Codon pair bias deoptimization of essential genes in infectious laryngotracheitis virus reduces protein expression.

Infectious laryngotracheitis virus (ILTV; an alphaherpesvirus) is a respiratory pathogen of chickens and causes significant economic losses in the poultry industry globally, in addition to severe animal health and welfare concerns. To date, studying the role of ILTV genes in viral infection, replication or pathogenesis has largely been limited to genes that can be deleted from the ILTV genome and the resultant deletion mutants characterized in vitro or in vivo. However, this approach is not suitable for the study of essential genes. This study trialled two different codon deoptimization techniques that aimed to separately disrupt and downregulate the expression of two ILTV genes, ICP8 and UL12, which are essential or very important in viral replication. The target genes were partially recoded using codon usage deoptimization (CUD) and codon pair bias deoptimization (CPBD) approaches and characterized in vitro. Viruses deoptimized via CPBD showed decreased protein expression as assessed by Western blotting and/or fluorescence microscopy to measure the intensity of the fluorescent marker fused to the target protein. Viruses deoptimized by CUD showed less consistent results, with some mutants that could not be generated or isolated. The results indicate that CPBD is an attractive and viable tool for the study of essential or critically important genes in ILTV. This is the first study, to our knowledge, that utilizes CPBD and CUD techniques for the study of ILTV genes.

Herpes simplex virus 1 ICP8 mutant lacking annealing activity is deficient for viral DNA replication.

Most DNA viruses that use recombination-dependent mechanisms to replicate their DNA encode a single-strand annealing protein (SSAP). The herpes simplex virus (HSV) single-strand DNA binding protein (SSB), ICP8, is the central player in all stages of DNA replication. ICP8 is a classical replicative SSB and interacts physically and/or functionally with the other viral replication proteins. Additionally, ICP8 can promote efficient annealing of complementary ssDNA and is thus considered to be a member of the SSAP family. The role of annealing during HSV infection has been difficult to assess in part, because it has not been possible to distinguish between the role of ICP8 as an SSAP from its role as a replicative SSB during viral replication. In this paper, we have characterized an ICP8 mutant, Q706A/F707A (QF), that lacks annealing activity but retains many other functions characteristic of replicative SSBs. Like WT ICP8, the QF mutant protein forms filaments in vitro, binds ssDNA cooperatively, and stimulates the activities of other replication proteins including the viral polymerase, helicase-primase complex, and the origin binding protein. Interestingly, the QF mutant does not complement an ICP8-null virus for viral growth, replication compartment formation, or DNA replication. Thus, we have been able to separate the activities of ICP8 as a replicative SSB from its annealing activity. Taken together, our data indicate that the annealing activity of ICP8 is essential for viral DNA replication in the context of infection and support the notion that HSV-1 uses recombination-dependent mechanisms during DNA replication.

Inhibition of herpes simplex virus-1 infection by MBZM-N-IBT: in silico and in vitro studies.

INTRODUCTION: The emergence of drug resistance and cross-resistance to existing drugs has warranted the development of new antivirals for Herpes simplex viruses (HSV). Hence, we have designed this study to evaluate the anti-viral activity of 1-[(2-methyl benzimidazole-1-yl) methyl]-2-oxo-indolin-3-ylidene] amino] thiourea (MBZM-N-IBT), against HSV-1. METHOD: Molecular docking was performed to assess the affinity of MBZM-N-IBT for HSV-1 targets. This was validated by plaque assay, estimation of RNA and protein levels as well as time of addition experiments in vitro. RESULT: Molecular docking analysis suggested the inhibitory capacity of MBZM-N-IBT against HSV-1. This was supported by the abrogation of the HSV-1 infectious viral particle formation with the IC50 value of 3.619 µM. Viral mRNA levels were also reduced by 72% and 84% for UL9 and gC respectively. MBZM-N-IBT also reduced the protein synthesis for gC and ICP8 significantly. While mRNA of ICP8 was not significantly affected, its protein synthesis was reduced by 47%. The time of addition experiment revealed the capacity of MBZM-N-IBT to inhibit HSV-1 at early as well as late stages of infection in the Vero cells. Similar effect of MBZM-N-IBT was also noticed in the Raw 264.7 and BHK 21 cells after HSV-1 infection. Supported by the in silico data, this can be attributed to possible interference with multiple HSV targets including the ICP8, ICP27, UL42, UL25, UL15 and gB proteins. CONCLUSION: These results along with the lack of acute oral toxicity and significant anti-inflammatory effects suggest its suitability for further evaluation as a non-nucleoside inhibitor of HSV.

