Replication and in vivo repair of the hepatitis A virus genome lacking the poly(A) tail

Kusov, Yuri; Gosert, Rainer; Gauss‐Müller, Verena

doi:10.1099/vir.0.80644-0

Cited by 20 publications

(18 citation statements)

References 26 publications

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“…The reduction of the infectious titer was in good agreement with the loss of HAV genome copies detected by real-time RT-PCR, indicating that siRNA genome degradation was more efficient after repeated siRNA treatments compared to a single transfection (not shown). Taken together, the results suggest that HAV RNAi was sustained after consecutive siRNA transfection and depended on the viral replication rate, which was influenced by the host cells (35). Substantially stronger inhibition of the acute HAV infection was achieved in human hepatoma cells than in BS-C-1 cells, and viral replication was significantly more suppressed when siRNAs targeting different HAV genomic regions were consecutively applied.…”

Section: Resultsmentioning

confidence: 61%

“…HAV replication is less efficient in BS-C-1 cells than in liver cells, which are the natural host for HAV replication in vivo and produce larger amounts of infectious virus (35,47) or viral replicon (16,57). To test whether the extent of RNAi depended on the host cell line and the rate of viral replication, the human hepatoma cell line Huh-7 was transfected with the same set of siRNAs and infected with HAV as described above.…”

Section: Resultsmentioning

confidence: 99%

“…The HAV capsid differs from that of other picornaviruses in its high physical stability, which efficiently protects the genome from degradation. This feature and the relative long half-life of the viral genome may hamper the efficacy of shortlived siRNAs (35). Finally, due to the high mutation rate during RNA virus replication, the HAV genome has the potential to escape the siRNA inhibitory pressure with the emergence of resistant mutants.…”

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confidence: 99%

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Silencing of Hepatitis A Virus Infection by Small Interfering RNAs

Kusov

Palmenberg

Sgro

et al. 2006

J Virol

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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...

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Section: Resultsmentioning

confidence: 61%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Silencing of Hepatitis A Virus Infection by Small Interfering RNAs

Kusov

Palmenberg

Sgro

et al. 2006

J Virol

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“…There is also evidence that completely deleted poly(A) tails can be repaired. For example, an engineered clone of cowpea mosaic virus regained a deleted poly(A) tail in vivo (Eggen et al, 1989) and similar examples exist for a number of other viruses (Chen & Frey, 1999;Guilford et al, 1991;Hill et al, 1997;Kusov et al, 2005;Riechmann et al, 1990;Tacahashi & Uyeda, 1999). It is possible that the poly(A) tails are restored by a cellular poly(A) polymerase activity and, in some cases, there is evidence for this: white clover mosaic virus can recover a deleted poly(A) tail, but this is dependent on an AAUAAA motif, which is a signal sequence for cellular poly(A) polymerase (Fitzgerald & Shenk, 1981;Guilford et al, 1991).…”

Section: Terminal Transferase and Poly(a) Tail Repair Mechanismsmentioning

confidence: 99%

How RNA viruses maintain their genome integrity

Barr

Fearns

2010

Journal of General Virology

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“…To combat these possible sources of error, many RNA viruses possess mechanisms that are able to perform genome repair. These mechanisms are diverse in their mode of action: For example some plant RNA viruses have subverted the activity of cellular tRNA nucleotidyl transferase to maintain a 59-CCA-39 motif required at their 39 termini (Rao et al 1989), whereas several mammalian viruses utilize either host-cell-encoded or virus-encoded terminal adenylyl transferases to maintain 39 poly(A) tails required for priming genome replication (Kusov et al 2005;van Leeuwen et al 2006;Rubach et al 2009). Other viruses make use of terminal redundancy within their complement of genome strands to supply missing nucleotides from one genome strand to another.…”

Section: Introductionmentioning

confidence: 99%

Bunyamwera virus can repair both insertions and deletions during RNA replication

Walter

Barr²

2010

RNA

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The genomic termini of RNA viruses contain essential cis-acting signals for such diverse functions as packaging, genome translation, mRNA transcription, and RNA replication, and thus preservation of their sequence integrity is critical for virus viability. Sequence alteration can arise due to cellular mechanisms that add or remove nucleotides from terminal regions, or, alternatively, from introduction of sequence errors through nucleotide misincorporation by the error-prone viral RNAdependent RNA polymerase (RdRp). To preserve template function, many RNA viruses utilize repair mechanisms to prevent accumulation of terminal alterations. Here we show that Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family of segmented negative-sense RNA viruses, also can repair its genomic termini. When an intact nontranslated region (NTR) was added to the anti-genomic 39 end, it was precisely removed, to restore both length and RNA synthesis function of the wild-type template. Furthermore, when nucleotides were removed from the anti-genome 39 end, and replaced with a duplicate and intact NTR, both the external NTR were removed, and the missing nucleotides were restored, thus, indicating that the BUNV RdRp can both remove and add nucleotides to the template. We show that the mechanism for repair of terminal extensions is likely that of internal entry of the viral RdRp during genome synthesis. Possible mechanisms for repair of terminal deletions are discussed.

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Replication and in vivo repair of the hepatitis A virus genome lacking the poly(A) tail

Cited by 20 publications

References 26 publications

Silencing of Hepatitis A Virus Infection by Small Interfering RNAs

Silencing of Hepatitis A Virus Infection by Small Interfering RNAs

How RNA viruses maintain their genome integrity

Bunyamwera virus can repair both insertions and deletions during RNA replication

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