Influenza Viral RNA Transcription Research Articles

Robust Sampling of Defective Pathways in Alzheimer's Disease. Implications in Drug Repositioning.

We present the analysis of the defective genetic pathways of the Late-Onset Alzheimer’s Disease (LOAD) compared to the Mild Cognitive Impairment (MCI) and Healthy Controls (HC) using different sampling methodologies. These algorithms sample the uncertainty space that is intrinsic to any kind of highly underdetermined phenotype prediction problem, by looking for the minimum-scale signatures (header genes) corresponding to different random holdouts. The biological pathways can be identified performing posterior analysis of these signatures established via cross-validation holdouts and plugging the set of most frequently sampled genes into different ontological platforms. That way, the effect of helper genes, whose presence might be due to the high degree of under determinacy of these experiments and data noise, is reduced. Our results suggest that common pathways for Alzheimer’s disease and MCI are mainly related to viral mRNA translation, influenza viral RNA transcription and replication, gene expression, mitochondrial translation, and metabolism, with these results being highly consistent regardless of the comparative methods. The cross-validated predictive accuracies achieved for the LOAD and MCI discriminations were 84% and 81.5%, respectively. The difference between LOAD and MCI could not be clearly established (74% accuracy). The most discriminatory genes of the LOAD-MCI discrimination are associated with proteasome mediated degradation and G-protein signaling. Based on these findings we have also performed drug repositioning using Dr. Insight package, proposing the following different typologies of drugs: isoquinoline alkaloids, antitumor antibiotics, phosphoinositide 3-kinase PI3K, autophagy inhibitors, antagonists of the muscarinic acetylcholine receptor and histone deacetylase inhibitors. We believe that the potential clinical relevance of these findings should be further investigated and confirmed with other independent studies.

RNA interference of avian influenza virus H5N1 by inhibiting viral mRNA with siRNA expression plasmids

Avian influenza virus H5N1 causes widespread infection in the birds and human respiratory tract, but existing vaccines and drug therapy are of limited value. Here we show that small interfering RNAs (siRNAs) specific for conserved regions of the viral genome can potently inhibit influenza virus production in cell lines, embryonated chicken eggs and BALB/c mice. siRNA expression plasmid pBabe-Super was chosen in the study, which directed the synthesis of small interfering RNAs in cells. The inhibition depended on the presence of a functional antisense strand in the small interfering RNA duplex, suggesting that viral mRNA is the target of RNA interference (RNAi). Among the three small interfering RNA expression plasmids we designed, we found that small interfering RNA for nucleocapsid protein (NP) had a specific effect in inhibiting the accumulation of RNAs in infected cells because of a critical requirement for newly synthesized nucleocapsid proteins in avian influenza viral RNA transcription and replication. The findings reveal that newly synthesized nucleocapsid, polymerase A (PA) and polymerase B1 (PB1) proteins are required for avian influenza virus transcription and replication and provide a basis for the development of small interfering RNAs as prophylaxis and therapy for avian influenza infection in birds and humans.

A unique cap(m 7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription

We propose a mechanism for the priming of influenza viral RNA transcription by capped RNAs in which specific 5′-terminal fragments are cleaved from the capped RNAs by a virion-associated endonuclease. These fragments would serve as the actual primers for the initiation of transcription by the initial incorporation of a G residue at their 3′ end. We show that virions and purified viral cores contain a unique endonuclease that cleaves RNAs containing a 5′ methylated cap structure (m 7GpppXm) preferentially at purine residues 10 to 14 nucleotides from the cap, generating fragments with 3′-terminal hydroxyl groups. RNAs containing the 5′-terminal structure GpppG could not be cleaved to produce these specific fragments. Consistent with our proposed mechanism, those capped fragments that function as primers could be linked to a G residue in transcriptase reactions containing α- 32P-GTP as the only ribonucleoside triphosphate. The pattern of G and C incorporation onto these primer fragments suggests that this incorporation is directed by the second and third bases at the 3′ end of the virion RNA template, which has the sequence 3′ UCG. Primer fragments with a 3′-terminal A residue were used more efficiently than those with a 3′-terminal G residue, indicating a preference for generating an AGC sequence in the viral mRNA complementary to the 3′ end of the virion RNA. Cleavage of the RNA primer and initiation of transcription are not necessarily coupled, because a 5′ fragment isolated from one reaction could be used as a primer when added to a second reaction. Uncapped ribopolymer inhibitors of viral RNA transcription inhibited the cleavage of capped RNAs.

