Inhibition of Influenza Virus Replication by α-Amanitin: Mode of Action

Mahy, Brian W. J.; Hastie, Nick; Armstrong, Sylvia J.

doi:10.1073/pnas.69.6.1421

Cited by 55 publications

(33 citation statements)

References 28 publications

(48 reference statements)

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“…2). A similar dependence was previously reported for members of the family Orthomyxoviridae (15,28), which use the cap structure of cellular mRNAs as primers for their own transcription (3,14). It remains unknown whether NYMV might use a similar mechanism to cap its transcripts or whether RNA polymerase II activity is required for a different step in the NYMV replication cycle.…”

Section: Discussionmentioning

confidence: 65%

“…Taken together, these results demonstrated that replication of the NYMV genome includes the nucleus. We next asked whether NYMV multiplication may be dependent on the activity of cellular RNA polymerase II as in the case of other RNA viruses that replicate in the nucleus such as influenza A virus and Thogoto virus (15,28). To check this, we infected Vero cells with NYMV in the presence of either plain medium or medium containing 5 g/ml of actinomycin D to block the activity of RNA polymerase II.…”

Section: Resultsmentioning

confidence: 99%

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Tick-Borne Nyamanini Virus Replicates in the Nucleus and Exhibits Unusual Genome and Matrix Protein Properties

Herrel

Hoefs

Staeheli

et al. 2012

J Virol

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Tick-borne Nyamanini virus (NYMV) is the prototypic member of a recently discovered genus in the order Mononegavirales , designated Nyavirus . The NYMV genome codes for six distinct genes. Sequence similarity and structural properties suggest that genes 1, 5, and 6 encode the nucleoprotein (N), the glycoprotein (G), and the viral polymerase (L), respectively. The function of the other viral genes has been unknown to date. We found that the third NYMV gene codes for a protein which, when coexpressed with N and L, can reconstitute viral polymerase activity, suggesting that it represents a polymerase cofactor. The second viral gene codes for a small protein that inhibits viral polymerase activity and further strongly enhances the formation of virus-like particles when coexpressed with gene 4 and the viral glycoprotein G. This suggests that two distinct proteins serve a matrix protein function in NYMV as previously described for members of the family Filoviridae . We further found that NYMV replicates in the nucleus of infected cells like members of the family Bornaviridae . NYMV is a poor inducer of beta interferon, presumably because the viral genome is 5′ monophosphorylated and has a protruding 3′ terminus as observed for bornaviruses. Taken together, our results demonstrate that NYMV possesses biological properties previously regarded as typical for filoviruses and bornaviruses, respectively.

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Section: Discussionmentioning

confidence: 65%

Section: Resultsmentioning

confidence: 99%

Tick-Borne Nyamanini Virus Replicates in the Nucleus and Exhibits Unusual Genome and Matrix Protein Properties

Herrel

Hoefs

Staeheli

et al. 2012

J Virol

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“…However, the structures of in vivo and in vitro synthesized mRNA do differ in that the in vitro mRNAs are shorter and lack a 5' methylated cap structure (Hay et al, 1977. Furthermore, primary transcription in vivo is sensitive to the drugs actinomycin D and alpha-amanitin (inhibitors of host cell DNA-dependent RNA polymerase ll), whereas transcription in vitro is insensitive to these drugs (Scholtissek & Rott, 1970;Penhoet et aL, 1971;Mahy et aL, 1972;Lamb & Choppin, 1977;Spooner & Barry, 1977). In addition, the influenza virion transcriptase was shown to possess another unusual property in that a number of guanosine related compounds, particularly the dinucleoside adenyl (3' to 5') guanosine (ApG), stimulate RNA synthesis in vitro by acting as primers for RNA synthesis (McGeoch & Kitron, 1975;Plotch & Krug, 1977).…”

mentioning

confidence: 99%

Evidence for the Involvement of Influenza A (Fowl Plague Rostock) Virus Protein P2 in ApG and mRNA Primed in vitro RNA Synthesis

Nichol

Penn

Mahy

1981

Journal of General Virology

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SUMMARYEleven temperature-sensitive (ts) mutants of influenza A (fowl plague, Rostock) virus were analysed for in vitro RNA transcriptase activity in reactions primed by ApG or globin rnRNA at 31 °C or at 40.5 °C, the restrictive temperature for ts mutant growth. Only those ts mutants studied which were defective in RNA segment 1, coding for the virion P2 protein, were defective in RNA transcriptase activity when compared to wild-type virus. Mutants having a defect in the P2 protein had no significant RNA transcriptase activity in reactions at 40.5 °C primed by globin mRNA. However, one mutant showed RNA transcriptase activity similar to wild-type virus at 40.5 °C when ApG (0.3 raM) was used as primer. The results suggest that influenza (fowl plague, Rostock) P2 protein is directly involved in the mRNA priming reaction, as well as in the RNA transcription reaction in vitro.The eight distinct negative-strand RNA segments of fowl plague virus, an avian influenza A virus, can be transcribed in vitro by the virion-bound RNA-dependent RNA polymerase (for reviews, see Barry & Mahy, 1979~ Hay & Skehel, 1979, Under optimal conditions the products of the influenza virion RNA-dependent RNA polymerase reaction have many of the properties of virus mRNA synthesized in infected cells, in that they are approx, full-length, polyadenylated transcripts of each genome segment (Plotch & Krug, 1977, 1978: Hay et al., 1977. However, the structures of in vivo and in vitro synthesized mRNA do differ in that the in vitro mRNAs are shorter and lack a 5' methylated cap structure (Hay et al., 1977. Furthermore, primary transcription in vivo is sensitive to the drugs actinomycin D and alpha-amanitin (inhibitors of host cell DNA-dependent RNA polymerase ll), whereas transcription in vitro is insensitive to these drugs (Scholtissek & Rott, 1970;Penhoet et aL, 1971;Mahy et aL, 1972;Lamb & Choppin, 1977;Spooner & Barry, 1977). In addition, the influenza virion transcriptase was shown to possess another unusual property in that a number of guanosine related compounds, particularly the dinucleoside adenyl (3' to 5') guanosine (ApG), stimulate RNA synthesis in vitro by acting as primers for RNA synthesis (McGeoch & Kitron, 1975;Plotch & Krug, 1977).Recently, an explanation for these findings became apparent when it was reported that various eukaryotic mRNAs stimulate the in vitro transcription reaction (Bouloy et al., 1978) and that the 5'-terminal cap structure plus 9 to 15 nucleotides is transferred to the 5' end of the transcript in vitro (Plotch et al., 1979;Bouloy et al., 1980. The authors suggested that capped host cell RNA molecules are used as 'primers' for synthesis of influenza mRNAs in vivo and that the continued synthesis of these primer molecules is the alpha-amanitin-sensitive step in influenza virus replication (Kruget aL, 1979). In an attempt to determine which virion proteins participate in this priming reaction, we have studied the ApG and globin mRNA-primed in vitro transcriptase activities of a number of genetically characteriz...

