The Interactomes of Influenza Virus NS1 and NS2 Proteins Identify New Host Factors and Provide Insights for ADAR1 Playing a Supportive Role in Virus Replication.

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IRGM Is a Common Target of RNA Viruses that Subvert the Autophagy Network

Autophagy is a conserved degradative pathway used as a host defense mechanism against intracellular pathogens. However, several viruses can evade or subvert autophagy to insure their own replication. Nevertheless, the molecular details of viral interaction with autophagy remain largely unknown. We have determined the ability of 83 proteins of several families of RNA viruses (Paramyxoviridae, Flaviviridae, Orthomyxoviridae, Retroviridae and Togaviridae), to interact with 44 human autophagy-associated proteins using yeast two-hybrid and bioinformatic analysis. We found that the autophagy network is highly targeted by RNA viruses. Although central to autophagy, targeted proteins have also a high number of connections with proteins of other cellular functions. Interestingly, immunity-associated GTPase family M (IRGM), the most targeted protein, was found to interact with the autophagy-associated proteins ATG5, ATG10, MAP1CL3C and SH3GLB1. Strikingly, reduction of IRGM expression using small interfering RNA impairs both Measles virus (MeV), Hepatitis C virus (HCV) and human immunodeficiency virus-1 (HIV-1)-induced autophagy and viral particle production. Moreover we found that the expression of IRGM-interacting MeV-C, HCV-NS3 or HIV-NEF proteins per se is sufficient to induce autophagy, through an IRGM dependent pathway. Our work reveals an unexpected role of IRGM in virus-induced autophagy and suggests that several different families of RNA viruses may use common strategies to manipulate autophagy to improve viral infectivity

Modulation of the type I interferon system by viruses : in particular by hepatitis C virus and influenza virus

Afin de se répliquer et de se propager efficacement, les virus ont développé de multiples stratégies leur permettant d’échapper au système de défense innée : le système IFN de type I. Ce travail de thèse a alors consisté à étudier les interactions entre protéines virales et protéines de ce système de défense afin de mieux comprendre les mécanismes de subversion virale et d’identifier d’éventuelles cibles cellulaires thérapeutiques. La reconstruction d’un réseau d’interactions entre ces protéines nous a permis d’identifier des stratégies différentielles de subversion pour 4 familles virales et de montrer un ciblage massif et significatif des protéines du système IFN de type I par les virus. Les protéines en interaction directe avec ces protéines sont également fortement touchées par les virus et sont de potentiels modulateurs du système IFN de type I. Parmi ces modulateurs, le processus biologique sur-représenté est le transport nucléocytoplasmique et la protéine KPNA1 impliquée dans ce processus a retenu notre attention. L’étude fonctionnelle de l’interaction entre la protéine KPNA1 et la protéine NS3 du VHC a montré que la protéine NS3 associée à son cofacteur NS4A inhibe partiellement la réponse IFN de type I en empêchant l’import nucléaire de STAT1. Ce phénotype pourrait résulter de la dégradation de KPNA1 par NS3/4A. Par ailleurs, l’identification de nouveaux inter-acteurs de la protéine NS1 du virus influenza par criblage double-hybride levure a révélé la protéine induite par les IFN de type I, ADAR1, comme partenaire de la protéine NS1 de multiples souches virales et nous avons montré qu'ADAR1 est un facteur pro-viral dont la fonction editing est activée par NS1To replicate and propagate efficiently, viruses have developed multiple strategies allowing them to escape the innatedefense system: the type I IFN system, This work of thesis then consisted in studying the interactions between viralproteins and proteins of this defence system in order to understand better the mechanisms of viral subversion andidentifY possible therapeutic cellular tatgets. The reconstruction of a network of interacting proteins involved in the typeI IFN system allowed us to identifY differentiai subversion strategies for 4 viral families and to show a massive andsignificant targeting of proteins of the type I IFN system by viruses. Proteins directly interacting with the type Iinterferon system network are also strongly targeted by viruses and are potential modulators of the type I IFN system.Among these modulators, the most tatgeted function conesponds to the transport of NLS-bearing substrates to thenucleus and the KPNAI protein involved in this process held our attention. The functional study of the interactionbetween KPNA1 and NS3 protein of the HCV showed that NS3 protein associated with its cofactor NS4A inhibitsprutially the type I IFN response by preventing the nuclear translocation of ST A Tl. This phenotype could result fromthe degradation of KPNAI by NS3/4A. Besides, the identification of new cellular prutners ofNS 1 prote in of influenzavirus by yeast two-hybrid screens revealed ADARI, an interferon-stimulated prote in, as partner of NS 1 of ali testedvirus strains and we showed that ADARI is an essential host factor for viral replication and its editing function isactivated by NS 1 protei

