Genetic Evidence That Captured Retroviral Envelope syncytins Contribute to Myoblast Fusion and Muscle Sexual Dimorphism in Mice.

Syncytins are envelope genes from endogenous retroviruses, "captured" for a role in placentation. They mediate cell-cell fusion, resulting in the formation of a syncytium (the syncytiotrophoblast) at the fetomaternal interface. These genes have been found in all placental mammals in which they have been searched for. Cell-cell fusion is also pivotal for muscle fiber formation and repair, where the myotubes are formed from the fusion of mononucleated myoblasts into large multinucleated structures. Here we show, taking advantage of mice knocked out for syncytins, that these captured genes contribute to myoblast fusion, with a >20% reduction in muscle mass, mean muscle fiber area and number of nuclei per fiber in knocked out mice for one of the two murine syncytin genes. Remarkably, this reduction is only observed in males, which subsequently show muscle quantitative traits more similar to those of females. In addition, we show that syncytins also contribute to muscle repair after cardiotoxin-induced injury, with again a male-specific effect on the rate and extent of regeneration. Finally, ex vivo experiments carried out on murine myoblasts demonstrate the direct involvement of syncytins in fusion, with a >40% reduction in fusion index upon addition of siRNA against both syncytins. Importantly, similar effects are observed with primary myoblasts from sheep, dog and human, with a 20-40% reduction upon addition of siRNA against the corresponding syncytins. Altogether, these results show a direct contribution of the fusogenic syncytins to myogenesis, with a demonstrated male-dependence of the effect in mice, suggesting that these captured genes could be responsible for the muscle sexual dimorphism observed in placental mammals.

NGR-bearing human adenovirus type 5 infects cells in flotillin- or caveolin-mediated manner depending on the NGR insertion site.

Human adenoviruses represent attractive candidates for the design of cancer gene therapy vectors. Modification of adenovirus tropism by incorporating a targeting ligand into the adenovirus capsid proteins allows retargeting of adenovirus towards the cells of interest. Human adenovirus type 5 (HAdV-C5) bearing NGR containing peptide (CNGRCVSGCAGRC) inserted into the fiber (AdFNGR) or the hexon (AdHNGR) protein demonstrated an increased transduction of endothelial cells showing expression of aminopeptidase N, also known as CD13, and αvß3 integrin both present on tumor vasculature, indicating that NGR-bearing adenoviruses could be used as tools for anti-angiogenic cancer therapy. Here we investigated how AdFNGR and AdHNGR infect cells lacking HAdV-C5 primary receptor, coxsackie and adenovirus receptor, and we showed that both AFNGR and AdHNGR enter cells by dynamin- and lipid raft-mediated endocytosis, while clathrin is not required for endocytosis of these viruses. We present evidence that productive infection of both AdFNGR and AdHNGR involves lipid rafts, with usage of flotillin-mediated cell entry for AdFNGR and limited role of caveolin in AdHNGR transduction efficiency. Lipid rafts play important role in angiogenesis and process of metastasis. Therefore, the ability of AdFNGR and AdHNGR to use lipid raft-dependent endocytosis, involving respectively flotillin- or caveolin-mediated pathway, could give them an advantage in targeting tumor cells lacking HAdV-C5 primary receptor.

Humoral Responses Elicited by Adenovirus Displaying Epitopes Are Induced Independently of the Infection Process and Shaped by the Toll-Like Receptor/MyD88 Pathway.

The use of serotype 5 adenovirus (Ad)-derived vectors in vaccination is confronted to preexisting anti-Ad immunity. Epitope display on Ad capsid is currently being investigated as an alternative approach of vaccination. The present study seeks to better understand virus- and host-related factors controlling the efficacy of this new vaccination approach. In contrast to an Ad vector expressing ovalbumin as a transgene, Ad displaying an ovalbumin-derived B-cell epitope inserted into the fiber protein was able to elicit antibody responses in both Ad-naive and Ad-immune mice. Moreover, introduction of a set of mutations abrogating Ad interaction with its receptors did not modify the virus capacity to elicit a humoral response against the inserted epitope while reducing its capacity to mount antibody responses against the transgene product. Taken as a whole these data indicate that the efficacy of Ad displaying epitopes requires neither Ad binding to its receptors nor the infection process. In addition, the use of genetically deficient mice demonstrated that both toll-like receptor (TLR)/MyD88 and RIG-I/mitochondrial antiviral-signaling (MAVS) innate immunity pathways were dispensable to mount anti-epitope antibody responses. However, they also revealed that TLR/MyD88 pathway but not RIG-I/MAVS pathway controls the nature of antibodies directed against the displayed epitope.

Combined therapy of colon carcinomas with an oncolytic adenovirus and valproic acid.

The anti-tumor potential of oncolytic adenoviruses (CRAds) has been demonstrated in preclinical and clinical studies. While these agents failed to eradicate tumors when used as a monotherapy, they may be more effective if combined with conventional treatments such as radiotherapy or chemotherapy. This study seeks to evaluate the combination of a CRAd bearing a ∆24 deletion in E1A with valproic acid (VPA), a histone deacetylase inhibitor, for the treatment of human colon carcinomas. This combination led to a strong inhibition of cell growth both in vitro and in vivo compared to treatment with CRAd or VPA alone. This effect did not stem from a better CRAd replication and production in the presence of VPA. Inhibition of cell proliferation and cell death were induced by the combined treatment. Moreover, whereas cells treated only with CRAd displayed a polyploidy (> 4N population), this phenotype was increased in cells treated with both CRAd and VPA. In addition, the increase in polyploidy triggered by combined treatment with CRAd and VPA was associated with the enhancement of H2AX phosphorylation (γH2AX), a hallmark of DNA damage, but also with a decrease of several DNA repair proteins. Finally, viral replication (or E1A expression) was shown to play a key role in the observed effects since no enhancement of polyploidy nor increase in γH2AX were found following cell treatment with a replication-deficient Ad and VPA. Taken together, our results suggest that CRAd and VPA could be used in combination for the treatment of colon carcinomas.

Pseudotyping Serotype 5 Adenovirus with the Fiber from Other Serotypes Uncovers a Key Role of the Fiber Protein in Adenovirus 5-Induced Thrombocytopenia.

Adenovirus (Ad) infection in humans is associated with inflammatory responses and thrombocytopenia. Although several studies were conducted in mice models to understand molecular and cellular mechanisms of Ad-induced inflammatory responses, only few of them turned their interest toward the mechanisms of Ad-induced thrombocytopenia. Using different depletion methods, the present study ruled out any significant role of spleen, macrophages, and vitamin K-dependent factor in Ad-induced thrombocytopenia. Interestingly, mice displaying thrombocytopenia expressed high levels of cytokines/chemokines after Ad administration. Most importantly, pseudotyping adenovirus with the fiber protein from other serotypes was associated with reduction of both cytokine/chemokine production and thrombocytopenia. Altogether, our results suggest that capsid fiber protein (and more precisely its shaft) of Ad serotype 5 triggers the cytokine production that leads to Ad-induced thrombocytopenia.