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“…These results strongly support our earlier hypothesis (2,3) that viral RNA transcription in vio requires priming by RNAs synthesized by the host cell nuclear RNA polymerase II-i.e., by host mRNAs or their precursors. The need for continued synthesis of these primer host mRNAs provides an explanation for the inhibition of in vivo viral RNA transcription by a-amanitin (4)(5)(6)(7)(8).…”

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confidence: 99%

Transfer of 5′-terminal cap of globin mRNA to influenza viral complementary RNA during transcription in vitro

Plotch

Bouloy

Krug

1979

Proc. Natl. Acad. Sci. U.S.A.

173

143

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We have recently demonstrated that globin mRNAs are effective primers for influenza viral RNA transcription in vitro catalyzed by the virion transcriptase [Bouloy, M., Plotch, S. J. & Krug, R. M. (1978) Proc. Nati. Acad. Sci. USA 75,[4886][4887][4888][4889][4890]. Here, we present direct evidence that the 5'-terminal methylated cap of the globin mRNAs is transferred to viral complementary RNA (cRNA) during transcription. Chemical (a-elimination) or enzymatic removal of the cap of globin mRNAs eliminated essentially all their priming activity. Much of this activity could be restored by recapping the ,-eliminated globin mRNAs with the vaccinia virus guanylyl and methyl transferases. Globin mRNAs containing 32P label only in the cap (m7G32pppm6A'l-) were prepared by recapping ,-eliminated globin mRNAs with the vaccinia virus enzymes, [a-32PJGTP, and unlabeled S-adenosylmethionine. By using this labeled globin mRNA as primer and unlabeled nucleoside triphosphates as precursors, the viral cRNA segments that were synthesized were shown to contain a 32P-labeled 5'-terminal cap structure. Gel electrophoretic analysis indicated that the globin mRNA-primed cRNA segnments were 10-15 nucleotides longer at their 5' end than ApG-primed cRNA segments, which initiate exactly at the 3' end of the virion RNA templates. This suggests that, in addition to the cap, about 10-15 other nucleotides are also transferred from the globin mRNA to viral cRNA. A mechanism for the priming of influenza viral cRNA synthesis by globin mRNA is proposed.Recently we demonstrated (1) We also presented suggestive evidence that the globin mRNA-primed transcripts contain a 5'-terminal methylated cap structure (1). The translation of this viral cRNA into virus-specific proteins was inhibited 75% by the cap analogue 7-methylguanosine 5'-phosphate, whereas under the same conditions the translation of uncapped viral cRNA (the cRNA primed by the dinucleotide ApG) was not inhibited. Because no detectable de novo synthesis of a methylated cap occurred during globin mRNA-primed viral RNA transcription, we proposed that the cap of the globin mRNA primer was transferred to the viral cRNA during in vitro transcription.In the present report, we provide the evidence that proves this proposal. We demonstrate directly that the 5'-methylated cap of the globin mRNA primer is transferred to newly synthesized viral cRNA. In addition, we show that the globin mRNA-primed cRNA segments are 10-15 nucleotides longer at their 5' end than the ApG-primed cRNA segments, which initiate exactly at the 3' end of the virion RNA (vRNA) segments (9, 10). We assume that these additional nucleotides are also transferred from the globin mRNA to viral cRNA during transcription. Based on these results, we propose a mechanism for the priming of influenza viral cRNA synthesis by globin mRNA.METHODS AND MATERIALS The procedures for the synthesis of viral cRNA by detergenttreated purified influenza virus, for the isolation of the poly(A)+ in vitro cRNA, and for the purification of 10S...

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Inhibition of Influenza Virus Replication by α-Amanitin: Mode of Action

Cited by 55 publications

References 28 publications

Tick-Borne Nyamanini Virus Replicates in the Nucleus and Exhibits Unusual Genome and Matrix Protein Properties

Tick-Borne Nyamanini Virus Replicates in the Nucleus and Exhibits Unusual Genome and Matrix Protein Properties

Evidence for the Involvement of Influenza A (Fowl Plague Rostock) Virus Protein P2 in ApG and mRNA Primed in vitro RNA Synthesis

Transfer of 5′-terminal cap of globin mRNA to influenza viral complementary RNA during transcription in vitro

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