Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 System from Neisseria meningitidis.

The microbial CRISPR systems enable adaptive defense against mobile elements and also provide formidable tools for genome engineering. The Cas9 proteins are type II CRISPR-associated, RNA-guided DNA endonucleases that identify double-stranded DNA targets by sequence complementarity and protospacer adjacent motif (PAM) recognition. Here we report that the type II-C CRISPR-Cas9 from Neisseria meningitidis (Nme) is capable of programmable, RNA-guided, site-specific cleavage and recognition of single-stranded RNA targets and that this ribonuclease activity is independent of the PAM sequence. We define the mechanistic feature and specificity constraint for RNA cleavage by NmeCas9 and also show that nuclease null dNmeCas9 binds to RNA target complementary to CRISPR RNA. Finally, we demonstrate that NmeCas9-catalyzed RNA cleavage can be blocked by three families of type II-C anti-CRISPR proteins. These results fundamentally expand the targeting capacities of CRISPR-Cas9 and highlight the potential utility of NmeCas9 as a single platform to target both RNA and DNA.

IFI16 Partners with KAP1 to Maintain Epstein-Barr Virus Latency.

Herpesviruses establish latency to ensure permanent residence in their hosts. Upon entry into a cell, these viruses are rapidly silenced by the host, thereby limiting the destructive viral lytic phase while allowing the virus to hide from the immune system. Notably, although the establishment of latency by the oncogenic herpesvirus Epstein-Barr virus (EBV) requires the expression of viral latency genes, latency can be maintained with a negligible expression of viral genes. Indeed, in several herpesviruses, the host DNA sensor IFI16 facilitated latency via H3K9me3 heterochromatinization. This silencing mark is typically imposed by the constitutive heterochromatin machinery (HCM). The HCM, in an antiviral role, also silences the lytic phase of EBV and other herpes viruses. We investigated if IFI16 restricted EBV lytic activation by partnering with the HCM and found that IFI16 interacted with core components of the HCM, including the KRAB-associated protein 1 (KAP1) and the site-specific DNA binding KRAB-ZFP SZF1. This partnership silenced the EBV lytic switch protein ZEBRA, encoded by the BZLF1 gene, thereby favoring viral latency. Indeed, IFI16 contributed to H3K9 trimethylation at lytic genes of all kinetic classes. In defining topology, we found that IFI16 coenriched with KAP1 at the BZLF1 promoter, and while IFI16 and SZF1 were each adjacent to KAP1 in latent cells, IFI16 and SZF1 were not. Importantly, we also found that disruption of latency involved rapid downregulation of IFI16 transcription. These findings revealed a previously unknown partnership between IFI16 and the core HCM that supports EBV latency via antiviral heterochromatic silencing. IMPORTANCE The interferon-gamma inducible protein 16 (IFI16) is a nuclear DNA sensor that mediates antiviral responses by activating the inflammasome, triggering an interferon response, and silencing lytic genes of herpesviruses. The last, which helps maintain latency of the oncoherpesvirus Epstein-Barr virus (EBV), is accomplished via H3K9me3 heterochromatinization through unknown mechanisms. Here, we report that IFI16 physically partners with the core constitutive heterochromatin machinery to silence the key EBV lytic switch protein, thereby ensuring continued viral latency in B lymphocytes. We also find that disruption of latency involves rapid transcriptional downregulation of IFI16. These findings point to hitherto unknown physical and functional partnerships between a well-known antiviral mechanism and the core components of the constitutive heterochromatin machinery.

KAP1/TRIM28 - antiviral and proviral protagonist of herpesvirus biology.

Dysregulation of the constitutive heterochromatin machinery (HCM) that silences pericentromeric regions and endogenous retroviral elements in the human genome has consequences for aging and cancer. By recruiting epigenetic regulators, Krüppel-associated box (KRAB)-associated protein 1 (KAP1/TRIM28/TIF1ß) is integral to the function of the HCM. Epigenetically silencing DNA genomes of incoming herpesviruses to enforce latency, KAP1 and HCM also serve in an antiviral capacity. In addition to gene silencing, newer reports highlight KAP1's ability to directly activate cellular gene transcription. Here, we discuss the many facets of KAP1, including recent findings that unexpectedly connect KAP1 to the inflammasome, reveal KAP1 cleavage as a novel mode of regulation, and argue for a pro-herpesviral KAP1 function that ensures transition from transcription to replication of the herpesvirus genome.

Inflammation and Epstein-Barr Virus at the Crossroads of Multiple Sclerosis and Post-Acute Sequelae of COVID-19 Infection.

Recent studies have strengthened the evidence for Epstein-Barr Virus (EBV) as an important contributing factor in the development of multiple sclerosis (MS). Chronic inflammation is a key feature of MS. EBV+ B cells can express cytokines and exosomes that promote inflammation, and EBV is known to be reactivated through the upregulation of cellular inflammasomes. Inflammation is a possible cause of the breakdown of the blood-brain barrier (BBB), which allows the infiltration of lymphocytes into the central nervous system. Once resident, EBV+ or EBV-specific B cells could both plausibly exacerbate MS plaques through continued inflammatory processes, EBV reactivation, T cell exhaustion, and/or molecular mimicry. Another virus, SARS-CoV-2, the cause of COVID-19, is known to elicit a strong inflammatory response in infected and immune cells. COVID-19 is also associated with EBV reactivation, particularly in severely ill patients. Following viral clearance, continued inflammation may be a contributor to post-acute sequelae of COVID-19 infection (PASC). Evidence of aberrant cytokine activation in patients with PASC supports this hypothesis. If unaddressed, long-term inflammation could put patients at risk for reactivation of EBV. Determining mechanisms by which viruses can cause inflammation and finding treatments for reducing that inflammation may help reduce the disease burden for patients suffering from PASC, MS, and EBV diseases.