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Antiviral Research (v.95, #1)

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

Prelim II: Editorial Board (pp. ii).

Serotype specificity of recombinant fusion protein containing domain III and capsid protein of dengue virus 2 by Alienys Izquierdo; Iris Valdés; Lázaro Gil; Lisset Hermida; Sheila Gutiérrez; Angélica García; Lidice Bernardo; Alekis Pavón; Gerardo Guillén; María G. Guzmán (pp. 1-8).

► We determined the serotype specificity of domain III-capsid recombinant protein. ► The antigenicity and the cross reactivity of immune response in mice was studied. ► The response is specific, so ADE and cross reactive T cells response are avoided. ► We conclude that this protein is a candidate potentially safe.Recombinant fusion protein containing domain III of the dengue envelope protein fused to capsid protein from dengue 2 virus was immunogenic and conferred protection in mice against lethal challenge in previously report. Here, the antigenic specificity of this recombinant protein using anti-dengue antibodies from mice and humans and the cross-reactive humoral and cellular response induced in immunized mice were evaluated. The homologous anti-dengue antibodies showed a higher reactivity to the recombinant protein compared to the wide cross-reactivity observed for viral antigen as determined by ELISA. The IgG anti-dengue and functional antibodies, induced by the recombinant proteins in mice, were highly serotype specific by ELISA, hemaglutination inhibition and plaque reduction neutralizing tests. Accordingly, the cellular immune response determined by the IFNγ and TNFα secretion, was serotype specific. The specificity of serotype associated to this recombinant protein in addition to its high antigenicity, immunogenicity and protecting capacity suggest its advantage as a possible functional and safe vaccine candidate against dengue in a future tetravalent formulation.

Keywords: Dengue; Recombinant protein; Serotype specificity; Vaccine; Domain III; Capsid

In-vivo selection of the mutation F121Y in a patient failing raltegravir containing salvage regimen by Jaqueline Souza Cavalcanti; André Minhoto Lança; João Leandro de Paula Ferreira; Margareth da Eira; Daniel Soares de Souza Dantas; Luís Fernando de Macedo Brígido (pp. 9-11).

► Three major pathways, G148H/R/K, N155H and Y143C/H/R are associated to resistance to raltegravir. ► The substitution F121Y is associated to resistance to raltegravir in vitro. ► The selection of F121Y in an patient failing raltegravir was followed by the emergence of Y143R. ► Changing regimen upon the identification of F121Y might avoid the evolution of raltegravir resistance.Raltegravir is an integrase inhibitor (INI) licensed for clinical use and other INI are in advanced stage of development. Different resistance mutations in HIV integrase from patients using these antiretroviral drugs have been described and G148H/R/K, N155H and less frequently Y143C/H/R are considered major resistant mutations to raltegravir. Both Stanford Database and Geno2Pheno list F121Y as conferring intermediate resistance “in vitro” both to raltegravir and elvitegravir. We report for the first time the “ in vivo” selection F121Y and evolution to Y143R in a 31years old male clade B HIV-1 infected patient failing a raltegravir-containing salvage regimen. Plasma samples nine months prior to raltegravir (RAL-Naïve) and at weeks 32, 40 and 88 after RAL-containing regimen were analyzed. Antiretroviral susceptibility was evaluated at Stanford and Geno2Pheno from sequences obtained with RT-PCR. After a Viral load at week 12 below 50 copies/mL, viremia raised at week 20 to 4.5log10. The emergence of F121Y was observed at week 32 and 40, alongside with L74I, T97A, Q137H and V151I. At week 88 F121Y was no longer detected, L74I and T97A were maintained and Y143R emerged. F121Y might be an alternative pathway to Y143R. Changing of RAL-containing regimen upon the identification of F121Y might avoid the evolution of raltegravir resistance.

Keywords: HIV-1; Brazil; Integrase; Raltegravir; Genetic resistance

A cell free protein fragment complementation assay for monitoring the core interaction of the human cytomegalovirus nuclear egress complex by Margit Schnee; Felicia M. Wagner; Ulrich H. Koszinowski; Zsolt Ruzsics (pp. 12-18).

