Epigenetic Restriction Factors (eRFs) in Virus Infection.

The ongoing arms race between viruses and their hosts is constantly evolving. One of the ways in which cells defend themselves against invading viruses is by using restriction factors (RFs), which are cell-intrinsic antiviral mechanisms that block viral replication and transcription. Recent research has identified a specific group of RFs that belong to the cellular epigenetic machinery and are able to restrict the gene expression of certain viruses. These RFs can be referred to as epigenetic restriction factors or eRFs. In this review, eRFs have been classified into two categories. The first category includes eRFs that target viral chromatin. So far, the identified eRFs in this category include the PML-NBs, the KRAB/KAP1 complex, IFI16, and the HUSH complex. The second category includes eRFs that target viral RNA or, more specifically, the viral epitranscriptome. These epitranscriptomic eRFs have been further classified into two types: those that edit RNA bases-adenosine deaminase acting on RNA (ADAR) and pseudouridine synthases (PUS), and those that covalently modify viral RNA-the N6-methyladenosine (m6A) writers, readers, and erasers. We delve into the molecular machinery of eRFs, their role in limiting various viruses, and the mechanisms by which viruses have evolved to counteract them. We also examine the crosstalk between different eRFs, including the common effectors that connect them. Finally, we explore the potential for new discoveries in the realm of epigenetic networks that restrict viral gene expression, as well as the future research directions in this area.

Identification of SARS-CoV-2 Spike Palmitoylation Inhibitors That Results in Release of Attenuated Virus with Reduced Infectivity.

The spike proteins of enveloped viruses are transmembrane glycoproteins that typically undergo post-translational attachment of palmitate on cysteine residues on the cytoplasmic facing tail of the protein. The role of spike protein palmitoylation in virus biogenesis and infectivity is being actively studied as a potential target of novel antivirals. Here, we report that palmitoylation of the first five cysteine residues of the C-terminal cysteine-rich domain of the SARS-CoV-2 S protein are indispensable for infection, and palmitoylation-deficient spike mutants are defective in membrane fusion. The DHHC9 palmitoyltransferase interacts with and palmitoylates the spike protein in the ER and Golgi and knockdown of DHHC9 results in reduced fusion and infection of SARS-CoV-2. Two bis-piperazine backbone-based DHHC9 inhibitors inhibit SARS-CoV-2 S protein palmitoylation and the resulting progeny virion particles released are defective in fusion and infection. This establishes these palmitoyltransferase inhibitors as potential new intervention strategies against SARS-CoV-2.