Selected host cell capped RNA fragments prime influenza viral RNA transcription in vivo.

Influenza viral RNA transcription in vitro is primed by capped RNA fragments cleaved from capped RNAs by a viral endonuclease. The present study was undertaken to determine whether the specificities of the viral endonuclease and transcriptase observed in in vitro studies are also observed in the infected cell. The NS (nonstructural) gene of influenza WSN virus was cloned in pBR322 by using a double-stranded DNA containing a cDNA copy of both virion RNA (vRNA) and in vivo viral mRNA. We determined the 5' terminal sequence of the particular NS viral mRNA molecule which was cloned and also the 5' terminal sequences of the entire population of in vivo NS viral mRNAs synthesized in two different cell lines. For the latter determination we used a restriction fragment from the cloned DNA for the reverse transcriptase-catalyzed extension of total in vivo viral mRNA. The results indicate that in vivo and in vitro viral RNA transcription are similar in two important respects: (i) transcription initiates not with an A residue directed by the 3' terminal U of the vRNA, but with a G residue directed by the 3' penultimate C of the vRNA; and (ii) capped RNA fragments containing a 3' terminal A residue are preferentially used as primers, therapy generating an AG sequence in the viral mRNA complementary to the 3' terminal UC of the vRNA. Actually, for in vivo transcription, a subset of A-terminated capped fragments, namely those containing a 3' penultimate C residue, are the preferred primers. The latter specificity had not been observed in previous in vitro studies.

Priming of influenza viral RNA transcription by capped heterologous RNAs.

Influenza virus is a negative-stranded RNA virus, i.e., the viral messenger RNA (mRNA) is complementary to the genome RNA (which is segmented), and the virion contains the enzyme system which transcribes the genome RNA into the viral mRNA. This virus employs a unique mechanism for the initiation of the synthesis of its mRNA. Specifically, the virion-associated transcriptase system needs to cannibalize capped heterologous RNAs, i.e., eukaryotic cellular mRNAs and/or their precursors, in order to synthesize the viral mRNA. This process involves the clipping off by a virion-associated endonuclease of a small piece of the eukaryotic mRNA near its 5’-end, followed by the utilization of this RNA fragment as a primer to initiate the synthesis of the viral mRNA. The methylated cap structure (m7GpppNm, where Nm = 2’ -O-methylated nucleoside) found at the 5J -end of all mammalian cellular mRNAs is part of the RNA fragment that is used as primer and transferred from the cellular to the viral mRNA, and this cap structure is absolutely necessary for the viral nuclease and the priming reaction. In fact, the 5;-methylated cap structure is more stringently required for priming influenza viral mRNA synthesis than for the process in which it had previously been shown to play a role, the translation of mRNAs in cell-free systems. This mechanism for viral mRNA synthesis explains why influenza virus requires the functioning of the host-cell nuclear RNA polymerase II in order to replicate: newly synthesized host mRNAs and/or their precursors are needed as primers for viral mRNA synthesis.

RNA primers and the role of host nuclear RNA polymerase II in influenza viral RNA transcription.

Influenza viral RNA transcription in the infected cell is inhibited by alpha-amanitin, a specific inhibitor of the host nuclear RNA polymerase II. Because viral RNA transcription in vitro catalysed by the virion-associated transcriptase 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 RNA polymerase II. In addition, because we did not detect any capping and methylating enzymes in virions, we have proposed that the 5' terminal methylated cap found on in-vivo viral messenger RNA (mRNA) is derived from the putative primer RNAs. Our recent experiments have proved these two hypotheses. Purified globin mRNAs were shown to stimulate viral RNA transcription in vitro very effectively. The resulting transcripts directed the synthesis of all the non-glycosylated virus-specific proteins in cell-free systems. Other eukaryotic mRNAs were also active as primers. The presence of a 5' terminal methylated cap structure in the priming mRNA was required for its priming activity. Thus, with globin mRNA, removal of the cap eliminated essentially all of its priming activity, and much of this activity could be restored by enzymically recapping the globin mRNA. Using globin mRNA containing 32P only in its cap, we demonstrated that the 5' cap of the globin mRNA primer was physically transferred to the viral RNA transcripts during transcription. Gel electrophoretic analysis suggested that, in addition to the cap, about 10-15 other nucleotides were also transferred from the globin mRNA to the viral RNA transcripts. A mechanism for the priming of influenza viral RNA transcription by globin mRNA is proposed. Initial experiments strongly suggest that priming by capped host mRNAs also occurs during the synthesis of viral mRNA in vivo.