Improvement of the Dengue Virus (DENV) Nonhuman Primate Model via a Reverse Translational Approach Based on Dengue Vaccine Clinical Efficacy Data against DENV-2 and -4

ABSTRACT Recent data obtained with the live-attenuated tetravalent dengue CYD-TDV vaccine showed higher protective efficacy against dengue virus type 4 (DENV-4) than against DENV-2. In contrast, results from previous studies in nonhuman primates predicted comparable high levels of protection against each serotype. Maximum viral loads achieved in macaques by subcutaneous inoculation of DENV are generally much lower than those observed in naturally dengue virus-infected humans. This may contribute to an overestimation of vaccine efficacy. Using more-stringent DENV infection conditions consisting of the intravenous inoculation of 10 7 50% cell culture infectious doses (CCID 50 ) in CYD-TDV-vaccinated macaques, complete protection (i.e., undetectable viral RNA) was achieved in all 6 monkeys challenged with DENV-4 and in 6/18 of those challenged with DENV-2, including transiently positive animals. All other infected macaques (12/18) developed sustained DENV-2 RNAemia (defined as detection of viral RNA in serum samples) although 1 to 3 log 10 units below the levels achieved in control animals. Similar results were obtained with macaques immunized with either CYD-TDV or monovalent (MV) CYD-2. This suggests that partial protection against DENV-2 was mediated mainly by CYD-2 and not by the other CYDs. Postchallenge induction of strong anamnestic responses, suggesting efficient vaccine priming, likely contributed to the reduction of DENV-2 RNAemia. Finally, an inverse correlation between DENV RNA titers postchallenge and vaccine-induced homotypic neutralizing antibody titers prechallenge was found, emphasizing the key role of these antibodies in controlling DENV infection. Collectively, these data show better agreement with reported data on CYD-TDV clinical vaccine efficacy against DENV-2 and DENV-4. Despite inherent limitations of the nonhuman primate model, these results reinforce its value in assessing the efficacy of dengue vaccines. IMPORTANCE The nonhuman primate (NHP) model is the most widely recognized tool for assessing the protective activity of dengue vaccine candidates, based on the prevention of postinfection DENV viremia. However, its use has been questioned after the recent CYD vaccine phase III trials, in which moderate protective efficacy against DENV-2 was reported, despite full protection against DENV-2 viremia previously being demonstrated in CYD-vaccinated monkeys. Using a reverse translational approach, we show here that the NHP model can be improved to achieve DENV-2 protection levels that show better agreement with clinical efficacy. With this new model, we demonstrate that the injection of the CYD-2 component of the vaccine, in either a monovalent or a tetravalent formulation, is able to reduce DENV-2 viremia in all immunized animals, and we provide clear statistical evidence that DENV-2-neutralizing antibodies are able to reduce viremia in a dose-dependent manner. </jats:p

Immunogenicity, protective efficacy, and non-replicative status of the HSV-2 vaccine candidate HSV529 in mice and guinea pigs.

HSV-2 vaccine is needed to prevent genital disease, latent infection, and virus transmission. A replication-deficient mutant virus (dl5-29) has demonstrated promising efficacy in animal models of genital herpes. However, the immunogenicity, protective efficacy, and non-replicative status of the highly purified clinical vaccine candidate (HSV529) derived from dl5-29 have not been evaluated. Humoral and cellular immune responses were measured in mice and guinea pigs immunized with HSV529. Protection against acute and recurrent genital herpes, mortality, latent infection, and viral shedding after vaginal HSV-2 infection was determined in mice or in naïve and HSV-1 seropositive guinea pigs. HSV529 replication and pathogenicity were investigated in three sensitive models of virus replication: severe combined immunodeficient (SCID/Beige) mice inoculated by the intramuscular route, suckling mice inoculated by the intracranial route, and vaginally-inoculated guinea pigs. HSV529 immunization induced HSV-2-neutralizing antibody production in mice and guinea pigs. In mice, it induced production of specific HSV-2 antibodies and splenocytes secreting IFNγ or IL-5. Immunization effectively prevented HSV-2 infection in all three animal models by reducing mortality, acute genital disease severity and frequency, and viral shedding. It also reduced ganglionic viral latency and recurrent disease in naïve and HSV-1 seropositive guinea pigs. HSV529 replication/propagation was not detected in the muscles of SCID/Beige mice, in the brains of suckling mice, or in vaginal secretions of inoculated guinea pigs. These results confirm the non-replicative status, as well as its immunogenicity and efficacy in mice and guinea pigs, including HSV-1 seropositive guinea pigs. In mice, HSV529 produced Th1/Th2 characteristic immune response thought to be necessary for an effective vaccine. These results further support the clinical investigation of HSV529 in human subjects as a prophylactic vaccine

HSV529 immunization protects mice from the effects of lethal HSV-2 vaginal challenge.

