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Antiviral Research (v.84, #3)
Solution characterization of [methyl-13C]methionine HIV-1 reverse transcriptase by NMR spectroscopy by Xunhai Zheng; Geoffrey A. Mueller; Eugene F. DeRose; Robert E. London (pp. 205-214).
HIV reverse transcriptase (RT) is a primary target for drug intervention in the treatment of AIDS. We report the first solution NMR studies of [methyl-13C]methionine HIV-1 RT, aimed at better understanding the conformational and dynamic characteristics of RT, both in the presence and absence of the non-nucleoside RT inhibitor (NNRTI) nevirapine. The selection of methionine as a structural probe was based both on its favorable NMR characteristics, and on the presence of two important active site methionine residues, M18466 and M23066. Observation of the M184 resonance is subunit dependent; in the p66 subunit the solvent-exposed residue produces a readily observed signal with a characteristic resonance shift, while in the globular p51 subunit, the M18451 resonance is shifted and broadened as M184 becomes buried in the protein interior. In contrast, although structural data indicates that the environment of M230 is also strongly subunit dependent, the M230 resonances from both subunits have very similar shift and relaxation characteristics. A comparison of chemical shift and intensity data with model-based predictions gives reasonable agreement for M18466, while M23066, located on the β-hairpin “primer grip”, is more mobile and solvent-exposed than suggested by crystal structures of the apo enzyme which have a “closed” fingers-thumb conformation. This mobility of the primer grip is presumably important for binding of non-nucleoside RT inhibitors (NNRTIs), since the NNRTI binding pocket is not observed in the absence of the inhibitors, requiring instead that the binding pocket be dynamically accessible. In the presence of the nevirapine, both the M18466 and M23066 resonances are significantly perturbed, while none of the methionine resonances in the p51 subunit is sensitive to this inhibitor. Site-directed mutagenesis indicates that both M16 and M357 produce two resonances in each subunit, and for both residues, the intensity ratio of the component peaks is strongly subunit dependent. Conformational features that might explain the multiple peaks are discussed.
Keywords: Abbreviations; DSS; 2,2-dimethylsilapentaned-5-sulfonic acid; HEPT; 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine; IPTG; Isopropyl thio-; d; -galactoside; HSQC; heteronuclear single-quantum coherence; NNRTI; non-nucleoside RT inhibitors; NMR; nuclear magnetic resonance; NOE; nuclear Overhauser effect; pdb; Protein Data Bank; RMSD; root mean square deviation; RT; reverse transcriptaseHIV-1 reverse transcriptase; Solution behavior; [Methyl-; 13; C]methionine labeled; NMR spectroscopy
Immunization by influenza virus-like particles protects aged mice against lethal influenza virus challenge by Zhiyuan Wen; Ling Ye; Yulong Gao; Lei Pan; Ke Dong; Zhigao Bu; Richard W. Compans; Chinglai Yang (pp. 215-224).
Influenza virus-like particles (VLPs) were produced in Sf9 insect cells by co-expressing the matrix protein M1 and the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) using the recombinant baculovirus expression system. The VLPs were morphologically similar to influenza virions. Both HA and NA proteins were incorporated into VLPs and these proteins retained their functional activities. Further, influenza VLPs but not inactivated influenza viruses (IIV) stimulated secretion of inflammatory cytokines from mouse bone marrow-derived dendritic cells (BMDC). Immunogenicity of influenza VLPs and their protective efficacies against lethal influenza virus challenge were evaluated in young and aged mice. Immunization with influenza VLPs induced strong antibody responses against HA that inhibited hemagglutination by influenza virus, similar to IIV vaccines. Compared to young mice, antibody responses in aged mice immunized with a low dose of either influenza VLPs or IIV vaccines exhibited markedly reduced avidity for HA. However, immunization of aged mice with a high dose of influenza VLPs induced antibody responses with high avidity similar to those in young mice. Furthermore, all vaccinated animals survived a lethal challenge by a mouse-adapted influenza virus (A/PR/8/34), indicating that influenza VLPs are highly efficacious for protection against influenza virus infection in both young and aged mice.
