Herpes simplex virus type 2 glycoprotein H interacts with integrin αvß3 to facilitate viral entry and calcium signaling in human genital tract epithelial cells.

UNLABELLED: Herpes simplex virus (HSV) entry requires multiple interactions at the cell surface and activation of a complex calcium signaling cascade. Previous studies demonstrated that integrins participate in this process, but their precise role has not been determined. These studies were designed to test the hypothesis that integrin αvß3 signaling promotes the release of intracellular calcium (Ca2+) stores and contributes to viral entry and cell-to-cell spread. Transfection of cells with small interfering RNA (siRNA) targeting integrin αvß3, but not other integrin subunits, or treatment with cilengitide, an Arg-Gly-Asp (RGD) mimetic, impaired HSV-induced Ca2+ release, viral entry, plaque formation, and cell-to-cell spread of HSV-1 and HSV-2 in human cervical and primary genital tract epithelial cells. Coimmunoprecipitation studies and proximity ligation assays indicated that integrin αvß3 interacts with glycoprotein H (gH). An HSV-2 gH-null virus was engineered to further assess the role of gH in the virus-induced signaling cascade. The gH-2-null virus bound to cells and activated Akt to induce a small Ca2+ response at the plasma membrane, but it failed to trigger the release of cytoplasmic Ca2+ stores and was impaired for entry and cell-to-cell spread. Silencing of integrin αvß3 and deletion of gH prevented phosphorylation of focal adhesion kinase (FAK) and the transport of viral capsids to the nuclear pore. Together, these findings demonstrate that integrin signaling is activated downstream of virus-induced Akt signaling and facilitates viral entry through interactions with gH by activating the release of intracellular Ca2+ and FAK phosphorylation. These findings suggest a new target for HSV treatment and suppression. IMPORTANCE: Herpes simplex viruses are the leading cause of genital disease worldwide, the most common infection associated with neonatal encephalitis, and a major cofactor for HIV acquisition and transmission. There is no effective vaccine. These epidemiological findings underscore the urgency to develop novel HSV treatment or prevention strategies. This study addresses this gap by further defining the signaling pathways the virus usurps to enter human genital tract epithelial cells. Specifically, the study defines the role played by integrins and by the viral envelope glycoprotein H in entry and cell-to-cell spread. This knowledge will facilitate the identification of new targets for the development of treatment and prevention.

Herpes simplex virus 2 (HSV-2) prevents dendritic cell maturation, induces apoptosis, and triggers release of proinflammatory cytokines: potential links to HSV-HIV synergy.

Herpes simplex virus 2 (HSV-2) may cause frequent recurrences, highlighting its ability to evade host defense. This study tested the hypothesis that HSV-2 interferes with dendritic cell (DC) function as an escape mechanism, which may contribute to enhanced HIV replication in coinfected populations. Immature monocyte-derived human DCs were exposed to live or UV-inactivated HSV-2 or lipopolysaccharide. Little or no increase in the maturation marker CD83 was observed in response to HSV-2 and HSV-2 exposed DCs were impaired in their ability to present antigen (influenza) to T cells. Exposure to UV-inactivated virus stimulated a modest, but significant increase in CD83, suggesting that viral gene expression contributes to the block in DC maturation. The functional impairment of HSV-2-exposed DCs could be partially attributed to the induction of apoptosis. Live and inactivated HSV-2 triggered an increase in the number of early and late apoptotic cells in both the infected and bystander cell populations; apoptosis was associated with a decrease in cellular FLICE-inhibitory protein (c-FLIP). Paradoxically, HSV-2 induced Akt phosphorylation, which typically promotes DC maturation and survival. Despite these aberrant responses, live and inactivated HSV-2 induced the release of cytokines into culture supernatants, which were sufficient to activate HIV-1 replication in latently infected U1 cells. Together, these findings suggest that in the presence of overt or subclinical HSV-2, the function of mucosal DCs would be impaired. These responses may allow HSV to escape immune surveillance but may also promote HIV infection and contribute to the epidemiological link between HIV and HSV.

HSV activates Akt to trigger calcium release and promote viral entry: novel candidate target for treatment and suppression.

