A vaccinia virus MVA-T7-mediated recovery of infectious hepatitis A virus from full-size cDNA or from two cDNAs, both by themselves unable to complete the virus life cycle

Kusov, Yuri; Shatirishvili, Gocha; Klinger, Matthias; Gauss‐Müller, Verena

doi:10.1016/s0168-1702(02)00115-6

Cited by 6 publications

(9 citation statements)

References 37 publications

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“…3 C). Note that the level of Renilla luciferase expression by the SUD-deleted replicon was also increased, probably because of more efficient mRNA transcription from a cryptic promoter or due to a helper effect of VV for replicon RNA synthesis, as we and others have noticed previously ( Sutter et al, 1995 , Kusov et al, 2002 ). However, none of the proteins, SUD ( Fig.…”

Section: Resultssupporting

confidence: 71%

“…Assuming that the inability to complement the SUD-deleted replicon by providing SUD or SUD-NM in trans was due to low levels of protein production, we increased the amount of expression using vaccinia virus (VV) MVA-T7 as a helper virus. Previously, we have successfully used MVA-T7 to efficiently express hepatitis A virus genes ( Kusov et al, 2002 ). Indeed, the transfection of constructs expressing SUD or SUD-NM followed by infection with the helper virus MVA-T7 (a procedure known as transinfection, Kusov et al, 2002 ) allowed immunological detection of SUD and SUD-NM using either polyclonal SARS-CoV anti-Nsp3 (Rockland; result not shown) or monoclonal anti-His 4 (Qiagen) ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The recombinant, non-cytopathic vaccinia virus (VV) MVA-T7 was used to produce SUD or its subdomains N+M (SUD-NM) in order to complement in trans the activity of the SARS-CoV replicon lacking SUD. MVA-T7 was propagated in BHK-21 baby hamster kidney cells and titrated as described previously ( Kusov et al, 2002 ). Other cell and culture conditions have been described in Almazán et al (2006) and Kusov et al (2006) .…”

Section: Methodsmentioning

confidence: 99%

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A G-quadruplex-binding macrodomain within the “SARS-unique domain” is essential for the activity of the SARS-coronavirus replication–transcription complex

et al. 2015

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The multi-domain non-structural protein 3 of SARS-coronavirus is a component of the viral replication/transcription complex (RTC). Among other domains, it contains three sequentially arranged macrodomains: the X domain and subdomains SUD-N as well as SUD-M within the “SARS-unique domain”. The X domain was proposed to be an ADP-ribose-1″-phosphatase or a poly(ADP-ribose)-binding protein, whereas SUD-NM binds oligo(G)-nucleotides capable of forming G-quadruplexes. Here, we describe the application of a reverse genetic approach to assess the importance of these macrodomains for the activity of the SARS-CoV RTC. To this end, Renilla luciferase-encoding SARS-CoV replicons with selectively deleted macrodomains were constructed and their ability to modulate the RTC activity was examined. While the SUD-N and the X domains were found to be dispensable, the SUD-M domain was crucial for viral genome replication/transcription. Moreover, alanine replacement of charged amino-acid residues of the SUD-M domain, which are likely involved in G-quadruplex-binding, caused abrogation of RTC activity.

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Section: Resultssupporting

confidence: 71%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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A G-quadruplex-binding macrodomain within the “SARS-unique domain” is essential for the activity of the SARS-coronavirus replication–transcription complex

et al. 2015

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“…Newly synthesized viral genomes of RNA 18f-A14 were found 15 days p.t. The poly(A)-tail length of the rescued virus was determined by a method described previously and consisted of about 60 residues, a tail length similar to that of HAV isolated from infected patients or cell culture (Siegl et al, 1981;Kusov et al, 2001Kusov et al, , 2002. The results implicate that, possibly owing to its reduced stability, the tailless HAV RNA was unable to initiate genome replication in quiescent cells.…”

mentioning

confidence: 99%

“…Infectious virus was rescued not only after transfection of in vitro RNA transcripts, but also after cDNA transfection into Huh-T7 cells, which express T7 RNA polymerase constitutively (Schultz et al, 1996). To compare the infectivity of HAV cDNAs in a reverse-genetics approach, plasmid DNAs encoding the HAV genome with and without a poly(A) tail (pT7-18f-A14 and pT7-18f-A0) were transfected into Huh-T7 cells (Gauss-Müller & Kusov, 2002;Kusov et al, 2001Kusov et al, , 2002 and the rescue of infectious virus was assessed by the accumulation of viral particles. In vivo transcripts with a tail of 14 adenosine residues produced viral antigen and infectious virus 10 days after pT7-18f-A14 cDNA transfection (Fig.…”

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confidence: 99%

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

Kusov

Gosert²,

Gauss‐Müller

2005

Journal of General Virology

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The precise role of the poly(A) tail at the 39 end of the picornavirus RNA genome and the cellular factors that control its homeostasis are unknown. To assess the importance of the poly(A) tail for virus replication, the genome of the slowly replicating hepatitis A virus (HAV) with and without a poly(A) tail was studied after transfection into cells maintained under various conditions. A tailless HAV genome had a shorter half-life than a poly(A)-containing genome and was unable to replicate in quiescent cells. In dividing cells, the tailless RNA gave rise to infectious virus with a restored poly(A) tail of up to 60 residues. Cells arrested at the G 0 and the G 2 /M phase produced lower amounts of infectious HAV than cells in the G 1 phase. These data suggest that the 39 poly(A) tail of HAV can be restored with the help of a cellular and/or viral function that is regulated during the cell cycle.

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Replication of a hepatitis A virus replicon detected by genetic recombination in vivo

Gauss‐Müller

Kusov

2002

Journal of General Virology

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Unlike other picornaviruses, hepatitis A virus (HAV) replicates so inefficiently in cell culture that the study of its RNA biosynthesis presents a major experimental challenge. To assess viral RNA replication independent of particle formation, a subgenomic replicon representing a selfreplicating RNA was constructed by replacing the P1 domain encoding the capsid proteins with the firefly luciferase sequence. Although translation of the HAV replicon was as efficient as a similar poliovirus replicon, the luciferase activity derived from replication of the HAV construct was more than 100-fold lower than that of poliovirus. The replication capacity of the HAV replicon was clearly demonstrated by its ability to recombine genetically with a non-viable, full-length HAV genome that served as capsid donor and thus to rescue a fully infectious virus. In contrast to a replicationdeficient replicon, co-expression of the genetically marked and replication-competent HAV replicon with several lethally mutated HAV genomes resulted in the successful rescue of infectious HAV with a unique genetic marker. Our data suggest : (i) that autonomous HAV RNA replication does not require sequences for the HAV structural proteins ; and (ii) that low-level genome replication can unequivocally be demonstrated by the rescue of infectious virus after co-expression with nonviable genomes.

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A vaccinia virus MVA-T7-mediated recovery of infectious hepatitis A virus from full-size cDNA or from two cDNAs, both by themselves unable to complete the virus life cycle

Cited by 6 publications

References 37 publications

A G-quadruplex-binding macrodomain within the “SARS-unique domain” is essential for the activity of the SARS-coronavirus replication–transcription complex

A G-quadruplex-binding macrodomain within the “SARS-unique domain” is essential for the activity of the SARS-coronavirus replication–transcription complex

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

Replication of a hepatitis A virus replicon detected by genetic recombination in vivo

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