Dinesh Jaishankar

Characterization of a Proteolytically Stable D-Peptide That Suppresses Herpes Simplex Virus 1 Infection: Implications for the Development of Entry-Based Antiviral Therapy

ABSTRACT Uncontrolled herpes simplex virus 1 (HSV-1) infection can advance to serious conditions, including corneal blindness or fatal encephalitis. Here, we describe a highly potent anti-HSV-1 peptide (DG2) that inhibits HSV-1 entry into host cells and blocks all aspects of infection. Importantly, DG2 is highly resistant to proteases and shows minimal toxicity, paving the way for prophylactic or therapeutic application of the peptide in vivo.

Extended Release of an Anti-Heparan Sulfate Peptide From a Contact Lens Suppresses Corneal Herpes Simplex Virus-1 Infection.

Purpose To prolong the release of a heparan sulfate binding peptide, G2-C, using a commercially available contact lens as a delivery vehicle and to demonstrate the ability of the released peptide to block herpes simplex virus-1 (HSV-1) infection using in vitro, ex vivo, and in vivo models of corneal HSV-1 infection. Methods Commercially available contact lenses were immersed in peptide solution for 5 days prior to determining the release of the peptide at various time points. Cytotoxicity of the released samples was determined by MTT and cell cycle analysis, and the functional activity of the released samples were assessed by viral entry, and viral spread assay using human corneal epithelial cells (HCE). The ability to suppress infection in human and pig cornea ex vivo and mouse in vivo models were also assessed. Results Peptide G2-C was released through the contact lens. Following release for 3 days, the peptide showed significant activity by inhibiting HSV-1 viral entry and spread in HCE cells. Significant suppression of infection was also observed in the ex vivo and in vivo experiments involving corneas. Conclusions Extended release of an anti–HS peptide through a commercially available contact lens can generate significant anti–HSV-1 activity and provides a new and effective way to control corneal herpes.

Cultured corneas show dendritic spread and restrict herpes simplex virus infection that is not observed with cultured corneal cells

Herpes simplex virus-1 (HSV-1) causes life-long morbidities in humans. While fever blisters are more common, occasionally the cornea is infected resulting in vision loss. A very intriguing aspect of HSV-1 corneal infection is that the virus spread is normally restricted to only a small fraction of cells on the corneal surface that connect with each other in a dendritic fashion. Here, to develop a comprehensive understanding of the susceptibility of human corneal epithelial (HCE) cells to HSV-1 infection, we infected HCE cells at three different dosages of HSV-1 and measured the outcomes in terms of viral entry, gene and protein expression, viral replication and cytokine induction. In cultured cells, infectivity and cytokine induction were observed even at the minimum viral dosage tested, while a more pronounced dose-restricted infectivity was seen in ex vivo cultures of porcine corneas. Use of fluorescent HSV-1 virions demonstrated a pattern of viral spread ex vivo that mimics clinical findings. We conclude that HCE cell cultures are highly susceptible to infection whereas the cultured corneas demonstrate a higher ability to restrict the infection even in the absence of systemic immune system. The restriction is helped in part by local interferon response and the unique cellular architecture of the cornea.

An off-target effect of BX795 blocks herpes simplex virus type 1 infection of the eye