Keywords: Influenza; Virus-like particle; Aging; Antibody response; Avidity
Naturally occurring mutations at residues 253 and 284 in VP2 contribute to the cell tropism and virulence of very virulent infectious bursal disease virus by Xiaole Qi; Honglei Gao; Yulong Gao; Liting Qin; Yongqiang Wang; Li Gao; Xiaomei Wang (pp. 225-233).
Infectious bursal disease virus (IBDV) is responsible for the highly contagious infectious bursal disease in chickens. Previously, by blind passage, a vvIBDV Gx strain was attenuated to the Gt strain, and a strain CEF-9 with intermediate characters was obtained during attenuation. Since CEF-9 exhibited only two interesting amino acid mutations (Q253H and A284T) on loops PDE and PFG at the tip of VP2 spikes, we hypothesized that, either function separately or in combination, they define the cell tropism and virulence of vvIBDV. To test this hypothesis, Q253H and A284T were introduced individually or in combination into VP2 of the Gx or Gt strain to obtain six modified clones. Using reverse genetics, combined mutations of Q253H and A284T could adapt vvIBDV to non-permissive CEF cells (rGx-F9VP2) but any single mutation could not. In vivo, rGx-F9VP2 did not cause mortality while the Gx strain induced 66.7% mortality. Dual evidence from natural and rescued strains identified that the cell tropism of vvIBDV to CEF cells was determined by the combined VP2 mutations Q253H and A284T, but not by single mutation. The two residues were mainly responsible for the virulence of vvIBDV. These findings may be helpful in the design of new tailored IBDV vaccines.
Keywords: Infectious bursal disease virus (IBDV); VP2; Cell tropism; Virulence
In silico screening of small molecule libraries using the dengue virus envelope E protein has identified compounds with antiviral activity against multiple flaviviruses by Thorsten Kampmann; Ragothaman Yennamalli; Phillipa Campbell; Martin J. Stoermer; David P. Fairlie; Bostjan Kobe; Paul R. Young (pp. 234-241).
The flaviviruses comprise a large group of related viruses, many of which pose a significant global human health threat, most notably the dengue viruses (DENV), West Nile virus (WNV) and yellow fever virus (YFV). Flaviviruses enter host cells via fusion of the viral and cellular membranes, a process mediated by the major viral envelope protein E as it undergoes a low pH induced conformational change in the endosomal compartment of the host cell. This essential entry stage in the flavivirus life cycle provides an attractive target for the development of antiviral agents. We performed an in silico docking screen of the Maybridge chemical database within a previously described ligand binding pocket in the dengue E protein structure that is thought to play a key role in the conformational transitions that lead to membrane fusion. The biological activity of selected compounds identified from this screen revealed low micromolar antiviral potency against dengue virus for two of the compounds. Our results also provide the first evidence that compounds selected to bind to this ligand binding site on the flavivirus E protein abrogate fusion activity. Interestingly, one of these compounds also has antiviral activity against both WNV (kunjin strain) and YFV.
Keywords: Abbreviations; BSA; bovine serum albumin; MTT; methyl thiazole tetrazolium; pfu; plaque-forming units; m.o.i.; multiplicity of infection; PBS; phosphate buffered saline; DENV; dengue viruses; YFV; yellow fever virus; WNV; West Nile virus; KUNV; kunjin virus; VS; virtual screeningDengue virus; Antiviral; Fusion inhibition; Flavivirus; Virtual screening
Detection of influenza A H1N1 and H3N2 mutations conferring resistance to oseltamivir using rolling circle amplification by Megan C. Steain; Dominic E. Dwyer; Aeron C. Hurt; Chenda Kol; Nitin K. Saksena; Anthony L. Cunningham; Bin Wang (pp. 242-248).