► A new β-lactamase based cell free protein fragment complementation assay (iPCA) was established. ► The iPCA showed that the core interaction of the nuclear egress complex of HCMV relies on a linear motif. ► The new pUL50–pUL53 interaction assay allowed identification of peptide inhibitors.Certain viral protein–protein interactions provide attractive targets for antiviral drug development. Recently, we described a β-lactamase based protein fragment complementation assay (PCA) to study the core interaction of the nuclear egress complex (NEC) of different herpesviruses in cells. Now, to have a cell free assay for inhibitor screens, we expressed split β-lactamase tagged interaction domains of the viral pUL50 and pUL53 proteins representing the NEC of human cytomegalovirus (HCMV) in bacteria. After validation and basic characterization of this NEC–PCA, we tested peptide inhibitors of the pUL50–pUL53 complex. We show that peptides resembling sequences of the first conserved region of pUL53 can inhibit the NEC–PCA. This, on one hand, indicated that the core interaction in the HCMV NEC is mediated by a linear motif. On the other hand it proved that this new pUL50–pUL53 interaction assay allows a simple cell free test for small molecular inhibitors.

Keywords: Abbreviations; HCMV; human cytomegalovirus; NEC; nuclear egress complex; PPI; Protein–protein interactions; CR1-4; conserved region one to four; Bla; β-lactamase; PCA; protein fragment complementation assayProtein–protein interaction; Herpesviruses; Viral morphogenesis; Primary envelopment; Antiviral agents

Brain injury caused by HIV protease inhibitors: Role of lipodystrophy and insulin resistance by Sunita Gupta; Alecia G. Knight; Boriss Y. Losso; Donald K. Ingram; Jeffrey N. Keller; Annadora J. Bruce-Keller (pp. 19-29).

► Lopinavir/ritonavir causes lipodystrophy and insulin resistance in mice. ► Lopinavir/ritonavir impairs cognition and increases depressive behavior. ► Lopinavir/ritonavir induces cerebrovascular injury and synapse loss. ► Cognitive impairment correlates with lipodystrophy and insulin resistance.HIV-associated neurocognitive disorders (HAND) remain prevalent even with widespread use of combination antiretroviral therapy (ART), suggesting a potential role for co-morbidities in neurologic decline. Indeed, it is well established that ART drugs, particularly HIV protease inhibitors, can induce hyperlipidemia, lipodystrophy, and insulin resistance; all of which are associated with neurologic impairment. This study was designed to determine how metabolic dysfunction might contribute to cognitive impairment and to reveal specific metabolic co-morbidities that could be targeted to preserve brain function. Adult male C57BL/6 mice were thus treated with clinically relevant doses of lopinavir/ritonavir for 4weeks, and subjected to thorough metabolic, neurobehavioral, and biochemical analyses. Data show that lopinavir/ritonavir resulted in manifestations of lipodystrophy, insulin resistance, and hyperlipidemia. Evaluation of neurologic function revealed cognitive impairment and increased learned helplessness, but not motor impairment following treatment with lopinavir/ritonavir. Further analyses revealed a significant linear relationship between cognitive performance and specific markers of lipodystrophy and insulin resistance. Finally, analysis of brain injury indicated that lopinavir/ritonavir treatment resulted in cerebrovascular injury associated with decreased synaptic markers and increased inflammation, and that the cerebral cortex was more vulnerable than the cerebellum or hippocampus. Collectively, these data reveal an intimate link between metabolic co-morbidities and cognitive impairment, and suggest that remediation of selective aspects of metabolic syndrome could potentially reduce the prevalence or severity HIV-associated neurocognitive disorders.

Keywords: HAND; HIV co-morbidities; HIV protease inhibitors; Insulin resistance; Lipodystrophy; Metabolic syndrome

Inhibition of herpes simplex virus infection by oligomeric stilbenoids through ROS generation by Xiaoqing Chen; Haishi Qiao; Taixiang Liu; Ziying Yang; Lanfang Xu; Yunxia Xu; Hui Ming Ge; Ren-Xiang Tan; Erguang Li (pp. 30-36).

