Silencing of Hepatitis A Virus Infection by Small Interfering RNAs

Abstract

Infection by hepatitis A virus (HAV) can cause acute hepatitis and, rarely, fulminant liver failure, in particular in patients chronically infected with hepatitis C virus. Based on our previous observation that small interfering RNAs (siRNAs) can silence translation and replication of the firefly luciferase-encoding HAV replicon, we now exploited this technology to demonstrate the effect of siRNAs on viral infection in Huh-7 cells. Freshly and persistently infected cells were transfected with siRNAs targeting various sites in the HAV nonstructural genes. Compared to a single application, consecutive siRNA transfections targeting multiple sequences in the viral genome resulted in a more efficient and sustained silencing effect than a single transfection. In most instances, multiple applications of a single siRNA led to the emergence of viral escape mutants with mutated target sites that rendered these genomes resistant to RNA interference (RNAi). Efficient and sustained suppression of the viral infectivity was achieved after consecutive applications of an siRNA targeting a computer-predicted hairpin structure. This siRNA holds promise as a therapeutic tool for severe courses of HAV infection. In addition, the results provide new insight into the structural bases for sequencespecific RNAi.Hepatitis A virus (HAV) has a single-stranded RNA genome of 7.5 kb with positive polarity. As a member of the picornavirus family, it is unique in its biological properties, in particular, in its highly protracted replicative cycle in cell cultures and inability to shut off the host cell metabolism. HAV causes a self-limiting infection of the liver with usually mild or no symptoms in young patients, whereas adult patients might suffer from severe symptoms. In immune-compromised or chronically infected patients, fulminant hepatic failure is often associated with a high mortality rate (15, 52). Albeit vaccines against hepatitis A virus can prevent the disease (3, 14, 31), they are futile for fulminant hepatitis. Therefore, therapeutic intervention strategies to prevent progression to a life-threatening liver disease are still in need. Given that the rapid degradation of the HAV genome can ameliorate viral infection and thus prevent fulminant failure, RNA interference (RNAi) based on sequence-specific genome degradation may present a novel and specific therapeutic approach in preventing severe disease progression.Initially demonstrated in Caenorabditis elegans and Drosophila melanogaster, RNAi is now recognized as a valuable tool to silence viral infections (reviewed in references 22, 48, and 50). Among the numerous viruses that have been successfully silenced by RNAi, there are five representatives of the picornavirus family: poliovirus (19,20), human rhinovirus (45), enterovirus 71 (37), foot-and-mouth disease virus (7, 9, 36), and coxsackievirus B3 (1, 40). Recently, we have shown that genome replication of an HAV replicon can be efficiently inhibited by small interfering RNAs (siRNAs) targeting either coding or noncoding regions...

Human Immunodeficiency Virus Type 1 Escape Is Restricted When Conserved Genome Sequences Are Targeted by RNA Interference

“…Aside from HIV-1, other human pathogens that have been targeted include poliovirus; hepatitis A, B, and C viruses; enteroviruses; coxsackievirus; rhinovirus; and influenza virus (3, 8, 10, 15, 17, 20-22, 25, 29, 30, 33, 40, 42, 43). Escape from transient siRNA treatment has been described for poliovirus (9,10), hepatitis C virus (42), and hepatitis A virus (20). These studies suggest that single point mutations can diminish or even abolish the RNAi effect.…”

“…These studies suggest that single point mutations can diminish or even abolish the RNAi effect. Repeated treatment with the same siRNA triggered the selection of multiple mutations in the viral target sequences, leading to increased levels of RNAi resistance (9,10,20,42).…”

Rna Interference as Antiviral Therapy: Dream or Reality?

“…Interestingly, viral escape to RNAi can also occur by a point mutation outside the target sequence of siRNA, if this mutation changes local RNA folding into a structure that reduces the accessibility of the target sequence (Westerhout et al 2005). Several viruses have been reported to escape suppression by effective siRNAs, which include HIV-1, HCV, HAV, peste des petits ruminants virus (PPRV) and Poliovirus (von Eije et al 2008;Wilson & Richardson 2005;Kusov et al 2006;Gitlin et al 2005). Studies of HIV-1 and HBV populations have shown that many resistant mutants may pre-exist before they have been exposed to inhibitors and the siRNAs may also exert selection pressure on these pre-existing resistant mutants (Najera et al 1995;Wu et al 2005).…”

RNA interference-based therapeutics for shrimp viral diseases

“…Usually, the viral structural proteins or polymerases are targeted for silencing. Promising results have been reported against HIV (Novina et al 2002), Hepatitis A (Kusov et al 2006) and B (Shlomai & Shaul 2003) in humans and foot-and-mouth-disease virus in cattle (Chen et al 2006a). Several RNAi-based drugs for human diseases are currently in the pre-clinical development stage and phase I or II clinical trials (www.…”