HSV triggers intracellular calcium release to promote viral entry. We hypothesized that Akt signaling induces the calcium responses and contributes to HSV entry. Exposure of human cervical and primary genital tract epithelial, neuronal, or keratinocyte cells to HSV serotype 2 resulted in rapid phosphorylation of Akt. Silencing of Akt with small interfering RNA prevented the calcium responses, blocked viral entry, and inhibited plaque formation by 90% compared to control siRNA. Susceptibility to infection was partially restored if Akt was reintroduced into silenced cells with an Akt-expressing plasmid. HSV-2 variants deleted in glycoproteins B or D failed to induce Akt phosphorylation, and coimmunoprecipitation studies indicated that Akt interacts with glycoprotein B. Cell-surface expression of Akt was rapidly induced in response to HSV exposure. Miltefosine (50 µM), a licensed drug that blocks Akt phosphorylation, inhibited HSV-induced calcium release, viral entry, and plaque formation following infection with acyclovir-sensitive and resistant clinical isolates. Miltefosine blocked amplification of HSV from explanted ganglia to epithelial cells; viral yields were significantly less in miltefosine compared to control-treated cocultures (P<0.01). Together, these findings identify a novel role for Akt in viral entry, link Akt and calcium signaling, and suggest a new target for HSV treatment and suppression.

Characterization of HCV-infected people who inject drugs (PWID) in the setting of clinical care in Canada (CAPICA): A retrospective study.

Background: People who use drugs (PWUD) are among the highest risk category for becoming infected with the hepatitis C virus (HCV) in Canada. There is a need for more information on the demographics of HCV-infected PWUD/PWID who have recently injected drugs or who are actively injecting drugs. Methods: CAPICA was a multicentre, retrospective database/chart review conducted from October 2015 to February 2016 that was designed to characterize HCV-infected people who inject drugs (PWID) and are enrolled in clinical care in Canada. The aim was to identify factors of health care engagement essential in the design systems of HCV care and treatment in this population. The study enrolled 420 patients with a history of injection drug use within the last 12 months who had been diagnosed with chronic viremic HCV infection and had been participants in an outpatient clinical care setting in the past 12 months. Patients who were co-infected with HIV/HCV were excluded. Results: Harm reduction programs were in place at 92% (11/12) of the sites, and 75% (9) of these sites offered opioid agonist therapy (OAT), with 48% of the patients currently taking OAT. HCV genotype 1a was most prevalent (56%), followed by G3 (34%), and the most common fibrosis score was F1 (34%). The average reinfection rate was about 5%. Seventeen percent of the patients were undergoing HCV treatment or had recently failed therapy, while 83% were not being treated. Conclusions: In a multivariate analysis, the following factors were significantly associated with treatment: increasing age (OR 1.10), a fibrosis score of F4 (OR 4.91), moderate alcohol consumption (OR 3.70), and not using a needle exchange program (OR 6.95).

The Z-Profile Study: a multicenter, retrospective cohort study to assess the real-world use and effectiveness of elbasvir/grazoprevir in Canadian adult patients with chronic hepatitis C.

Background: Canada was the first country to approve elbasvir/grazoprevir (EBR/GZR) for the treatment of chronic HCV infection for genotypes 1 and 4 with or without ribavirin and genotype 3 with sofosbuvir, with no recommendation for baseline resistance testing. The aim of this study was to describe the effectiveness of EBR/GZR and the profile of patients selected for treatment in a Canadian real-world setting. Methods: This multicenter retrospective study of HCV-infected patients treated with EBR/GZR took place among selected Canadian health care providers, with no exclusion criteria. Primary outcome measures included parameters associated with patient profile and sustained virologic response at 12 weeks (SVR12) and 24 weeks after treatment. Results: A total of 408 patients were included; 244 had available SVR12 information (per-protocol population [PP]). Genotype distribution included 1a (54.7%), 1b (17.2%), 3 (11.8%), 4 (10.0%), and other (6.4%). The majority (88.7%) of participants were treated for 12 weeks without ribavirin. Fifty-nine (14.5%) participants, predominantly with genotype 1a (49/59) infection, were tested for baseline resistance-associated substitutions (bRAS). SVR12 was achieved by 95.9% of the PP. In an exploratory analysis assessing potential predictors of SVR12, participants who had undergone bRAS testing (OR 0.14, 95% CI 0.03-0.64) and participants who had undergone liver transplant (OR 0.05, 95% CI 0.00-0.68) had significantly lower odds of achieving SVR12. Conclusions: This study supports the real-world effectiveness of EBR/GZR-including a broad range of genotypes and diverse fibrosis stages-in the absence of bRAS testing and in special populations.