Identification of the RNA region transferred from a representative primer, β-globin mRNA, to influenza mRNA during in vitro transcription

Capped eukaryotic mRNAs strongly stimulate influenza viral RNA transcription in vitro and donate their cap and also additional nucleotides to the viral transcripts (1). To identify which bases of a given primer mRNA are transferred, we synthesized influenza viral mRNA using a primer rabbit globin mRNA (enriched in beta-globin mRNA) which had been labeled in vitro to high specific activity with 125I. We show that during transcription the same 125I-labeled oligonucleotides were transferred to the 5' termini of each of the eight viral mRNA segments. The predominant sequence, representing 75 percent of the transferred oligonucleotides, was identical to the first 13 nucleotides at the 5' end of beta-globin mRNA (m7G5'ppp5'm6AmC(m)ACUUGCUUUUG). Because only the C-residues are labeled with 125I, these results indicate that either the first 12, 13 or 14 5' terminal bases of beta-globin mRNA were transferred to the viral mRNAs. 125I-labeled oligonucleotides recovered from the viral mRNA in minor yields indicated that shorter 5' terminal pieces of beta-globin mRNA were sometimes transferred and that G was probably the first base inserted by transcription.

Cap and internal nucleotides of reovirus mRNA primers are incorporated into influenza viral complementary RNA during transcription in vitro.

Reovirus mRNA's containing a 5'-terminal methylated cap structure (m(7)GpppG(m)) were shown to be effective primers for influenza viral RNA transcription in vitro catalyzed by the influenza virion transcriptase. Priming activity required the presence of methyl groups in the cap since reovirus mRNA's with 5'-terminal GpppG were inactive as primers. Both the cap and internal nucleotides were physically transferred from radiolabeled reovirus mRNA to influenza viral complementary RNA (cRNA) during transcription in vitro. By using reovirus mRNA's with methyl-(3)H-labeled caps as primers, we showed that the influenza viral cRNA synthesized in the presence of unlabeled nucleoside triphosphates contained [methyl-(3)H]m(7)GpppG(m), identical to that found in the reovirus mRNA primer. To demonstrate transfer of internal residues, reovirus mRNA's synthesized in the presence of all four alpha-(32)P-labeled ribonucleoside triphosphates were used as primers. The resulting influenza viral cRNA was (32)P-labeled. Diethyl-aminoethyl-Sephadex chromatography of the RNase T2 digest of this cRNA demonstrated (32)P radiolabel in both internal residues (charge -2) and the cap (charge -4.6). Approximately 25 internal nucleotides along with the cap of reovirus mRNA were transferred to each chain of influenza viral cRNA. Gel electrophoretic analysis indicated that the segments of influenza viral cRNA primed by reovirus mRNA were approximately the same size as those primed by a different mRNA, globin mRNA, strongly suggesting that the influenza virion transcriptase complex transfers approximately the same number of nucleotides plus the cap from different mRNA primers to the 5' end of influenza viral RNA transcripts.

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

Eucaryotic mRNAs serve as primers for influenza viral RNA transcription in vitro and donate their 5′ terminal methylated cap and a short stretch of internal nucleotides to the resulting viral RNA transcripts (Plotch, Bouloy and Krug, 1979). If a similar mechanism operates in vivo, then the viral mRNA synthesized in the infected cell would contain a short stretch of nucleotides at its 5′ end, including the cap, which is not viral-coded. This study was undertaken to establish whether such a nonviral sequence exists in in vivo viral mRNA. Gel electrophoresis analysis indicated that the segments of in vivo viral mRNA were 10–15 nucleotides longer at their 5′ end than the segments of ApG-primed complementary RNA synthesized in vitro. As the latter segments initiate exactly at the 3′ end of the virion RNA templates, this result indicates that the segments of in vivo viral mRNA contain 10–15 nucleotides at their 5′ end which are not viral-coded. In addition, when 3H-methyl-labeled in vivo viral mRNA was hybridized to virion RNA, the 5′ terminal cap structure of the mRNA was not protected against pancreatic or T1 RNAase digestion. One of the three 6-methyladenosine (m 6A) residues found per chain of viral mRNA was also not protected. These results strongly suggest that host cell mRNAs serve as primers for viral RNA transcription in the infected cell, and that they donate their cap and 10–15 internal nucleotides, one of which is m 6A, to the resulting viral mRNA molecules.