http://llama.mshri.on.ca/courses/Biophysics205/Papers/Braas_2009.pdf

The IFITM Proteins Mediate Cellular Resistance to Influenza A H1N1 Virus, West Nile Virus, and Dengue Virus

Many scientists, if not all, feel that their particular plant virus should appear in any list of the most important plant viruses. However, to our knowledge, no such list exists. The aim of this review was to survey all plant virologists with an association with Molecular Plant Pathology and ask them to nominate which plant viruses they would place in a 'Top 10' based on scientific/economic importance. The survey generated more than 250 votes from the international community, and allowed the generation of a Top 10 plant virus list for Molecular Plant Pathology. The Top 10 list includes, in rank order, (1) Tobacco mosaic virus, (2) Tomato spotted wilt virus, (3) Tomato yellow leaf curl virus, (4) Cucumber mosaic virus, (5) Potato virus Y, (6) Cauliflower mosaic virus, (7) African cassava mosaic virus, (8) Plum pox virus, (9) Brome mosaic virus and (10) Potato virus X, with honourable mentions for viruses just missing out on the Top 10, including Citrus tristeza virus, Barley yellow dwarf virus, Potato leafroll virus and Tomato bushy stunt virus. This review article presents a short review on each virus of the Top 10 list and its importance, with the intent of initiating discussion and debate amongst the plant virology community, as well as laying down a benchmark, as it will be interesting to see in future years how perceptions change and which viruses enter and leave the Top 10.

/pdf/top-10-plant-viruses-in-molecular-plant-pathology-2jkmwvdzqw.pdf

Top 10 plant viruses in molecular plant pathology

A unique cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription

The influenza virus polymerase, a heterotrimer composed of three subunits, PA, PB1 and PB2, is responsible for replication and transcription of the eight separate segments of the viral RNA genome in the nuclei of infected cells. The polymerase synthesizes viral messenger RNAs using short capped primers derived from cellular transcripts by a unique 'cap-snatching' mechanism. The PB2 subunit binds the 5' cap of host pre-mRNAs, which are subsequently cleaved after 10-13 nucleotides by the viral endonuclease, hitherto thought to reside in the PB2 (ref. 5) or PB1 (ref. 2) subunits. Here we describe biochemical and structural studies showing that the amino-terminal 209 residues of the PA subunit contain the endonuclease active site. We show that this domain has intrinsic RNA and DNA endonuclease activity that is strongly activated by manganese ions, matching observations reported for the endonuclease activity of the intact trimeric polymerase. Furthermore, this activity is inhibited by 2,4-dioxo-4-phenylbutanoic acid, a known inhibitor of the influenza endonuclease. The crystal structure of the domain reveals a structural core closely resembling resolvases and type II restriction endonucleases. The active site comprises a histidine and a cluster of three acidic residues, conserved in all influenza viruses, which bind two manganese ions in a configuration similar to other two-metal-dependent endonucleases. Two active site residues have previously been shown to specifically eliminate the polymerase endonuclease activity when mutated. These results will facilitate the optimisation of endonuclease inhibitors as potential new anti-influenza drugs.

The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit

The expression of pattern-recognition receptors (PRRs) by immune and tissue cells provides the host with the ability to detect and respond to infection by viruses and other microorganisms. Significant progress has been made from studying this area, including the identification of PRRs, such as Toll-like receptors and RIG-I-like receptors, and the description of the molecular basis of their signalling pathways, which lead to the production of interferons and other cytokines. In parallel, common mechanisms used by viruses to evade PRR-mediated responses or to actively subvert these pathways for their own benefit are emerging. Accumulating evidence on how viral infection and PRR signalling pathways intersect is providing further insights into the function of the pathways involved, their constituent proteins and ways in which they could be manipulated therapeutically.