Oligonucleotide-based therapeutic options against hepatitis C virus infection.

The hepatitis C virus (HCV) is the cause of a silent pandemic that, due to the chronic nature of the disease and the absence of curative therapy, continues to claim an ever-increasing number of lives. Current antiviral regimens have proven largely unsatisfactory for patients with HCV drug-resistant genotypes. It is therefore important to explore alternative therapeutic stratagems whose mode of action allows them to bypass viral resistance. Antisense oligonucleotides, ribozymes, small interfering RNAs, aptamers and deoxyribozymes constitute classes of oligonucleotide-based compounds designed to target highly conserved or functionally crucial regions contained within the HCV genome. The therapeutic expectation for such compounds is the elimination of HCV from infected individuals. Progress in oligonucleotide-based HCV antivirals towards clinical application depends on development of nucleotide designs that bolster efficacy while minimizing toxicity, improvement in liver-targeting delivery systems, and refinement of small-animal models for preclinical testing.

HSV usurps eukaryotic initiation factor 3 subunit M for viral protein translation: novel prevention target.

Prevention of genital herpes is a global health priority. B5, a recently identified ubiquitous human protein, was proposed as a candidate HSV entry receptor. The current studies explored its role in HSV infection. Viral plaque formation was reduced by approximately 90% in human cells transfected with small interfering RNA targeting B5 or nectin-1, an established entry receptor. However, the mechanisms were distinct. Silencing of nectin-1 prevented intracellular delivery of viral capsids, nuclear transport of a viral tegument protein, and release of calcium stores required for entry. In contrast, B5 silencing had no effect on these markers of entry, but inhibited viral protein translation. Specifically, viral immediate early genes, ICP0 and ICP4, were transcribed, polyadenylated and transported from the nucleus to the cytoplasm, but the viral transcripts did not associate with ribosomes or polysomes in B5-silenced cells. In contrast, immediate early gene viral transcripts were detected in polysome fractions isolated from control cells. These findings are consistent with sequencing studies demonstrating that B5 is eukaryotic initiation factor 3 subunit m (eIF3m). Although B5 silencing altered the polysome profile of cells, silencing had little effect on cellular RNA or protein expression and was not cytotoxic, suggesting that this subunit is not essential for host cellular protein synthesis. Together these results demonstrate that B5 plays a major role in the initiation of HSV protein translation and could provide a novel target for strategies to prevent primary and recurrent herpetic disease.

Reduction in intracellular HCV RNA and virus protein expression in human hepatoma cells following treatment with 2'-O-methyl-modified anti-core deoxyribozyme.

HCV RNA is gaining greater consideration as a principal target for newer HCV antivirals because its destruction has the potential of eliminating the virus. These newer antivirals include deoxyribozymes (Dz), which are small single-stranded DNA molecules that cleave homologous RNA targets. Using a liver cell model containing functional genomic-length HCV-1b RNA we tested whether 2'-O-methyl-modified Dz, designed to recognize a highly-conserved RNA sequence located within the core-E1 coding region, could recognize and cleave its target sequence in the structural context of a functional HCV RNA molecule. Dz858-4-OMe contains four 2'-O-methyl nucleotide derivatives consecutively located on the distal ends of its two annealing arms. Intracellular HCV RNA, core protein and HCV antigen expression were reduced by 63%, 87% and 84%, respectively, when HCV RNA was challenged 6 h post-transfection with Dz858-4-OMe. The observed reduction of intracellular HCV RNA and protein by Dz858-4-OMe suggests that it may constitute an attractive HCV antiviral.