The genomes of influenza viruses consist of multiple segments of single-stranded negative-sense RNA. Each of these segments is bound by the heterotrimeric viral RNA-dependent RNA polymerase and ...multiple copies of nucleoprotein, which form viral ribonucleoprotein (vRNP) complexes. It is in the context of these vRNPs that the viral RNA polymerase carries out transcription of viral genes and replication of the viral RNA genome. In this Review, we discuss our current knowledge of the structure of the influenza virus RNA polymerase, and insights that have been gained into the molecular mechanisms of viral transcription and replication, and their regulation by viral and host factors. Furthermore, we discuss how advances in our understanding of the structure and function of polymerases could help in identifying new antiviral targets.
Eukaryotes and bacteria can be infected with a wide variety of RNA viruses. On average, these pathogens share little sequence similarity and use different replication and transcription strategies. ...Nevertheless, the members of nearly all RNA virus families depend on the activity of a virally encoded RNA-dependent polymerase for the condensation of nucleotide triphosphates. This review provides an overview of our current understanding of the viral RNA-dependent polymerase structure and the biochemistry and biophysics that is involved in replicating and transcribing the genetic material of RNA viruses.
The mechanism of resistance to favipiravir in influenza Goldhill, Daniel H.; te Velthuis, Aartjan J. W.; Fletcher, Robert A. ...
Proceedings of the National Academy of Sciences - PNAS,
11/2018, Letnik:
115, Številka:
45
Journal Article
Recenzirano
Odprti dostop
Favipiravir is a broad-spectrum antiviral that has shown promise in treatment of influenza virus infections. While emergence of resistance has been observed for many antiinfluenza drugs, to date, ...clinical trials and laboratory studies of favipiravir have not yielded resistant viruses. Here we show evolution of resistance to favipiravir in the pandemic H1N1 influenza A virus in a laboratory setting. We found that two mutations were required for robust resistance to favipiravir. We demonstrate that a K229R mutation in motif F of the PB1 subunit of the influenza virus RNA-dependent RNA polymerase (RdRP) confers resistance to favipiravir in vitro and in cell culture. This mutation has a cost to viral fitness, but fitness can be restored by a P653L mutation in the PA subunit of the polymerase. K229R also conferred favipiravir resistance to RNA polymerases of other influenza A virus strains, and its location within a highly conserved structural feature of the RdRP suggests that other RNA virusesmight also acquire resistance through mutations in motif F. The mutations identified here could be used to screen influenza virus-infected patients treated with favipiravir for the emergence of resistance.
RNA viruses include many important human and animal pathogens, such as the influenza viruses, respiratory syncytial virus, Ebola virus, measles virus and rabies virus. The genomes of these viruses ...consist of single or multiple RNA segments that assemble with oligomeric viral nucleoprotein into ribonucleoprotein complexes. Replication and transcription of the viral genome is performed by ~250-450 kDa viral RNA-dependent RNA polymerases that also contain capping or cap-snatching activity. In this Review, we compare recent high-resolution X-ray and cryoelectron microscopy structures of RNA polymerases of negative-sense RNA viruses with segmented and non-segmented genomes, including orthomyxoviruses, peribunyaviruses, phenuiviruses, arenaviruses, rhabdoviruses, pneumoviruses and paramyxoviruses. In addition, we discuss how structural insights into these enzymes contribute to our understanding of the molecular mechanisms of viral transcription and replication, and how we can use these insights to identify targets for antiviral drug design.
Transcription and replication of the influenza virus RNA genome is catalyzed by the viral heterotrimeric RNA-dependent RNA polymerase in the context of viral ribonucleoprotein (vRNP) complexes. ...Atomic resolution structures of the viral RNA synthesis machinery have offered insights into the initiation mechanisms of viral transcription and genome replication, and the interaction of the viral RNA polymerase with host RNA polymerase II, which is required for the initiation of viral transcription. Replication of the viral RNA genome by the viral RNA polymerase depends on host ANP32A, and host-specific sequence differences in ANP32A underlie the poor activity of avian influenza virus polymerases in mammalian cells. A failure to faithfully copy the viral genome segments can lead to the production of aberrant viral RNA products, such as defective interfering (DI) RNAs and mini viral RNAs (mvRNAs). Both aberrant RNA types have been implicated in innate immune responses against influenza virus infection. This review discusses recent insights into the structure-function relationship of the viral RNA polymerase and its role in determining host range and virulence.
An RNA-dependent RNA polymerase (RdRp) is the central catalytic subunit of the RNA-synthesizing machinery of all positive-strand RNA viruses. Usually, RdRp domains are readily identifiable by ...comparative sequence analysis, but biochemical confirmation and characterization can be hampered by intrinsic protein properties and technical complications. It is presumed that replication and transcription of the approximately 30-kb severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) RNA genome are catalyzed by an RdRp domain in the C-terminal part of nonstructural protein 12 (nsp12), one of 16 replicase subunits. However, thus far full-length nsp12 has proven refractory to expression in bacterial systems, which has hindered both the biochemical characterization of coronavirus RNA synthesis and RdRp-targeted antiviral drug design. Here, we describe a combined strategy involving bacterial expression of an nsp12 fusion protein and its in vivo cleavage to generate and purify stable SARS-CoV nsp12 (106 kDa) with a natural N-terminus and C-terminal hexahistidine tag. This recombinant protein possesses robust in vitro RdRp activity, as well as a significant DNA-dependent activity that may facilitate future inhibitor studies. The SARS-CoV nsp12 is primer dependent on both homo- and heteropolymeric templates, supporting the likeliness of a close enzymatic collaboration with the intriguing RNA primase activity that was recently proposed for coronavirus nsp8.
