach, we demonstrate that a bulk of B. burgdorferi SodA directly associates with manganese, and a smaller pool of inactive enzyme accumulates as apoprotein. Other metalloproteins may have similarly adapted to using manganese as co-factor, including the BB0366 aminopeptidase. Whereas B. burgdorferi SodA has evolved in a manganese-rich, iron-poor environment, the opposite is true for Mn-SODs of organisms such as Escherichia coli and bakers' yeast. These Mn-SODs still capture manganese in an iron-rich cell, and we tested whether the same is true for Borrelia SodA. When expressed in the iron-rich mitochondria of Saccharomyces cerevisiae, B. burgdorferi SodA was inactive. Activity was only possible when cells accumulated extremely high levels of manganese that exceeded cellular iron. Moreover, there was no evidence for iron inactivation of the SOD. B. burgdorferi SodA shows strong overall homology with other members of the Mn-SOD family, but computer-assisted modeling revealed some unusual features of the hydrogen bonding network near the enzyme's active site. The unique properties of B. burgdorferi SodA may represent adaptation to expression in the manganese-rich and iron-poor environment of the spirochete.

Dynamic changes in the expression of MicroRNA-31 during inflammatory bowel disease-associated neoplastic transformation.

BACKGROUND: Patients with inflammatory bowel disease (IBD) are at increased risk of developing colorectal cancer. Aberrant microRNA (miR) expression has been linked to carcinogenesis; however, no reports document a relationship between IBD-related neoplasia (IBDN) and altered miR expression. In the current study we sought to identify specific miR dysregulation along the normal-inflammation-cancer axis. METHODS: miR microarrays and quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) were used to detect dysregulated miRs. Receiver operating characteristic curve analysis was employed to test for potential usefulness of miR-31 as a disease marker of IBDNs. In silico prediction analysis, Western blot, and luciferase activity measurement were employed for target identification. RESULTS: Several dysregulated miRs were identified between chronically inflamed mucosae and dysplasia arising in IBD. MiR-31 expression increases in a stepwise fashion during progression from normal to IBD to IBDN and accurately discriminated IBDNs from normal or chronically inflamed tissues in IBD patients. Finally, we identified factor inhibiting hypoxia inducible factor 1 as a direct target of miR-31. CONCLUSIONS: Our study reveals specific miR dysregulation as chronic inflammation progresses to dysplasia. MiR-31 expression levels increase with disease progression and accurately discriminates between distinct pathological entities that coexist in IBD patients. The novel effect of miR-31 on regulating factor inhibiting hypoxia inducible factor 1 expression provides a new insight on the pathogenesis of IBDN.