► Stilbenoid polyphenols inhibit HSV-2 infection at micromolar concentrations. ► Those compounds inhibit viral infection through ROS generation. ► Trimeric and tetrameric stilbenoids exhibit strongest antiviral activity.Stilbenoids including resveratrol contain the basic structural unit of 1,2-diphenylethylene. Naturally occurring stilbenoids have broad structural features due to oligomerization and modifications and some have demonstrated potent biological activities. In an effort to identify bioactive stilbenoids, we screened a group of dimeric and oligomeric stilbenoids against HSV-1 and HSV-2 infection. Several trimeric and tetrameric derivatives showed anti-herpetic activity at single digit micromolar concentrations. HSV-1 and HSV-2 replication requires for NF-κB and MAPK activation. The compounds showed no inhibitory activity against NF-κB and Erk/MAPK activation, instead those compounds promoted rapid and transient release of reactive oxygen species (ROS). Addition of N-acetylcysteine (NAC), a scavenger of ROS, reversed the inhibitory effect of those compounds against HSV replication. In addition to the identification of resveratrol derivatives with potent anti-HSV activity, our results uncover a mechanism of polyphenol-mediated anti-HSV response, linking anti-herpetic activity of oligomeric stilbenoids to innate immunity.

Keywords: Herpes simplex virus; Resveratrol; Stilbenoid; Reactive oxygen species

In vitro antiviral activity of dehydroepiandrosterone, 17 synthetic analogs and ERK modulators against herpes simplex virus type 1 by Nicolás I. Torres; Viviana Castilla; Andrea C. Bruttomesso; Javier Eiras; Lydia R. Galagovsky; Mónica B. Wachsman (pp. 37-48).

► DHEA, EA, three synthetic EA analogs and two synthetic DHEA analogs rendered antiherpetic activity. ► Raf/MEK/ERK signaling pathway modulators can inhibit or enhance viral multiplication. ► DHEA and the synthetic analog E2 may block HSV-1 particle formation and release. ► DHEA’s antiherpetic activity is not due to its Raf/MEK/ERK pathway modulator activity.In the present study the in vitro antiviral activity of dehydroepiandrosterone (DHEA) and 17 synthetic derivatives against herpes simplex type 1 (HSV-1) was determined. DHEA, epiandrosterone (EA), two synthetic DHEA analogs and three synthetic EA analogs showed a selective inhibitory effect on HSV in vitro multiplication. DHEA and E2, a synthetic derivative of EA, were not found to be virucidal to cell-free HSV-1 and did not impair virus adsorption or penetration. We determined that treatment with both compounds decreased viral protein synthesis. Moreover, inhibitory effect of DHEA and E2 on extracellular viral titer was stronger than the inhibition found on total viral infectivity, suggesting that the antiherpetic activity of these compounds may also be in part due to an inhibition in virus formation and release.Since DHEA is a known Raf/MEK/ERK signaling pathway activator, we studied the role of this pathway on HSV-1 infection. ERK1/2 phosphorylation was stimulated in HSV-1 infected cultures. UO126, a Raf/MEK/ERK signaling pathway inhibitor, impaired viral multiplication, while anisomycin, an activator of this pathway, enhanced it.Treatment with DHEA 6h before infection enhanced HSV-1 multiplication. On the contrary, pre-treatment with E2, which does not modulate Raf/MEK/ERK signaling pathway, did not produce an increase of viral replication. Taking together these results, the antiviral activity of DHEA seems to occur via a mechanism independent of its ability to modulate ERK phosphorylation.

Keywords: Antiviral; DHEA; DHEA analogs; Herpes simplex virus; ERK

Dasatinib therapy results in decreased B cell proliferation, splenomegaly, and tumor growth in a murine model of lymphoma expressing Myc and Epstein-Barr virus LMP2A by Jamie L. Dargart; Kamonwan Fish; Leo I. Gordon; Richard Longnecker; Osman Cen (pp. 49-56).

