Hepatitis C virus (HCV), a member of the Flaviviridae family, is a single-stranded positive-sense RNA virus that infects >170 million people worldwide and causes acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Despite its ability to block the innate host response in infected hepatocyte cell lines in vitro, HCV induces a strong type 1 interferon (IFN) response in the infected liver. The source of IFN in vivo and how it is induced are currently undefined. Here we report that HCV-infected cells trigger a robust IFN response in plasmacytoid dendritic cells (pDCs) by a mechanism that requires active viral replication, direct cell-cell contact, and Toll-like receptor 7 signaling, and we show that the activated pDC supernatant inhibits HCV infection in an IFN receptor-dependent manner. Importantly, the same events are triggered by HCV subgenomic replicon cells but not by free virus particles, suggesting the existence of a novel cell-cell RNA transfer process whereby HCV-infected cells can activate pDCs to produce IFN without infecting them. These results may explain how HCV induces IFN production in the liver, and they reveal a heretofore unsuspected aspect of the innate host response to viruses that can subvert the classical sensing machinery in the cells they infect, and do not infect or directly activate pDCs.

Stress granules (SGs) are cytoplasmic structures that are induced in response to environmental stress, including viral infections. Here we report that hepatitis C virus (HCV) triggers the appearance of SGs in a PKR- and interferon (IFN)-dependent manner. Moreover, we show an inverse correlation between the presence of stress granules and the induction of IFN-stimulated proteins, i.e., MxA and USP18, in HCV-infected cells despite high-level expression of the corresponding MxA and USP18 mRNAs, suggesting that interferon-stimulated gene translation is inhibited in stress granule-containing HCV-infected cells. Finally, in short hairpin RNA (shRNA) knockdown experiments, we found that the stress granule proteins T-cell-restricted intracellular antigen 1 (TIA-1), TIA1-related protein (TIAR), and RasGAP-SH3 domain binding protein 1 (G3BP1) are required for efficient HCV RNA and protein accumulation at early time points in the infection and that G3BP1 and TIA-1 are required for intracellular and extracellular infectious virus production late in the infection, suggesting that they are required for virus assembly. In contrast, TIAR downregulation decreases extracellular infectious virus titers with little effect on intracellular RNA content or infectivity late in the infection, suggesting that it is required for infectious particle release. Collectively, these results illustrate that HCV exploits the stress granule machinery at least two ways: by inducing the formation of SGs by triggering PKR phosphorylation, thereby downregulating the translation of antiviral interferon-stimulated genes, and by co-opting SG proteins for its replication, assembly, and egress.

Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, are urgently needed. We developed two COVID-19 vaccines based on modified vaccinia virus Ankara (MVA) vectors expressing the entire SARS-CoV-2 spike (S) protein (MVA-CoV2-S); their immunogenicity was evaluated in mice using DNA/MVA or MVA/MVA prime/boost immunizations. Both vaccines induced robust, broad, and polyfunctional S-specific CD4⁺ (mainly Th1) and CD8⁺ T-cell responses, with a T effector memory phenotype. DNA/MVA immunizations elicited higher T-cell responses. All vaccine regimens triggered high titers of IgG antibodies specific for the S, as well as for the receptor-binding domain; the predominance of the IgG2c isotype was indicative of Th1 immunity. Notably, serum samples from vaccinated mice neutralized SARS-CoV-2 in cell cultures, and those from MVA/MVA immunizations showed a higher neutralizing capacity. Remarkably, one or two doses of MVA-CoV2-S protect humanized K18-hACE2 mice from a lethal dose of SARS-CoV-2. In addition, two doses of MVA-CoV2-S confer full inhibition of virus replication in the lungs. These results demonstrate the robust immunogenicity and full efficacy of MVA-based COVID-19 vaccines in animal models and support its translation to the clinic. IMPORTANCE The continuous dissemination of the novel emerging SARS-CoV-2 virus, with more than 78 million infected cases worldwide and higher than 1,700,000 deaths as of 23 December 2020, highlights the urgent need for the development of novel vaccines against COVID-19. With this aim, we have developed novel vaccine candidates based on the poxvirus modified vaccinia virus Ankara (MVA) strain expressing the full-length SARS-CoV-2 spike (S) protein, and we have evaluated their immunogenicity in mice using DNA/MVA or MVA/MVA prime/boost immunization protocols. The results showed the induction of a potent S-specific T-cell response and high titers of neutralizing antibodies. Remarkably, humanized K18-hACE2 mice immunized with one or two doses of the MVA-based vaccine were 100% protected from SARS-CoV-2 lethality. Moreover, two doses of the vaccine prevented virus replication in lungs. Our findings prove the robust immunogenicity and efficacy of MVA-based COVID-19 vaccines in animal models and support its translation to the clinic.