Kaposi's Sarcoma-Associated Herpesvirus Infection Induces the Expression of Neuroendocrine Genes in Endothelial Cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with endothelial Kaposi's sarcoma (KS) in immunocompromised individuals. KS lesion cells exhibit many similarities to neuroendocrine (NE) cancers, such as highly vascular and red/purple tumor lesions, spindle-shaped cells, an insignificant role for classic oncogenes in tumor development, the release of bioactive amines, and indolent growth of the tumors. However, the mechanistic basis for the similarity of KS lesion endothelial cells to neuroendocrine tumors remains unknown. Next-generation sequencing and bioinformatics analysis in the present study demonstrate that endothelial cells latently infected with KSHV express several neuronal and NE genes. De novo infection of primary dermal endothelial cells with live and UV-inactivated KSHV demonstrated that viral gene expression is responsible for the upregulation of five selected NE genes (adrenomedullin 2 [ADM2], histamine receptor H1 [HRH1], neuron-specific enolase [NSE] [ENO2], neuronal protein gene product 9.5 [PGP9.5], and somatostatin receptor 1 [SSTR1]). Immunofluorescence and immunohistochemistry examinations demonstrated the robust expression of the NE genes HRH1 and NSE/ENO2 in KSHV-infected KS tissue samples and KS visceral tissue microarrays. Further analysis demonstrated that KSHV latent open reading frame K12 (ORFK12) gene (kaposin A)-mediated decreased host REST/NRSF (RE1-silencing transcription factor/neuron-restrictive silencer factor) protein, a neuronal gene transcription repressor protein, is responsible for NE gene expression in infected endothelial cells. The NE gene expression observed in KSHV-infected cells was recapitulated in uninfected endothelial cells by the exogenous expression of ORFK12 and by the treatment of cells with the REST inhibitor X5050. When the neuroactive ligand-activating receptor HRH1 and inhibitory SSTR1 were knocked out by CRISPR, HRH1 knockout (KO) significantly inhibited cell proliferation, while SSTR1 KO induced cell proliferation, thus suggesting that HRH1 and SSTR1 probably counteract each other in regulating KSHV-infected endothelial cell proliferation. These results demonstrate that the similarity of KS lesion cells to neuroendocrine tumors is probably a result of KSHV infection-induced transformation of nonneuronal endothelial cells into cells with neuroendocrine features. These studies suggest a potential role of neuroendocrine pathway genes in the pathobiological characteristics of KSHV-infected endothelial cells, including a potential mechanism of escape from the host immune system by the expression of immunologically privileged neuronal-site NE genes, and NE genes could potentially serve as markers for KSHV-infected KS lesion endothelial cells as well as novel therapeutic targets to control KS lesions.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) manipulates several cellular pathways for its survival advantage during its latency in the infected human host. Here, we demonstrate that KSHV infection upregulates the expression of genes related to neuronal and neuroendocrine (NE) functions that are characteristic of NE tumors, both in vitro and in KS patient tissues and the heterogeneity of neuroendocrine receptors having opposing roles in KSHV-infected cell proliferation. Induction of NE genes by KSHV could also provide a potential survival advantage, as the expression of proteins at immunologically privileged sites such as neurons on endothelial cells may be an avenue to escape host immune surveillance functions. The NE gene products identified here could serve as markers for KSHV-infected cells and could potentially serve as therapeutic targets to combat KSHV-associated KS.

IFI16, a nuclear innate immune DNA sensor, mediates epigenetic silencing of herpesvirus genomes by its association with H3K9 methyltransferases SUV39H1 and GLP.

IFI16, an innate immune DNA sensor, recognizes the nuclear episomal herpes viral genomes and induces the inflammasome and interferon-ß responses. IFI16 also regulates cellular transcription and act as a DNA virus restriction factor. IFI16 knockdown disrupted the latency of Kaposi's sarcoma associated herpesvirus (KSHV) and induced lytic transcripts. However, the mechanism of IFI16's transcription regulation is unknown. Here, we show that IFI16 is in complex with the H3K9 methyltransferase SUV39H1 and GLP and recruits them to the KSHV genome during de novo infection and latency. The resulting depositions of H3K9me2/me3 serve as a docking site for the heterochromatin-inducing HP1α protein leading into the IFI16-dependent epigenetic modifications and silencing of KSHV lytic genes. These studies suggest that IFI16's interaction with H3K9MTases is one of the potential mechanisms by which IFI16 regulates transcription and establish an important paradigm of an innate immune sensor's involvement in epigenetic silencing of foreign DNA.

Novel 5-arylthio-5H-chromenopyridines as a new class of anti-fibrotic agents.

Liver fibrosis is a critical wound healing response to chronic liver injury such as hepatitis C virus (HCV) infection. If persistent, liver fibrosis can lead to cirrhosis and hepatocellular carcinoma (HCC). The development of new therapies for preventing liver fibrosis and its progression to cancer associated with HCV infection remains a critical challenge. Identification of novel anti-fibrotic compounds will provide opportunities for innovative therapeutic intervention of HCV-mediated liver fibrosis. We designed and synthesized a focused set of 5-arylthio-5H-chromenopyridines as a new class of anti-fibrotic agents. Liver fibrosis assays demonstrated that the compounds 3a and 3c show inhibitory activity towards human hepatic stellate cells (LX2) activation at 10µM. The HCV NS3 and NS5A proteins in HCV subgenome-expressing cells were also significantly reduced in cells treated with 3a and 3c, suggesting the possible inhibitory role of the compounds in HCV translation/replication activities. We have also examined the reactivity of these compounds with medicinally-relevant metal compounds such as platinum and gold. The reactivity of these complexes with metals and during Mass Spectrometry suggests that CS bond cleavage is relatively facile.