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Antiviral Research (v.69, #1)

Prelim II: Editorial Board (pp. ii).

Prelim V: Guide for Authors (pp. v-xi).

Inside Back Cover: Notes to Authors (pp. co3).

Inhibition of drug-resistant HIV-1 by RNA interference by Peter M. Huelsmann; Pia Rauch; Kristina Allers; Matthias J. John; Karin J. Metzner (pp. 1-8).

RNA interference is a powerful tool used to inhibit human immunodeficiency virus type 1 (HIV-1) replication in vitro. Almost all HIV-1 genes have been targets for small interfering RNA (siRNA) molecules, and HIV-1 replication can be specifically and successfully inhibited by this technique. RNA interference has been proposed as an alternative strategy to inhibit replication of drug-resistant viruses that emerge during suboptimal antiretroviral therapy for HIV-1. To investigate specific inhibition of drug-resistant HIV-1 by RNA interference, we designed siRNA molecules that recognize codons 181–188 of the reverse transcriptase (RT) gene of wild-type HIV-1 and HIV-1 carrying the M184V mutation, which confers high-level resistance to the RT inhibitor lamivudine. Using viral variants with single point mutations at codon 184, we measured the impact of these mutations on virus replication. We have demonstrated that siRNA targeting either wild-type HIV-1 or M184V variants inhibits replication of the corresponding virus, but does not influence replication of virus with a mismatch in the targeted region. Combining two effective siRNAs did not show synergistic inhibitory effect on HIV-1 replication. However, a combination of lamivudine and siRNA–M184V was very effective in inhibiting replication of both wild-type and variant M184V viruses in mixed infection experiments. Taken together, these results demonstrate that RNA interference might be useful in the treatment of drug-resistant HIV-1 infection.

Keywords: RNA interference; HIV-1; Drug resistance; M184V; Lamivudine

Chemical library screen for novel inhibitors of Kaposi's sarcoma-associated herpesvirus processive DNA synthesis by Dorjbal Dorjsuren; Angela Burnette; Glenn N. Gray; Xulin Chen; Weimin Zhu; Paula E. Roberts; Michael J. Currens; Robert H. Shoemaker; Robert P. Ricciardi; Shizuko Sei (pp. 9-23).

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma and certain lymphoproliferative disorders. The role of KSHV lytic replication has been implicated in the tumor pathogenesis. A highly specific molecular complex formed by the KSHV DNA polymerase (POL8) and processivity factor (PF8) is indispensable for lytic viral DNA synthesis and may serve as an excellent molecular anti-KSHV target. The majority of conventional nucleoside-based anti-herpetic DNA synthesis inhibitors require intracellular phosphorylation/activation before they can exert inhibitory activity as competitive substrates for viral DNA polymerases. Novel and more potent inhibitors of KSHV DNA synthesis may be discovered through POL8/PF8-targeted high throughput screening (HTS) of small molecule chemical libraries. We developed a microplate-based KSHV POL8/PF8-mediated DNA synthesis inhibition assay suitable for HTS and screened the NCI Diversity Set that comprised 1992 synthetic compounds. Twenty-eight compounds exhibited greater than 50% inhibition. The inhibitory activity was confirmed for 25 of the 26 hit compounds available for further testing, with the 50% inhibitory concentrations ranging from 0.12±0.07μM (mean±S.D.) to 10.83±4.19μM. Eighteen of the confirmed active compounds efficiently blocked KSHV processive DNA synthesis in vitro. One of the hit compounds, NSC 373989, a pyrimidoquinoline analog, was shown to dose-dependently reduce the levels of KSHV virion production and KSHV DNA in lytically induced KSHV-infected BCBL-1 cells, suggesting that the compound blocked lytic KSHV DNA synthesis. HTS for KSHV POL8/PF8 inhibitors is feasible and may lead to discovery of novel non-nucleoside KSHV DNA synthesis inhibitors.

Keywords: Kaposi's sarcoma-associated herpesvirus; DNA polymerase; Processivity factor; Small molecule inhibitor screen

A genotype 2b NS5B polymerase with novel substitutions supports replication of a chimeric HCV 1b:2b replicon containing a genotype 1b NS3-5A background by Donald J. Graham; Mark Stahlhut; Osvaldo Flores; David B. Olsen; Daria J. Hazuda; Robert L. LaFemina; Steven W. Ludmerer (pp. 24-30).