Central nervous system (CNS) disease is the most common extra-respiratory tract complication of influenza A virus infections in humans. Remarkably, zoonotic highly pathogenic avian influenza (HPAI) ...H5N1 virus infections are more often associated with CNS disease than infections with seasonal influenza viruses. Evolution of avian influenza viruses has been extensively studied in the context of respiratory infections, but evolutionary processes in CNS infections remain poorly understood. We have previously observed that the ability of HPAI A/Indonesia/5/2005 (H5N1) virus to replicate in and spread throughout the CNS varies widely between individual ferrets. Based on these observations, we sought to understand the impact of entrance into and replication within the CNS on the evolutionary dynamics of virus populations. First, we identified and characterized three substitutions-PB1 E177G and A652T and NP I119M - detected in the CNS of a ferret infected with influenza A/Indonesia/5/2005 (H5N1) virus that developed a severe meningo-encephalitis. We found that some of these substitutions, individually or collectively, resulted in increased polymerase activity in vitro. Nevertheless, in vivo, the virus bearing the CNS-associated mutations retained its capacity to infect the CNS but showed reduced dispersion to other anatomical sites. Analyses of viral diversity in the nasal turbinate and olfactory bulb revealed the lack of a genetic bottleneck acting on virus populations accessing the CNS via this route. Furthermore, virus populations bearing the CNS-associated mutations showed signs of positive selection in the brainstem. These features of dispersion to the CNS are consistent with the action of selective processes, underlining the potential for H5N1 viruses to adapt to the CNS.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
RNA viruses include respiratory viruses, such as coronaviruses and influenza viruses, as well as vector-borne viruses, like dengue and West Nile virus. RNA viruses like these encounter various ...environments when they copy themselves and spread from cell to cell or host to host.
differences, such as geographical location and humidity, affect their stability and transmission, while
differences, such as pH and host gene expression, impact viral receptor binding, viral replication, and the host immune response against the viral infection. A critical factor affecting RNA viruses both
and
, and defining the outcome of viral infections and the direction of viral evolution, is temperature. In this minireview, we discuss the impact of temperature on viral replication, stability, transmission, and adaptation, as well as the host innate immune response. Improving our understanding of how RNA viruses function, survive, and spread at different temperatures will improve our models of viral replication and transmission risk analyses.
Influenza A viruses cause a mild-to-severe respiratory disease that affects millions of people each year. One of the many determinants of disease outcome is the innate immune response to the viral ...infection. While antiviral responses are essential for viral clearance, excessive innate immune activation promotes lung damage and disease. The influenza A virus RNA polymerase is one of viral proteins that affect innate immune activation during infection, but the mechanisms behind this activity are not well understood. In this review, we discuss how the viral RNA polymerase can both activate and suppress innate immune responses by either producing immunostimulatory RNA species or directly targeting the components of the innate immune signalling pathway, respectively. Furthermore, we provide a comprehensive overview of the polymerase residues, and their mutations, associated with changes in innate immune activation, and discuss their putative effects on polymerase function based on recent advances in our understanding of the influenza A virus RNA polymerase structure.
The RNA-dependent RNA polymerase (RdRp) of the influenza A virus replicates and transcribes the viral genome segments in the nucleus of the host cell. To transcribe these viral genome segments, the ...RdRp "snatches" capped RNA oligonucleotides from nascent host cell mRNAs and aligns these primers to the ultimate or penultimate nucleotide of the segments for the initiation of viral mRNA synthesis. It has been proposed that this initiation process is not processive and that the RdRp uses a prime-realign mechanism during transcription. Here we provide
evidence for the existence of this transcriptional prime-realign mechanism but show that it functions efficiently only for primers that are short or cannot stably base pair with the template. In addition, we demonstrate that transcriptional elongation is dependent on the priming loop of the PB1 subunit of the RdRp. We propose that the prime-realign mechanism may be used to rescue abortive transcription initiation events or cope with sequence variation among primers. Overall, these observations advance our mechanistic understanding of how influenza A virus initiates transcription correctly and efficiently.
Influenza A virus causes severe disease in humans and is considered a major global health threat. The virus replicates and transcribes its genome by using an enzyme called the RNA polymerase. To ensure that the genome is amplified faithfully and abundant viral mRNAs are made for viral protein synthesis, the viral RNA polymerase must transcribe the viral genome efficiently. In this report, we characterize a structure inside the polymerase that contributes to the efficiency of viral mRNA synthesis.
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