► Dasatinib inhibits B-cell colony formation by bone marrow cells from Tg-LMP2A mice. ► Dasatinib specifically decreases the spleen size of LMP2A-transgenic mice. ► Dasatinib specifically inhibits Lyn phosphorylation in LMP2A/MYC lymphomas. ► Dasatinib inhibits splenomegaly and lymphoma in LMP2A/MYC double transgenic mice.Epstein-Barr virus (EBV) infection and latency has been associated with malignant diseases including nasopharyngeal carcinoma, Hodgkin lymphoma, Burkitt lymphoma, and immune deficiency associated lymphoproliferative diseases. EBV-encoded latent membrane protein 2A (LMP2A) recruits Lyn and Syk kinases via its SH2-domain binding motifs, and modifies their signaling pathways. LMP2A transgenic mice develop hyperproliferative bone marrow B cells and immature peripheral B cells through modulation of Lyn kinase signaling. LMP2A/λ-MYC double transgenic mice develop splenomegaly and cervical lymphomas starting at 8weeks of age. We reasoned that targeting Lyn in LMP2A-expressing B cells with dasatinib would provide a therapeutic option for EBV-associated malignancies. Here, we show that dasatinib inhibits B cell colony formation by LMP2A transgenic bone marrow cells, and reverses splenomegaly and tumor growth in both a pre-tumor and a syngeneic tumor transfer model of EBV-associated Burkitt lymphoma. Our data support the idea that dasatinib may prove to be an effective therapeutic molecule for the treatment of EBV-associated malignancies.

Keywords: Abbreviations; BCR; B cell receptor; Das; dasatinib, a small molecule inhibitor of Src and Abl kinases; EBV; Epstein-Barr virus; λ-MYC; transgenic mice expressing MYC under the regulatory DNA sequence of immunoglobulin λ gene; LMP2A; EBV-encoded latent membrane protein 2A; PTLD; post-transplant lymphoproliferative disorders; Rag1 KO; recombinase activating gene-1 knockout mice; Tg6, TgE; two independently derived transgenic mouse lines expressing LMP2A in B cellsBurkitt lymphoma; Dasatinib; Epstein-Barr virus (EBV); Latent membrane protein 2A (LMP2A); Lyn; Post-transplant lymphoproliferative diseases (PTLD)

IFN-λ inhibits HIV-1 integration and post-transcriptional events in vitro, but there is only limited in vivo repression of viral production by Ren-Rong Tian; Hong-Xiong Guo; Ji-Fu Wei; Chuan-Kun Yang; Shao-Heng He; Jian-Hua Wang (pp. 57-65).

► Lambda interferons suppress HIV-1 integration and post-transcriptional events in vitro. ► IL-29 negatively correlates with IFN-γ and positively correlates with IL-10 in vivo. ► Lambda interferons show limited in vivo repression of viral production.The lambda interferons (IL-28a, 28b, and IL-29) inhibit the replication of many viruses, but their role in the inhibition of HIV-1 infection remains unclear. During this study, we monitored IL-29 production in HIV-1 infected individuals and analyzed the in vitro and in vivo inhibition of HIV-1 production. Prior treatment with IL-28a or IL-29 induced an antiviral state in cultured primary T-cells, which suppressed HIV-1 integration and post-transcriptional events. The antiviral factors MxA, OAS, and PKR were up-regulated. In HIV-1 infected patients, IL-29 level was increased along with the depletion of CD4⁺ T-cells in peripheral blood, while the elevated IL-29 did not show a significantly negative correlation with viral load. Further analysis of HIV-1 infected individuals showed that IL-29 was positively correlated with IFN-β and anti-inflammatory cytokine IL-10, and was negatively correlated with IFN-γ, which might suggest that IFN-λ participates in modulating antiviral immune responses during HIV-1 infection in vivo. Together, although IFN-λ impeded HIV-1 infection of T-cells in vitro, IFN-λ showed only limited in vivo repression of viral production. The modulation of IFN-λ on inflammatory factors might be worthy for further concentrating on for better understanding the host immune response during HIV-1 infection.

Keywords: HIV-1; Lambda interferon; Antiviral factor

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Antiviral Research (v.95, #1)