2'-C-methylated nucleotides terminate virus RNA synthesis by preventing active site closure of the viral RNA-dependent RNA polymerase.

The 2'-C-methyl ribonucleosides are nucleoside analogs representing an important class of antiviral agents, especially against positive-strand RNA viruses. Their value is highlighted by the highly successful anti-hepatitis C drug sofosbuvir. When appropriately phosphorylated, these nucleotides are successfully incorporated into RNA by the virally encoded RNA-dependent RNA polymerase (RdRp). This activity prevents further RNA extension, but the mechanism is poorly characterized. Previously, we had identified NMR signatures characteristic of formation of RdRp-RNA binary and RdRp-RNA-NTP ternary complexes for the poliovirus RdRp, including an open-to-closed conformational change necessary to prepare the active site for catalysis of phosphoryl transfer. Here we used these observations as a framework for interpreting the effects of 2'-C-methyl adenosine analogs on RNA chain extension in solution-state NMR spectroscopy experiments, enabling us to gain additional mechanistic insights into 2'-C-methyl ribonucleoside-mediated RNA chain termination. Contrary to what has been proposed previously, poliovirus RdRp that was bound to RNA with an incorporated 2'-C-methyl nucleotide could still bind to the next incoming NTP. Our results also indicated that incorporation of the 2'-C-methyl nucleotide does not disrupt RdRp-RNA interactions and does not prevent translocation. Instead, incorporation of the 2'-C-methyl nucleotide blocked closure of the RdRp active site upon binding of the next correct incoming NTP, which prevented further nucleotide addition. We propose that other nucleotide analogs that act as nonobligate chain terminators may operate through a similar mechanism.

Hijacking of multiple phospholipid biosynthetic pathways and induction of membrane biogenesis by a picornaviral 3CD protein.

RNA viruses induce specialized membranous structures for use in genome replication. These structures are often referred to as replication organelles (ROs). ROs exhibit distinct lipid composition relative to other cellular membranes. In many picornaviruses, phosphatidylinositol-4-phosphate (PI4P) is a marker of the RO. Studies to date indicate that the viral 3A protein hijacks a PI4 kinase to induce PI4P by a mechanism unrelated to the cellular pathway, which requires Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1, GBF1, and ADP ribosylation factor 1, Arf1. Here we show that a picornaviral 3CD protein is sufficient to induce synthesis of not only PI4P but also phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidylcholine (PC). Synthesis of PI4P requires GBF1 and Arf1. We identified 3CD derivatives: 3CDm and 3CmD, that we used to show that distinct domains of 3CD function upstream of GBF1 and downstream of Arf1 activation. These same 3CD derivatives still supported induction of PIP2 and PC, suggesting that pathways and corresponding mechanisms used to induce these phospholipids are distinct. Phospholipid induction by 3CD is localized to the perinuclear region of the cell, the outcome of which is the proliferation of membranes in this area of the cell. We conclude that a single viral protein can serve as a master regulator of cellular phospholipid and membrane biogenesis, likely by commandeering normal cellular pathways.

Multiple poliovirus-induced organelles suggested by comparison of spatiotemporal dynamics of membranous structures and phosphoinositides.

At the culmination of poliovirus (PV) multiplication, membranes are observed that contain phosphatidylinositol-4-phosphate (PI4P) and appear as vesicular clusters in cross section. Induction and remodeling of PI4P and membranes prior to or concurrent with genome replication has not been well studied. Here, we exploit two PV mutants, termed EG and GG, which exhibit aberrant proteolytic processing of the P3 precursor that substantially delays the onset of genome replication and/or impairs virus assembly, to illuminate the pathway of formation of PV-induced membranous structures. For WT PV, changes to the PI4P pool were observed as early as 30 min post-infection. PI4P remodeling occurred even in the presence of guanidine hydrochloride, a replication inhibitor, and was accompanied by formation of membrane tubules throughout the cytoplasm. Vesicular clusters appeared in the perinuclear region of the cell at 3 h post-infection, a time too slow for these structures to be responsible for genome replication. Delays in the onset of genome replication observed for EG and GG PVs were similar to the delays in virus-induced remodeling of PI4P pools, consistent with PI4P serving as a marker of the genome-replication organelle. GG PV was unable to convert virus-induced tubules into vesicular clusters, perhaps explaining the nearly 5-log reduction in infectious virus produced by this mutant. Our results are consistent with PV inducing temporally distinct membranous structures (organelles) for genome replication (tubules) and virus assembly (vesicular clusters). We suggest that the pace of formation, spatiotemporal dynamics, and the efficiency of the replication-to-assembly-organelle conversion may be set by both the rate of P3 polyprotein processing and the capacity for P3 processing to yield 3AB and/or 3CD proteins.