Viral evasion and subversion of pattern-recognition receptor signalling

Because influenza viral RNA transcription in vitro is greatly enhanced by the addition of a primer dinucleotide, ApG or GpG, we have proposed that viral RNA transcription in vivo requires initiation by primer RNAs synthesized by the host cell, specifically by RNA polymerase II, thereby explaining the α-amanitin sensitivity of viral RNA transcription in vivo. Here, we identify such primer RNAs, initially in reticulocyte extracts, where they are shown to be globin mRNAs. Purified globin mRNAs very effectively stimulated viral RNA transcription in vitro, and the resulting transcripts directed the synthesis of all the nonglycosylated virus-specific proteins in micrococcal nuclease-treated L cell extracts. The viral RNA transcripts synthesized in vitro primed by ApG also directed the synthesis of the nonglycosylated virus-specific proteins, but the globin mRNA-primed transcripts were translated about 3 times more efficiently. The translation of the globin mRNA-primed, but not the ApG-primed, viral RNA transcripts was inhibited by 7-methylguanosine 5′-phosphate in the presence of S-adenosylhomocysteine, suggesting that the globin mRNA-primed transcripts contained a 5′-terminal methylated cap structure. We propose that this cap was transferred from the globin mRNA primer to the newly synthesized viral RNA transcripts, because no detectable de novo synthesis of a methylated cap occurred during globin mRNA-primed viral RNA transcription. Preliminary experiments indicate that other purified eukaryotic mRNAs also stimulate influenza viral RNA transcription in vitro.

https://www.pnas.org/content/pnas/75/10/4886.full.pdf

Globin mRNAs are primers for the transcription of influenza viral RNA in vitro

Are the 5′ ends of influenza viral mrnas synthesized in vivo donated by host mRNAs?

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. Natl. Acad. Sci. USA 75, 4886-4890]. 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 (β-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 β-eliminated globin mRNAs with the vaccinia virus guanylyl and methyl transferases. Globin mRNAs containing 32P label only in the cap (m7G32pppm6Am-) were prepared by recapping β-eliminated globin mRNAs with the vaccinia virus enzymes, [α-32P]GTP, 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 segments 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.

https://www.pnas.org/content/pnas/76/4/1618.full.pdf

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

The ability of eukaryotic mRNAs to serve as primers for influenza virus RNA transcription depends on the presence of a 5'-terminal methylated can structure, the absence of which eliminates essentially all priming activity [Plotch, S. J., Bouloy, M. & Krug, R. M. (1979) Proc. Natl. Acad. Sci. USA 76, 1618-1622]. The present study was undertaken to determine the extent to which each of the methyl groups in the cap influences the priming activity of a mRNA. To assess the importance of the 2'-O-methyl group on the penultimate base of the cap, we used several plant viral RNAs containing the monomethylated cap 0 structure, m7GpppG. Brome mosaic virus (BMV) RNA 4 stimulated influenza virus RNA transcription only about 10-15% as effectively as did globin mRNA, which has a cap with a 2'-O-methyl group. When the cap of BMV RNA 4 was enzymatically 2'-O-methylated, its priming activity was increased 14-fold. Qualitatively similar results were obtained with other plant virus RNAs. To assess the importance of the terminal 7-methyl group, BMV RNA 4 containing the cap structure GpppGm was prepared by a series of chemical and enzymatic steps. These molecules were found to be only about 15% as active in priming as BMV RNA 4 molecules containing the fully methylated cap, m7GpppGm, indicating that the terminal 7-methyl group also strongly enhances priming activity. These results indicate that the cap 1 structure (m7GpppXm) found in all mammalian cellular mRNAs is more stringently required for priming influenza virus RNA transcription than for translation in cell-free systems.

https://www.pnas.org/content/pnas/77/7/3952.full.pdf

Michele Bouloy

Papers

A unique cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription

Globin mRNAs are primers for the transcription of influenza viral RNA in vitro

Are the 5′ ends of influenza viral mrnas synthesized in vivo donated by host mRNAs?

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

Both the 7-methyl and the 2'-O-methyl groups in the cap of mRNA strongly influence its ability to act as primer for influenza virus RNA transcription