The SP100 component of ND10 enhances accumulation of PML and suppresses replication and the assembly of HSV replication compartments.

Nuclear domain 10 (ND10) bodies are small (0.1-1 µM) nuclear structures containing both constant [e.g., promyelocytic leukemia protein (PML), SP100, death domain-associated protein (Daxx)] and variable proteins, depending on the function of the cells or the stress to which they are exposed. In herpes simplex virus (HSV)-infected cells, ND10 bodies assemble at the sites of DNA entering the nucleus after infection. In sequence, the ND10 bodies become viral replication compartments, and ICP0, a viral E3 ligase, degrades both PML and SP100. The amounts of PML and SP100 and the number of ND10 structures increase in cells exposed to IFN-ß. Earlier studies have shown that PML has three key functions. Thus, (i) the interaction of PML with viral components facilitates the initiation of replication compartments, (ii) viral replication is significantly less affected by IFN-ß in PML-/- cells than in parental PML+/+ cells, and (iii) viral yields are significantly lower in PML-/- cells exposed to low ratios of virus per cell compared with parental PML+/+ cells. This report focuses on the function of SP100. In contrast to PML-/- cells, SP100-/- cells retain the sensitivity of parental SP100+/+ cells to IFN-ß and support replication of the ΔICP0 virus. At low multiplicities of infection, wild-type virus yields are higher in SP100-/- cells than in parental HEp-2 cells. In addition, the number of viral replication compartments is significantly higher in SP100-/- cells than in parental SP100+/+ cells or in PML-/- cells.

The HIV Integrase Inhibitor Raltegravir Inhibits Felid Alphaherpesvirus 1 Replication by Targeting both DNA Replication and Late Gene Expression.

Alphaherpesvirus-associated ocular infections in humans caused by human alphaherpesvirus 1 (HHV-1) remain challenging to treat due to the frequency of drug application required and the potential for the selection of drug-resistant viruses. Repurposing on-the-market drugs is a viable strategy to accelerate the pace of drug development. It has been reported that the human immunodeficiency virus (HIV) integrase inhibitor raltegravir inhibits HHV-1 replication by targeting the DNA polymerase accessory factor and limits terminase-mediated genome cleavage of human betaherpesvirus 5 (HHV-5). We have previously shown, both in vitro and in vivo, that raltegravir can also inhibit the replication of felid alphaherpesvirus 1 (FeHV-1), a common ocular pathogen of cats with a pathogenesis similar to that of HHV-1 ocular disease. In contrast to what was reported for HHV-1, we were unable to select for a raltegravir-resistant FeHV-1 strain in order to define any basis for drug action. A candidate-based approach to explore the mode of action of raltegravir against FeHV-1 showed that raltegravir did not impact FeHV-1 terminase function, as described for HHV-5. Instead, raltegravir inhibited DNA replication, similarly to HHV-1, but by targeting the initiation of viral DNA replication rather than elongation. In addition, we found that raltegravir specifically repressed late gene expression independently of DNA replication, and both activities are consistent with inhibition of ICP8. Taken together, these results suggest that raltegravir could be a valuable therapeutic agent against herpesviruses.IMPORTANCE The rise of drug-resistant herpesviruses is a longstanding concern, particularly among immunocompromised patients. Therefore, therapies targeting viral proteins other than the DNA polymerase that may be less likely to lead to drug-resistant viruses are urgently needed. Using FeHV-1, an alphaherpesvirus closely related to HHV-1 that similarly causes ocular herpes in its natural host, we found that the HIV integrase inhibitor raltegravir targets different stages of the virus life cycle beyond DNA replication and that it does so without developing drug resistance under the conditions tested. This shows that the drug could provide a viable strategy for the treatment of herpesvirus infections.

Visualizing the replicating HSV-1 virus using STED super-resolution microscopy.