A Narrative Review of Alternative Symptomatic Treatments for Herpes Simplex Virus.

Herpes simplex virus-1 (HSV-1) and -2 (HSV-2) are large, spherically shaped, double-stranded DNA viruses that coevolved with Homo sapiens for over 300,000 years, having developed numerous immunoevasive mechanisms to survive the lifetime of their human host. Although in the continued absence of an acceptable prophylactic and therapeutic vaccine, approved pharmacologics (e.g., nucleoside analogs) hold benefit against viral outbreaks, while resistance and toxicity limit their universal application. Against these shortcomings, there is a long history of proven and unproven home remedies. With the breadth of purported alternative therapies, patients are exposed to risk of harm without proper information. Here, we examined the shortcomings of the current gold standard HSV therapy, acyclovir, and described several natural products that demonstrated promise in controlling HSV infection, including lemon balm, lysine, propolis, vitamin E, and zinc, while arginine, cannabis, and many other recreational drugs are detrimental. Based on this literature, we offered recommendations regarding the use of such natural products and their further investigation.

Herpes simplex virus infected cell protein 8 is required for viral inhibition of the cGAS pathway.

DNA virus infection triggers an antiviral type I interferon (IFN) response in cells that suppresses infection of surrounding cells. Consequently, viruses have evolved mechanisms to inhibit the IFN response for efficient replication. The cellular cGAS protein binds to double-stranded DNA and synthesizes the small molecule cGAMP to initiate DNA-dependent type I IFN production. We showed previously that cGAMP production is relatively low during HSV-1 infection compared to plasmid DNA transfection. Therefore, we hypothesized that HSV-1 produces antagonists of the cGAS DNA sensing pathway. In this study, we found that the HSV-1 ICP8 protein is required for viral inhibition of the cGAS pathway by reducing cGAMP levels stimulated by double-stranded DNA transfection. ICP8 alone inhibited the cGAMP response and may inhibit cGAS action by direct interaction with DNA, cGAS, or other infected cell proteins. Our results reveal another cGAS antiviral pathway inhibitor and highlight the importance of countering IFN for efficient viral replication.

Screening Method for CRISPR/Cas9 Inhibition of a Human DNA Virus: Herpes Simplex Virus.

The efficiency of cleavage of individual CRISPR/Cas9-sgRNAs remains difficult to predict based on the CRISPR target sequence alone. Different intracellular environments (dependent on cell type or cell cycle state for example) may affect sgRNA efficiency by altering accessibility of genomic DNA through DNA modifications such as epigenetic marks and DNA-binding proteins (e.g., histones) as well as alteration of the chromatin state of genomic DNA within the nucleus. We recently reported a multi-step screening method for the identification of efficient sgRNAs targeting the Herpes simplex virus (HSV-1) genome and reported a differential mechanism for viral inhibition by CRISPR-Cas9 in the latent versus lytic phase. The screening platform detailed in this protocol allows step-by-step testing of the efficiency of cleavage in a cell-free system and in the context of viral target cells such as human foreskin fibroblasts followed by functional testing of the effects of CRISPR/sgRNA on viral protein expression, replication, and reactivation. This strategy could be readily applied to other target cells such as pluripotent stem cell-derived human sensory neurons or other human DNA viruses.