In the event of an influenza pandemic, the use of oseltamivir (OTV) will undoubtedly increase and therefore it is more likely that OTV-resistant influenza strains will also arise. OTV-resistance genotyping using sequence-based testing on viruses isolated in cell culture is time consuming and less likely to detect the low-level presence of drug-resistant virus populations. We have developed a novel rolling circle amplification (RCA) method to achieve the sensitive detection of OTV-resistant viruses from clinical specimens. Using artificially created templates, RCA could detect the presence of OTV-resistant mutations (N2: 119V, 292K, N1: 274Y) even if the population carrying the mutations was <1% of the total. By applying RCA to clinical samples, we identified the emergence of the 274Y mutation in one OTV-treated patient, as well as in seven individuals who were treatment-naïve (confirming community transmission of 274Y-containing resistant influenza A H1N1). These results were further confirmed by neuraminidase region sequencing. In conclusion, RCA technology can provide rapid (<24h), high-throughput diagnosis of OTV resistance mutations with a high specificity and sensitivity.
Keywords: Influenza; Oseltamivir; Drug resistance; Rolling circle amplification
HD-03/ES: A promising herbal drug for HBV antiviral therapy by Premashis Kar; Mohammad Asim; Manash Pratim Sarma; Pralhad S. Patki (pp. 249-253).
The present study was designed to study the genotypes associated with different groups of chronic liver disease and to see their response to HD-03/ES (an antiviral herbal molecule) on chronic HBV patients.A total of 51 patients of chronic liver disease were recruited in the study and were given HD-03/ES, two capsules twice daily for 6 months. Liver function tests were done every month after initiating treatment. Serum was analyzed for HBsAg, HBeAg and HBV DNA and quantitative estimation of HBV at baseline, 4 and 6 months after therapy. The genotype of all the cases was also determined by PCR-RFLP method.After 6 months of therapy with HD-03/ES, a significant reduction of ALT values from 71.2±16.3 to 36.4±6.8 and a significant HBeAg loss (27.4%) and HBV DNA loss (27.4%) was observed. Adverse effects were mild. Genotype D was found in 39 (76.5%) while genotype A was found in 12 (33.5%) cases, respectively. The mean reduction in viral load was observed from log107.1±1.8copies/ml to log104.4±1.1copies/ml. However, a sharp decline in viral load was observed in patients infected with genotype A (log106.8±2.5 to log104.9±1.8; P<0.01) compared to genotype D (log107.0±2.6 to log105.9±3.5; P=0.074).The study had shown that majority of the patients of chronic HBV related liver disease had genotype D. In additional, the molecule HD03/ES had a better therapeutic capability of lowering the HBV viral load in patients with genotype A, which needs to be validated in larger studies.
Keywords: HBV; Viral load; Genotype; Chronic liver disease
Antiviral evaluation of octadecyloxyethyl esters of ( S)-3-hydroxy-2-(phosphonomethoxy)propyl nucleosides against herpesviruses and orthopoxviruses by Nadejda Valiaeva; Mark N. Prichard; R. Mark Buller; James R. Beadle; Caroll B. Hartline; Kathy A. Keith; Jill Schriewer; Julissa Trahan; Karl Y. Hostetler (pp. 254-259).
Our previous studies showed that esterification of 9-( S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine (HPMPA) or 1-( S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]cytosine (HPMPC) with alkoxyalkyl groups such as hexadecyloxypropyl (HDP) or octadecyloxyethyl (ODE) resulted in large increases in antiviral activity and oral bioavailability. The HDP and ODE esters of HPMPA were shown to be active in cells infected with human immunodeficiency virus, type 1 (HIV-1), while HPMPA itself was virtually inactive. To explore this approach in greater detail, we synthesized four new compounds in this series, the ODE esters of 9-( S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]guanine (HPMPG), 1-( S)-[3-hydroxy-2-(phosphonomethoxy)propyl]thymine (HPMPT), 9-( S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine (HPMPDAP) and 9-( S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-2-amino-6-cyclopropylaminopurine (HPMP-cPrDAP) and evaluated their antiviral activity against herpes simplex virus, type 1 (HSV-1), human cytomegalovirus (HCMV), and vaccinia, cowpox and ectromelia. Against HSV-1, subnanomolar EC50 values were observed with ODE–HPMPA and ODE–HPMPC while ODE–HPMPG had intermediate antiviral activity with an EC50 of 40nM. In HFF cells infected with HCMV, the lowest EC50 values were observed with ODE–HPMPC, 0.9nM. ODE–HPMPA was highly active with an EC50 of 3nM, while ODE–HPMPG and ODE–HPMPDAP were also highly active with EC50s of 22 and 77nM, respectively. Against vaccinia and cowpox viruses, ODE–HPMPG and ODE–HPMPDAP were the most active and selective compounds with EC50 values of 20–60nM and selectivity index values of 600–3500. ODE–HPMPG was also active against ectromelia virus with an EC50 value of 410nM and a selectivity index value of 166. ODE–HPMPG and ODE–HPMPDAP are proposed for further preclinical evaluation as possible candidates for treatment of HSV, HCMV or orthopoxvirus diseases.
