SUMOylation promotes PML degradation during encephalomyocarditis virus infection.

The promyelocytic leukemia (PML) protein is expressed in the diffuse nuclear fraction of the nucleoplasm and in matrix-associated structures, known as nuclear bodies (NBs). PML NB formation requires the covalent modification of PML to SUMO. The noncovalent interactions of SUMO with PML based on the identification of a SUMO-interacting motif within PML seem to be required for further recruitment within PML NBs of SUMOylated proteins. RNA viruses whose replication takes place in the cytoplasm and is inhibited by PML have developed various strategies to counteract the antiviral defense mediated by PML NBs. We show here that primary fibroblasts derived from PML knockout mice are more sensitive to infection with encephalomyocarditis virus (EMCV), suggesting that the absence of PML results in an increase in EMCV replication. Also, we found that EMCV induces a decrease in PML protein levels both in interferon-treated cells and in PMLIII-expressing cells. Reduction of PML was carried out by the EMCV 3C protease. Indeed, at early times postinfection, EMCV induced PML transfer from the nucleoplasm to the nuclear matrix and PML conjugation to SUMO-1, SUMO-2, and SUMO-3, leading to an increase in PML body size where the viral protease 3C and the proteasome component were found colocalizing with PML within the NBs. This process was followed by PML degradation occurring in a proteasome- and SUMO-dependent manner and did not involve the SUMO-interacting motif of PML. Together, these findings reveal a new mechanism evolved by EMCV to antagonize the PML pathway in the interferon-induced antiviral defense.

Delivery of mengovirus-derived RNA replicons into tumoural liver enhances the anti-tumour efficacy of a peripheral peptide-based vaccine.

Hepatocellular carcinoma is a deadly cancer with growing incidence for which immunotherapy is one of the most promising therapeutic approach. Peptide-based vaccines designed to induce strong, sustained CD8+ T cell responses are effective in animal models and cancer patients. We demonstrated the efficacy of curative peptide-based immunisation against a unique epitope of SV40 tumour antigen, through the induction of a strong CD8+ T cell-specific response, in our liver tumour model. However, as in human clinical trials, most tumour antigen epitopes did not induce a therapeutic effect, despite inducing strong CD8+ T cell responses. We therefore modified the tumour environment to enhance peptide-based vaccine efficacy by delivering mengovirus (MV)-derived RNA autoreplicating sequences (MV-RNA replicons) into the liver. The injection of replication-competent RNA replicons into the liver converted partial tumour regression into tumour eradication, whereas non-replicating RNA had no such effect. Replicating RNA replicon injection induced local recruitment of innate immunity effectors (NK and NKT) to the tumour and did not affect specific CD8+ T cell populations or other myelolymphoid subsets. The local delivery of such RNA replicons into tumour stroma is therefore a promising strategy complementary to the use of peripheral peptide-based vaccines for treating liver tumours.

Recombinant influenza A viruses harboring optimized dicistronic NA segment with an extended native 5' terminal sequence: induction of heterospecific B and T cell responses in mice.

We generated novel recombinant influenza A viruses (vNA38) harboring dicistronic NA segments with an extended native 5' terminal sequence of 70 nucleotides comprised of the last 42 nucleotides of the NA ORF and the 5' noncoding region (5' NCR). vNA38 viruses replicated stably and more efficiently than vNA35 viruses with a dicistronic NA segment comprised of the native 5' NCR only, that we described previously (Vieira Machado, A., Naffakh, N., van der Werf, S., Escriou, N., 2003. Expression of a foreign gene by stable recombinant influenza viruses harboring a dicistronic genomic segment with an internal promoter. Virology 313, 235-249). In addition, vNA38 viruses drove the expression of higher levels of encoded heterologous proteins than corresponding vNA35 viruses, both in cell culture and in the pulmonary tissue of infected mice. These data demonstrate that a sequence overlapping 5' coding and noncoding regions of the NA segment determines efficient replication and/or propagation of the vRNA. Intranasal immunization of mice with live vNA38 viruses induced B and T cell responses specific for the heterologous protein expressed, establishing the usefulness of such recombinant influenza viruses with a dicistronic segment for the development of live bivalent vaccines.

Expression of a membrane-anchored glycoprotein, the influenza virus hemagglutinin, by dicistronic replicons derived from the poliovirus genome.

Mono- and dicistronic poliovirus replicons were constructed to express the influenza virus hemagglutinin, retaining its signal peptide and transmembrane region. Picornavirus genomes do not normally encode glycoproteins, and only the dicistronic replicon, in which the foreign and poliovirus sequences were separated by the encephalomyocarditis virus internal ribosomal entry site, replicated and expressed glycosylated hemagglutinin.

Naked RNA immunization with replicons derived from poliovirus and Semliki Forest virus genomes for the generation of a cytotoxic T cell response against the influenza A virus nucleoprotein.

The potential of RNA-based vaccines was evaluated for the generation of a protective immune response in the mouse model of influenza type A virus infection using the internal nucleoprotein (NP) as antigen. This antigen is of particular interest, since it has the potential to elicit protective cytotoxic T lymphocytes (CTL) against heterologous strains of influenza A virus. In view of the short half-life of RNA, self-replicating RNAs or replicons of the positive-stranded genomes of Semliki Forest virus (SFV) and poliovirus were engineered to synthesize the influenza A virus NP in place of their structural proteins. NP expression was demonstrated by immunoprecipitation after transfection of cells with RNA from the SFV (rSFV-NP) and poliovirus (rDeltaP1-E-NP) genome-derived replicons transcribed in vitro. C57BL/6 mice were injected intramuscularly with these synthetic RNAs in naked form. Both replicons, rSFV-NP and rDeltaP1-E-NP, induced antibodies against the influenza virus NP, but only mice immunized with the rSFV-NP replicon developed a CTL response against the immunodominant H-2D(b) epitope NP366. Finally, the protective potential of the CTL response induced by immunization of mice with rSFV-NP RNA was demonstrated by the reduction of virus load in the lungs after challenge infection with mouse-adapted influenza A/PR/8/34 virus and was comparable to the protective potential of the response induced by plasmid DNA immunization. These results demonstrate that naked RNA immunization with self-replicating molecules can effectively induce both humoral and cellular immune responses and constitutes an alternative strategy to DNA immunization.