Influenza Virus Endoribonuclease

Klumpp, Klaus; Hooker, Lisa; Handa, B.K.

doi:10.1016/s0076-6879(01)42566-3

Cited by 15 publications

(34 citation statements)

References 32 publications

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“…This result is consistent with previous studies showing that IAV RdRp generally cleaves host mRNA 10–15 nt downstream of the cap9111538474849. Interestingly, the host-derived sequences in both PB1 and PB2 mRNAs were smaller by one nucleotide (peaking at 11 nt) when compared to the other viral mRNAs.…”

Section: Discussionsupporting

confidence: 92%

“…Because the G residue just downstream of the 3′ end of the heterogeneous sequences might correspond to the conserved G2, it is possible that a large number of cap-snatched fragments have a 3′ end corresponding to CAG and that the IAV RdRp endonucleolytic cleavage occurs between G and the downstream C. This is consistent with studies showing that IAV RdRp usually cleaves after a purine residue71049, that IAV RdRp endonucleolytic cleavage is highly efficient for substrates that carry GC motifs56, and that the purified N-terminal domain of PA selectively cleaves after G residues of 5′-GC-3′ motifs38. However, because the vRNA template is required to activate endonuclease cleavage and transcription35, our results cannot exclude the possibility that the observed enrichment of this G residue is determined by the complementarity of the host primer to the 3′ end of the vRNA template.…”

Section: Discussionsupporting

confidence: 81%

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Deep sequencing reveals the eight facets of the influenza A/HongKong/1/1968 (H3N2) virus cap-snatching process

Sikora

Rocheleau

Brown

et al. 2014

Sci Rep

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The influenza A virus RNA polymerase cleaves the 5′ end of host pre-mRNAs and uses the capped RNA fragments as primers for viral mRNA synthesis. We performed deep sequencing of the 5′ ends of viral mRNAs from all genome segments transcribed in both human (A549) and mouse (M-1) cells infected with the influenza A/HongKong/1/1968 (H3N2) virus. In addition to information on RNA motifs present, our results indicate that the host primers are divergent between the viral transcripts. We observed differences in length distributions, nucleotide motifs and the identity of the host primers between the viral mRNAs. Mapping the reads to known transcription start sites indicates that the virus targets the most abundant host mRNAs, which is likely caused by the higher expression of these genes. Our findings suggest negligible competition amongst RdRp:vRNA complexes for individual host mRNA templates during cap-snatching and provide a better understanding of the molecular mechanism governing the first step of transcription of this influenza strain.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionsupporting

confidence: 81%

Deep sequencing reveals the eight facets of the influenza A/HongKong/1/1968 (H3N2) virus cap-snatching process

Sikora

Rocheleau

Brown

et al. 2014

Sci Rep

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“…Glycerol gradient centrifugation was performed using vRNPs isolated from cell-free virions. Free, non-specifically incorporated polymerase is predicted to remain at the top of the gradient, whereas polymerase associated with vRNPs migrates into the gradient (Klumpp et al, 2001). Similar amounts of lysed viral cores were separated on a linear 30–60% glycerol gradient and fractions were analyzed by western blot (Figure 5C).…”

Section: Resultsmentioning

confidence: 99%

“…Recombinant virus was also produced in the presence of PB2-TAP by transfecting 293T cells with pTM-polI-WSN-All, pCAGGS-WSN-NP 0/12, pCDNA-PA, pCDNA-PB1, pCDNA-PB2, and pCDNA-PB2-TAP WT or K627E. Virus was harvested 24 hr later and vRNPs were fractionated on a linear 30–60% glycerol gradient as described (Klumpp et al, 2001). …”

Section: Methodsmentioning

confidence: 99%

An Inhibitory Activity in Human Cells Restricts the Function of an Avian-like Influenza Virus Polymerase

Mehle

Doudna

2008

Cell Host & Microbe

163

209

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SUMMARY Transmission of highly pathogenic avian influenza across species into human populations can result in lethal infections and increases the potential of a pandemic outbreak. A major determinant of species tropism is the influenza polymerase subunit PB2. We show here that a dominant inhibitory activity in human cells potently and selectively restricts the function of polymerase containing an avian-like PB2 with glutamic acid at residue 627. This inhibitory activity dramatically reduces virus production by polymerases containing PB2 K627E without reducing their relative infectivity. Restricted polymerases fail to assemble into ribonucleoprotein complexes, resulting in decreased genome transcription, replication, and virus production. However, both wild-type and mutant PB2 bind to viral nucleoprotein when expressed alone, suggesting that species-specific conformational alterations within the polymerase trimer disrupt ribonucleoprotein assembly. Understanding the molecular basis for influenza virus host cell specificity and pathogenesis will enable rational strategies to treat and prevent future avian influenza outbreaks in humans.

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“…The canonical two-metal-ion mechanism of phosphodiester bond hydrolysis was initially developed from studies of E. coli exonuclease I [45], but has since been proposed for a number of additional nucleases, based on biochemical and structural studies. Influenza RNA endonuclease, restriction endonucleases BamHI, BglI, PvuII and double stranded RNA specific RNase III are examples for this group of enzymes [32,[46][47][48][49][50][51]. Fig.…”

Section: Mechanism Of Rna Hydrolysis Cata-lyzed By Hiv-rhmentioning

confidence: 99%

Recent Progress in the Design of Small Molecule Inhibitors of HIV RNase H

Klumpp¹,

Mirzadegan²

2006

CPD

Self Cite

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DNA polymerase and RNase H (RH) activities of HIV reverse transcriptase (RT) have been recognized as potential targets for antiretroviral therapy for more than 15 years. The development of medicines targeting the DNA polymerase activity has been highly successful, with currently 12 drugs approved for the treatment of HIV infection and more candidates in preclinical and clinical development. In contrast, the discovery of potent and selective inhibitors of HIV RH has been slow, and inhibitors of this enzyme function have yet to reach the clinical development stage. Selective HIV RH inhibitors are likely to provide significant clinical benefit in combination therapies, considering the high prevalence of HIV strains resistant to currently available antiretroviral therapies. Recent progress in a number of key areas has provided new impetus to the discovery of HIV RH inhibitors. High throughput assay systems based on fluorescence detection have been developed, which facilitate screening of inhibitor candidates. Substantial progress has been made in expression, purification, crystallisation and solution studies of HIV RT and RH, in particular with regards to aspects of structural dynamics. Crystal structures of active site binding and allosteric HIV RH inhibitors bound to HIV RT and RH have been obtained. Finally, an improved understanding of similarities and differences in enzymatic mechanisms between related nuclease enzymes has provided new concepts for achieving inhibitor selectivity. Together, these developments provide promising new starting points for the rational design of selective HIV RH inhibitors.

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Influenza Virus Endoribonuclease

Cited by 15 publications

References 32 publications

Deep sequencing reveals the eight facets of the influenza A/HongKong/1/1968 (H3N2) virus cap-snatching process

Deep sequencing reveals the eight facets of the influenza A/HongKong/1/1968 (H3N2) virus cap-snatching process

An Inhibitory Activity in Human Cells Restricts the Function of an Avian-like Influenza Virus Polymerase

Recent Progress in the Design of Small Molecule Inhibitors of HIV RNase H

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