Keywords: Herpes simplex virus; Human cytomegalovirus; Murine cytomegalovirus; Vaccinia virus; Cowpox virus; Ectromelia virus; Acyclic nucleoside phosphonates; Alkoxyalkyl prodrugs; Drug delivery
A small molecule fusion inhibitor of dengue virus by Mee Kian Poh; Andy Yip; Summer Zhang; John P. Priestle; Ngai Ling Ma; Jolanda M. Smit; Jan Wilschut; Pei-Yong Shi; Markus R. Wenk; Wouter Schul (pp. 260-266).
The dengue virus envelope protein plays an essential role in viral entry by mediating fusion between the viral and host membranes. The crystal structure of the envelope protein shows a pocket (located at a “hinge” between Domains I and II) that can be occupied by ligand n-octyl-β-d-glucoside (βOG). Compounds blocking the βOG pocket are thought to interfere with conformational changes in the envelope protein that are essential for fusion. Two fusion assays were developed to examine the anti-fusion activities of compounds. The first assay measures the cellular internalization of propidium iodide upon membrane fusion. The second assay measures the protease activity of trypsin upon fusion between dengue virions and trypsin-containing liposomes. We performed an in silico virtual screening for small molecules that can potentially bind to the βOG pocket and tested these candidate molecules in the two fusion assays. We identified one compound that inhibits dengue fusion in both assays with an IC50 of 6.8μM and reduces viral titers with an EC50 of 9.8μM. Time-of-addition experiments showed that the compound was only active when present during viral infection but not when added 1h later, in agreement with a mechanism of action through fusion inhibition.
Keywords: Dengue virus; Antiviral; Fusion inhibitor
Cistus incanus (CYSTUS052) for treating patients with infection of the upper respiratory tract by Ulrich Kalus; Alexandre Grigorov; Oliver Kadecki; Jan-Peter Jansen; Holger Kiesewetter; Hartmut Radtke (pp. 267-271).
In this prospective, randomized, placebo-controlled clinical study we aimed to investigate the clinical effect of a Cistus extract (CYSTUS052) in 160 patients with infections of the upper respiratory tract. The extract contains a high percentage of highly polymeric polyphenols. In cell culture and in a mouse model it exerts antiviral and antimicrobial activities. Principal active constituents of the genus Cistus are polyphenolic compounds. Plant-derived polyphenols have been shown to be strong antioxidants with potential health benefits. Various reports have appeared on the antiviral and antibacterial potential, including several reports describing the antiviral activity of polyphenols against influenza virus. Clinical studies on the effectiveness of Cistus incanus are scarce. Only one controlled application observation study demonstrated the effectiveness of a Cistus extract. The present randomised, placebo-controlled clinical study was designed to compare the symptom scores in patients with common cold treated either with CYSTUS052 or with placebo. A score of subjective symptoms decreased significantly over the course of treatment with Cistus, whereas treatment with placebo resulted in a less distinct decrease of symptoms. Among the inflammatory markers investigated, the C-reactive protein was mostly affected by Cistus and decreased significantly in the treatment group.
Keywords: Cistus incanus; CYSTUS052; Infection of the upper respiratory tract; Score of subjective symptoms