<p>BALB/c mice were immunized with HSV529 (10⁶ CCID₅₀) or PBS by the i.m. route on days 0 and 21. On day 48, mice received medroxyprogesterone (2 mg, s.c.) to prevent reproductive cycling. The next day, mice were challenged with an intravaginal inoculation of HSV-2 (strain G; 10⁵ CCID₅₀). (A) Mean body weight change after HSV-2 challenge. (B) Mean vaginal lesion score after HSV-2 challenge. (C) Percent survival after HSV-2 challenge. (D) HSV-2 viral shedding after challenge. *Dead or euthanized animal. Error bars represent the standard error of the mean.</p

HSV529 does not propagate in the brains of 4−6-day-old suckling mice.

<p>Four- to 6-day-old sucking mice received an intracranial injection of vaccine buffer (gray squares), HSV529 (gray triangles, 5 x 10⁵ CCID₅₀), or wild-type (wt) HSV-2 186 syn+-1 (black circles, 10 CCID₅₀). Brains were collected on p.i. days 0 (4 hours p.i.), 2, 4, 6, and 14, and from animals that died during the experiment. The titer of each animal is represented by an individual symbol and the mean titer is represented by a horizontal bar. Virus titers were determined on AV529-19 cells.</p

HSV529 immunization protects HSV-1-primed guinea pigs from effects of HSV-2 vaginal challenge.

<p>Guinea pigs were inoculated with HSV-1 (KOS strain; 10⁶ CCID₅₀; n = 30) or PBS (n = 18) by the intranasal route on day 0. All animal inoculated with HSV-1 were positive for HSV-1 at week 5. At weeks 7 and 10, animals inoculated with HSV-1 were immunized with HSV529 (10⁶ CCID₅₀; n = 15) or PBS (n = 14) by the i.m. route. At week 14, all animals except 3 PBS controls were challenged with an intravaginal inoculation of HSV-2 (G strain, 2 x 10⁶ CCID₅₀). (A) Mean body weight change after HSV-2 challenge. (B) Mean vaginal lesion score after HSV-2 challenge. (C) Percent survival after HSV-2 challenge. (D) HSV-2 viral shedding after challenge. (E) Cumulative number of recurrent lesions per animal. *Dead or euthanized animal. Error bars represent standard error of the mean.</p

Mice immunized with HSV529 produce HSV-2-specific IgG1 and IgG2a antibodies, neutralizing antibodies, and HSV-2-specific splenic lymphocytes secreting IFNγ and IL-5.

<p>BALB/c mice (n = 10/group) were immunized with HSV529 (10⁴ CCID₅₀, 10⁵ CCID₅₀, or 10⁶ CCID₅₀) or PBS by the i.m. route on days 0 and 21. Sera were collected on days 21 (D21; n = 10) and 41 (D41; n = 5). (A) HSV-2-specific IgG1 and IgG2a antibody titers in the sera were determined by ELISA using a lysate prepared from HSV-2-infected Vero cells and secondary antibodies specific for mouse IgG1 and IgG2a. (B) HSV-2 neutralizing antibodies in the sera were measured by preincubating dilutions of heat-inactivated sera with 100 CCID₅₀ of live HSV-2 (strain G) virus for 1 hour prior to infection of Vero cell cultures. Infected cells were detected with anti-HSV glycoprotein D antibodies. The serum dilution that neutralized 50% of the virus (SN₅₀) was determined by plotting the neutralization activity versus the serum dilutions. Splenic lymphocytes secreting IFNγ (C) or IL-5 (D) in response to <i>ex vivo</i> stimulation with heat-inactivated HSV-2 (strain G) were counted using an ELISPOT assay. Error bars represent standard error of the mean.</p

Guinea pigs immunized with HSV529 produce antibodies that neutralize HSV-2 infection <i>in vitro</i>.

<p>Guinea pigs were immunized with HSV529 (10⁴ CCID₅₀, 10⁵ CCID₅₀, or 10⁶ CCID₅₀) by the intramuscular (IM; n = 5 each) or subcutaneous (SC; n = 5 each) route or with PBS (n = 3) by the intramuscular route on days 0 (D0) and 21 (D21). Sera were collected from all animals on days 21 and 29 (D29) and measured for HSV-2 neutralizing activity by preincubating dilutions of heat-inactivated sera with 100 CCID₅₀ of live HSV-2 virus for 1 hour prior to infection of Vero cell cultures. Infected cells were detected with anti-HSV glycoprotein D antibodies. The serum dilution that neutralized 50% of the virus (SN50) was determined by plotting the neutralization activity versus the serum dilutions. Error bars represent standard error of the mean.</p