SignificanceThe clustered, regularly interspaced, short palindromic repeats associated endonuclease, Cas9, has quickly become a revolutionary tool in genome engineering. Utilizing small guiding RNAs, Cas9 can be targeted to specific DNA sequences of interest, where it catalyzes DNA cleavage. We now demonstrate that Cas9 from the Gram-negative bacterium Francisella novicida (FnCas9) can be reprogrammed to target a specific RNA substrate, the genome of the +ssRNA virus, hepatitis C virus, in eukaryotic cells. Further, this targeting results in inhibition of viral protein production. Overall, programmable Cas9-mediated viral RNA targeting likely represents one of myriad potential applications of FnCas9 in RNA targeting in eukaryotic cells.

Summary Resolution of acute and chronic viral infections requires activation of innate cells to initiate and maintain adaptive immune responses. Here we report that infection with acute Armstrong (ARM) or chronic Clone 13 (C13) strains of lymphocytic choriomeningitis virus (LCMV) led to two distinct phases of innate immune response. During the first 72hr of infection, dendritic cells upregulated activation markers, and stimulated anti-viral CD8+ T cells, independent of viral strain. Seven days after infection, there was an increase in Ly6Chi monocytic and Gr-1hi neutrophilic cells in lymphoid organs and blood. This expansion in cell numbers was enhanced and sustained in C13 infection, whereas it occurred only transiently with ARM infection. These cells resembled myeloid-derived suppressor cells, and potently suppressed T cell proliferation. The reduction of monocytic cells in Ccr2−/− mice or after Gr-1 antibody depletion enhanced anti-viral T cell function. Thus, innate cells have an important immunomodulatory role throughout chronic infection.

Globally, about 1% of pregnant women are persistently infected with the hepatitis C virus (HCV)1. Vertical transmission occurs in 3–5% of cases2 and accounts for most new childhood HCV infections1,3. HCV-specific CD8+ cytotoxic T-lymphocytes (CTLs) play a vital role in the clearance of acute infections4–6, but in the 60–80% of infections that persist these cells become functionally exhausted or select mutant viruses that escape T-cell recognition7–9. Increased HCV replication during pregnancy10,11 suggests that maternofetal immune tolerance mechanisms12 may further impair HCV-specific CTLs, limiting their selection pressure on persistent viruses. To assess this possibility, we characterized the circulating viral quasispecies during and after consecutive pregnancies. This revealed a loss of some escape mutations in class I epitopes in pregnancy associated with emergence of more fit viruses13. CTL selection pressure was reimposed after childbirth, when escape mutations in these epitopes again predominated in the quasispecies and viral load dropped sharply14. Importantly, viruses transmitted perinatally were those with enhanced fitness due to reversion of escape mutations. Our findings indicate that immunoregulatory changes of pregnancy reduce CTL selection pressure on HCV class I epitopes, thereby facilitating vertical transmission of viruses with optimized replicative fitness.

Clustered, regularly interspaced, short palindromic repeats - CRISPR associated (CRISPR-Cas) systems are sequence specific RNA-directed endonuclease complexes that bind and cleave nucleic acids. These systems evolved within prokaryotes as adaptive immune defenses to target and degrade nucleic acids derived from bacteriophages and other foreign genetic elements. The antiviral function of these systems has now been exploited to combat eukaryotic viruses throughout the viral life cycle. Here we discuss current advances in CRISPR-Cas9 technology as a eukaryotic antiviral defense.