BACKGROUND: Replication of viral genome is the central event during the lytic infectious cycle of herpes simplex virus 1 (HSV-1). However, the details of HSV-1 replication process are still elusive due to the limitations of current molecular and conventional fluorescent microscopy methods. Stimulated emission depletion (STED) microscopy is one of the recently available super-resolution techniques allowing observation at sub-diffraction resolution. METHODS: To gain new insight into HSV-1 replication, we used a combination of stimulated emission depletion microscopy, fluorescence in situ hybridization (FISH) and immunofluorescence (IF) to observe the HSV-1 replication process. RESULTS: Using two colored probes labeling the same region of HSV-1 genome, the two probes highly correlated in both pre-replication and replicating genomes. In comparison, when probes from different regions were used, the average distance between the two probes increased after the virus enters replication, suggesting that the HSV-1 genome undergoes dynamic structure changes from a compact to a relaxed formation and occupies larger space as it enters replication. Using FISH and IF, viral single strand binding protein ICP8 was seen closely positioned with HSV-1 genome. In contrast, ICP8 and host RNA polymerase II were less related. This result suggests that ICP8 marked regions of DNA replication are spatially separated from regions of active transcription, represented by the elongating form of RNA polymerase II within the viral replication compartments. Comparing HSV-1 genomes at early stage of replication with that in later stage, we also noted overall increases among different values. These results suggest stimulated emission depletion microscopy is capable of investigating events during HSV-1 replication. CONCLUSION: 1) Replicating HSV-1 genome could be observed by super-resolution microscopy; 2) Viral genome expands spatially during replication; 3) Viral replication and transcription are partitioned into different sub-structures within the replication compartments.

Ligand induced stabilization of the melting temperature of the HSV-1 single-strand DNA binding protein using the thermal shift assay.

We have adapted the thermal shift assay to measure the ligand binding properties of the herpes simplex virus-1 single-strand DNA binding protein, ICP8. By measuring SYPRO Orange fluorescence in microtiter plates using a fluorescence-enabled thermal cycler, we have quantified the effects of oligonucleotide ligands on the melting temperature of ICP8. We found that single-stranded oligomers raise the melting temperature of ICP8 in a length- and concentration-dependent manner, ranging from 1°C for (dT)5 to a maximum of 9°C with oligomers ⩾10 nucleotides, with an apparent Kd of <1µM for (dT)20. Specifically, the results indicate that ICP8 is capable of interacting with oligomers as short as 5 nucleotides. Moreover, the observed increases in melting temperature of up to 9°C, indicates that single-strand DNA binding significantly stabilizes the structure of ICP8. This assay may be applied to investigate the ligand binding proteins of other single-strand DNA binding proteins and used as a high-throughput screen to identify compounds with therapeutic potential that inhibit single-strand DNA binding. As proof of concept, the single-strand DNA binding agent ciprofloxacin reduces the ligand induced stabilization of the melting temperature of ICP8 in a dose-dependent manner.

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.

Two-Metal Ion-Dependent Enzymes as Potential Antiviral Targets in Human Herpesviruses.

The majority of drug discovery efforts against herpesviruses have focused on nucleoside analogs that target viral DNA polymerases, agents that are associated with dose-limiting toxicity and/or a narrow spectrum of activity. We are pursuing a strategy based on targeting two-metal ion-dependent (TMID) viral enzymes. This family of enzymes consists of structurally related proteins that share common active sites containing conserved carboxylates predicted to coordinate divalent cations essential for catalysis. Compounds that target TMID enzymes, such as HIV integrase and influenza endoribonuclease, have been successfully developed for clinical use. HIV integrase inhibitors have been reported to inhibit replication of herpes simplex virus (HSV) and other herpesviruses; however, the molecular targets of their antiviral activities have not been identified. We employed a candidate-based approach utilizing several two-metal-directed chemotypes and the potential viral TMID enzymatic targets in an effort to correlate target-based activity with antiviral potency. The panel of compounds tested included integrase inhibitors, the anti-influenza agent baloxavir, three natural products previously shown to exhibit anti-HSV activity, and two 8-hydroxyquinolines (8-HQs), AK-157 and AK-166, from our in-house program. The integrase inhibitors exhibited weak overall anti-HSV-1 activity, while the 8-HQs were shown to inhibit both HSV-1 and cytomegalovirus (CMV). Target-based analysis demonstrated that none of the antiviral compounds acted by inhibiting ICP8, contradicting previous reports. On the other hand, baloxavir inhibited the proofreading exonuclease of HSV polymerase, while AK-157 and AK-166 inhibited the alkaline exonuclease UL12. In addition, AK-157 also inhibited the catalytic activity of the HSV polymerase, which provides an opportunity to potentially develop dual-targeting agents against herpesviruses. IMPORTANCE Human herpesviruses (HHVs) establish lifelong latent infections, which undergo periodic reactivation and remain a major cause of morbidity and mortality, especially in immunocompromised individuals. Currently, HHV infections are treated primarily with agents that target viral DNA polymerase, including nucleoside analogs; however, long-term treatment can be complicated by the development of drug resistance. New therapies with novel modes of action would be important not only for the treatment of resistant viruses but also for use in combination therapy to reduce dose-limiting toxicities and potentially eliminate infection. Since many essential HHV proteins are well conserved, inhibitors of novel targets would ideally exhibit broad-spectrum activity against multiple HHVs.