A kinase inhibitor shows promise as a topical antiviral against ocular herpes. Herpes tanked by a kinase inhibitor Interferon (IFN) responses are a cornerstone of antiviral immunity. As TANK-binding kinase 1 (TBK1) promotes IFN, Jaishankar et al. thought that using the TBK1 inhibitor BX795 would allow for increased herpes virus replication. Somewhat surprisingly, they found that this inhibitor reduced replication and may be a promising antiviral candidate. They tested BX795 in different herpes simplex virus type 1 (HSV-1) ocular infection models and examined the pathway through which it was working, which was not TBK1. BX795 may have advantages over traditional nucleoside analogs currently used to combat herpes infections and should be investigated further. Herpes simplex virus type 1 (HSV-1) causes recurrent mucocutaneous lesions in the eye that may advance to corneal blindness. Nucleoside analogs exemplified by acyclovir (ACV) form the primary class of antiherpetic drugs, but this class suffers limitations due to the emergence of viral resistance and other side effects. While studying the molecular basis of ocular HSV-1 infection, we observed that BX795, a commonly used inhibitor of TANK-binding kinase 1 (TBK1), strongly suppressed infection by multiple strains of HSV-1 in transformed and primary human cells, cultured human and animal corneas, and a murine model of ocular infection. Our investigations revealed that the antiviral activity of BX795 relies on targeting Akt phosphorylation in infected cells, leading to the blockage of viral protein synthesis. This small-molecule inhibitor, which was also effective against an ACV-resistant HSV-1 strain, shows promise as an alternative to existing drugs and as an effective topical therapy for ocular herpes infection. Collectively, our results obtained using multiple infection models and virus strains establish BX795 as a promising lead compound for broad-spectrum antiviral applications in humans.

Herpesvirus-encoded microRNAs detected in human gingiva alter host cell transcriptome and regulate viral infection

MicroRNAs (miRNAs) are small, non-coding RNAs of ~18-25 nucleotides that have gained extensive attention as critical regulators in complex gene networks including immune cell lineage commitment, differentiation, maturation, and maintenance of immune homeostasis and function. Many viruses encode miRNAs that directly downregulate the expression of factors of the innate immune system, which includes proteins involved in promoting apoptosis and recruitment. In this study, we examined the expression profiles of three previously identified viral miRNAs (v-miRs) from the human herpesvirus (HHV) family, HSV-1 (miR-H1), KSHV (miR-K12-3-3p), and HCMV (miR-US4) in healthy and diseased periodontal tissues and observed increased levels of v-miRs in diseased tissues. To understand the significance of this increase, we overexpressed v-miRs in human oral keratinocytes (HOK), a common target for various HHV, and analyzed the impact of miR-H1 and miR-K12-3-3p on the host transcriptome. More than 1300 genes were altered in HOK overexpressing miR-H1 and miR-K12-3-3p. Global pathway analysis of deregulated genes identified several key cellular pathways that may favor viral persistence. Using bioinformatic analysis, we predicted hundreds of potential v-miR binding sites on genes downregulated by miR-H1 and miR-K12-3-3p and validated three novel target v-miR sites suggesting widespread direct and indirect modulation of numerous host genes/pathways by a single v-miR. Finally, in vitro HSV-1 infection assays showed that miR-H1 can regulate viral entry and infection in human oral keratinocytes (HOK). Overall, our results demonstrate clinical and functional relevance of pathogenic viral molecules viz., v-miRs that regulate both host and viral functions and may contribute to the pathogenesis of inflammatory oral diseases.

Genital Herpes: Insights into Sexually Transmitted Infectious Disease

Etiology, transmission and protection: Herpes simplex virus-2 (HSV-2) is a leading cause of sexually transmitted infections with recurring manifestations throughout the lifetime of infected hosts. Currently no effective vaccines or prophylactics exist that provide complete protection or immunity from the virus, which is endemic throughout the world. Pathology/Symptomatology: Primary and recurrent infections result in lesions and inflammation around the genital area and the latter accounts for majority of genital herpes instances. Immunocompromised patients including neonates are susceptible to additional systemic infections including debilitating consequences of nervous system inflammation. Epidemiology, incidence and prevalence: More than 500 million people are infected worldwide and most reported cases involve the age groups between 16-40 years, which coincides with an increase in sexual activity among this age group. While these numbers are an estimate, the actual numbers may be underestimated as many people are asymptomatic or do not report the symptoms. Treatment and curability: Currently prescribed medications, mostly nucleoside analogs, only reduce the symptoms caused by an active infection, but do not eliminate the virus or reduce latency. Therefore, no cure exists against genital herpes and infected patients suffer from periodic recurrences of disease symptoms for their entire lives. Molecular mechanisms of infection: The last few decades have generated many new advances in our understanding of the mechanisms that drive HSV infection. The viral entry receptors such as nectin-1 and HVEM have been identified, cytoskeletal signaling and membrane structures such as filopodia have been directly implicated in viral entry, host motor proteins and their viral ligands have been shown to facilitate capsid transport and many host and HSV proteins have been identified that help with viral replication and pathogenesis. New understanding has emerged on the role of autophagy and other innate immune mechanisms that are subverted to enhance HSV pathogenesis. This review summarizes our current understanding of HSV-2 and associated diseases and available or upcoming new treatments.