HCV diversity suggests that evaluation of HCV inhibitors for broad genotypic efficacy is warrented. The replicon system enables cell-culture compound efficacy evaluation against an active replication complex, and a functional replicon dependent upon a genotype 2b polymerase would augment existing cell-culture efficacy studies that are presently limited to genotype 1a, 1b, and 2a replicons. We made a chimeric Neo^r 1b:2b replicon where genotype 2b NS5B was inserted into a genotype 1b NS3-5A background and transfected replicon RNA to generate Neo^r cell lines. All cell lines contained novel substitutions within NS5B which were subsequently engineered into the parental 1b:2b replicon and shown to enhance replication to various degrees. A single NS5B M31I substitution enhanced replication to levels sufficiently robust to quantify sensitivity to HCV inhibitors in a transient replication assay. The M31I 1b:2b replicon was similarly sensitive to an active-site nucleoside inhibitor of NS5B as genotype 1b replicons, but was insensitive to two non-nucleoside inhibitors which were otherwise efficacious against the genotype 1b replicons. This work describes a novel HCV replicon sustained by a genotype 2b polymerase that is sufficiently robust for quantifiable analysis in a transient replication assay, and demonstrates its utility in characterizing anti-HCV compounds for cross-genotypic efficacy.

Keywords: HCV; Replicon; NS5B; Polymerase; Inhibitor

Antiviral effect of the heparan sulfate mimetic, PI-88, against dengue and encephalitic flaviviruses by Eva Lee; Megan Pavy; Nicolie Young; Craig Freeman; Mario Lobigs (pp. 31-38).

Many viruses, including flaviviruses, display affinity for cell surface heparan sulfate (HS) proteoglycans with biological relevance in virus attachment/entry. This raises the possibility of the application of HS mimetics in antiviral therapy. We have evaluated the antiviral effect of the sulfated polysaccharides, suramin, pentosan polysulfate (PPS) and PI-88, which are currently approved or in trial for clinical use, against dengue virus (DEN) and the encephalitic flaviviruses, Japanese encephalitis virus, West Nile virus, and Murray Valley encephalitis virus. A flow cytometry-based method for the measurement of inhibition of virus infectivity was developed, which showed the in vitro antiviral activity of the three compounds, albeit with differences in efficiency which were virus-dependent. The 50% effective concentration (EC₅₀) values for DEN inhibition were in the order: PPS

Keywords: Flavivirus; Heparan sulfate mimetic; Japanese encephalitis virus; West Nile virus

Anti-influenza virus activity of peramivir in mice with single intramuscular injection by Shanta Bantia; C. Shane Arnold; Cynthia D. Parker; Ramanda Upshaw; Pooran Chand (pp. 39-45).

In the event of an influenza outbreak, antivirals including the neuraminidase (NA) inhibitors, peramivir, oseltamivir, and zanamivir may provide valuable benefit when vaccine production is delayed, limited, or cannot be used. Here we demonstrate the efficacy of a single intramuscular injection of peramivir in the mouse influenza model. Peramivir potently inhibits the neuraminidase enzyme N9 from H1N9 virus in vitro with a 50% inhibitory concentration (IC₅₀) of 1.3±0.4nM. On-site dissociation studies indicate that peramivir remains tightly bound to N9 NA ( t_1/2>24h), whereas, zanamivir and oseltamivir carboxylate dissociated rapidly from the enzyme ( t_1/2=1.25h). A single intramuscular injection of peramivir (10mg/kg) significantly reduces weight loss and mortality in mice infected with influenza A/H1N1, while oseltamivir demonstrates no efficacy by the same treatment regimen. This may be due to tight binding of peramivir to the N1 NA enzymes similar to that observed for N9 enzyme. Additional efficacy studies indicate that a single injection of peramivir (2–20mg/kg) was comparable to a q.d.×5 day course of orally administered oseltamivir (2–20mg/kg/day) in preventing lethality in H3N2 and H1N1 influenza models. A single intramuscular injection of peramivir may successfully treat influenza infections and provide an alternate option to oseltamivir during an influenza outbreak.

Keywords: Peramivir; Influenza; Mouse model; Intramuscular

Avian metapneumovirus phosphoprotein targeted RNA interference silences the expression of viral proteins and inhibits virus replication by Shirin Munir; Kuljeet Kaur; Vivek Kapur (pp. 46-51).

Avian metapneumovirus (aMPV) is one of the major causes of serious respiratory infections of poultry and leads to considerable economic losses to food animal production worldwide. Here, we show that double stranded short interfering RNA (siRNA) molecules corresponding to aMPV phosphoprotein (P) gene silence P RNA and protein expression. These siRNAs broadly reduced the expression of other viral proteins in addition to P, but did not have a discernable effect on cellular protein expression. The exposure of cells to P-specific siRNAs also led to inhibition of virus replication as evidenced by marked reduction in the progeny virion titers. Taken together, the findings suggest that exogenous P silencing siRNAs can inhibit aMPV replication with potential implications in the design of novel siRNA based prophylactics.

Keywords: Avian metapneumovirus; aMPV; RNA interference; siRNA; Phosphoprotein; Post-transcriptional gene silencing

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Antiviral Research (v.69, #1)