Targeting Herpes Simplex Virus-1 gD by a DNA Aptamer Can Be an Effective New Strategy to Curb Viral Infection

Herpes simplex virus type 1 (HSV-1) is an important factor for vision loss in developed countries. A challenging aspect of the ocular infection by HSV-1 is that common treatments, such as acyclovir, fail to provide effective topical remedies. Furthermore, it is not very clear whether the viral glycoproteins, required for HSV-1 entry into the host, can be targeted for an effective therapy against ocular herpes in vivo. Here, we demonstrate that HSV-1 envelope glycoprotein gD, which is essential for viral entry and spread, can be specifically targeted by topical applications of a small DNA aptamer to effectively control ocular infection by the virus. Our 45-nt-long DNA aptamer showed high affinity for HSV-1 gD (binding affinity constant [Kd] = 50 nM), which is strong enough to disrupt the binding of gD to its cognate host receptors. Our studies showed significant restriction of viral entry and replication in both in vitro and ex vivo studies. In vivo experiments in mice also resulted in loss of ocular infection under prophylactic treatment and statistically significant lower infection under therapeutic modality compared to random DNA controls. Thus, our studies validate the possibility that targeting HSV-1 entry glycoproteins, such as gD, can locally reduce the spread of infection and define a novel DNA aptamer-based approach to control HSV-1 infection of the eye.

Emerging Roles of Heparanase in Viral Pathogenesis

Heparan sulfate (HS) is ubiquitously expressed on mammalian cells. It is a polysaccharide that binds growth factors, cytokines, and chemokines, and thereby controls several important physiological functions. Ironically, many human pathogens including viruses interact with it for adherence to host cells. HS functions can be regulated by selective modifications and/or selective cleavage of the sugar chains from the cell surface. In mammals, heparanase (HPSE) is the only known enzyme capable of regulating HS functions via a selective endoglycosidase activity that cleaves polymeric HS chains at internal sites. During homeostasis, HPSE expression and its endoglycosidase activity are tightly regulated; however, under stress conditions, including infection, its expression may be upregulated, which could contribute directly to the onset of several disease pathologies. Here we focus on viral infections exemplified by herpes simplex virus, dengue virus, human papillomavirus, respiratory syncytial virus, adenovirus, hepatitis C virus, and porcine respiratory and reproductive syncytial virus to summarize recent advances in understanding the highly significant, but emerging roles, of the enzyme HPSE in viral infection, spread and pathogenesis.

Liposome-mediated herpes simplex virus uptake is glycoprotein-D receptor-independent but requires heparan sulfate

Cationic liposomes are widely used to facilitate introduction of genetic material into target cells during transfection. This study describes a non-receptor mediated herpes simplex virus type-1 (HSV-1) entry into the Chinese hamster ovary (CHO-K1) cells that naturally lack glycoprotein D (gD)-receptors using a commercially available cationic liposome: lipofectamine. Presence of cell surface heparan sulfate (HS) increased the levels of viral entry indicating a potential role of HS in this mode of entry. Loss of viral entry in the presence of actin de-polymerizing or lysosomotropic agents suggests that this mode of entry results in the endocytosis of the lipofectamine-virus mixture. Enhancement of HSV-1 entry by liposomes was also demonstrated in vivo using a zebrafish embryo model that showed stronger infection in the eyes and other tissues. Our study provides novel insights into gD receptor independent viral entry pathways and can guide new strategies to enhance the delivery of viral gene therapy vectors or oncolytic viruses.