Publications
30 results found
Sheppard C, Barclay W, Goldhill D, et al., 2023, An Influenza A virus can evolve to use human ANP32E through altering polymerase dimerization, Nature Communications, Vol: 14, ISSN: 2041-1723
Human ANP32A and ANP32B are essential but redundant host factors for influenza virus genome replication. While most influenza viruses cannot replicate in edited human cells lacking both ANP32A and ANP32B, some strains exhibit limited growth. Here, we experimentally evolve such an influenza A virus in these edited cells and unexpectedly, after 2 passages, we observe robust viral growth. We find two mutations in different subunits of the influenza polymerase that enable the mutant virus to use a novel host factor, ANP32E, an alternative family member, which is unable to support the wild type polymerase. Both mutations reside in the symmetric dimer interface between two polymerase complexes and reduce polymerase dimerization. These mutations have previously been identified as adapting influenza viruses to mice. Indeed, the evolved virus gains the ability to use suboptimal mouse ANP32 proteins and becomes more virulent in mice. We identify further mutations in the symmetric dimer interface which we predict allow influenza to adapt to use suboptimal ANP32 proteins through a similar mechanism. Overall, our results suggest a balance between asymmetric and symmetric dimers of influenza virus polymerase that is influenced by the interaction between polymerase and ANP32 host proteins.
Idoko-Akoh A, Goldhill DH, Sheppard CM, et al., 2023, Creating resistance to avian influenza infection through genome editing of the ANP32 gene family., Nat Commun, Vol: 14
Chickens genetically resistant to avian influenza could prevent future outbreaks. In chickens, influenza A virus (IAV) relies on host protein ANP32A. Here we use CRISPR/Cas9 to generate homozygous gene edited (GE) chickens containing two ANP32A amino acid substitutions that prevent viral polymerase interaction. After IAV challenge, 9/10 edited chickens remain uninfected. Challenge with a higher dose, however, led to breakthrough infections. Breakthrough IAV virus contained IAV polymerase gene mutations that conferred adaptation to the edited chicken ANP32A. Unexpectedly, this virus also replicated in chicken embryos edited to remove the entire ANP32A gene and instead co-opted alternative ANP32 protein family members, chicken ANP32B and ANP32E. Additional genome editing for removal of ANP32B and ANP32E eliminated all viral growth in chicken cells. Our data illustrate a first proof of concept step to generate IAV-resistant chickens and show that multiple genetic modifications will be required to curtail viral escape.
Peacock T, Sheppard C, Lister M, et al., 2023, Mammalian ANP32A and ANP32B proteins drive differential polymerase adaptations in avian influenza virus, Journal of Virology, Vol: 97, Pages: 1-13, ISSN: 0022-538X
ANP32 proteins, which act as influenza polymerase cofactors, vary between birds and mammals. In mammals, ANP32A and ANP32B have been reported to serve essential but redundant roles to support influenza polymerase activity. The well-known mammalian adaptation PB2-E627K enables influenza polymerase to use mammalian ANP32 proteins. However, some mammalian-adapted influenza viruses do not harbor this substitution. Here, we show that alternative PB2 adaptations, Q591R and D701N, also allow influenza polymerase to use mammalian ANP32 proteins, whereas other PB2 mutations, G158E, T271A, and D740N, increase polymerase activity in the presence of avian ANP32 proteins as well. Furthermore, PB2-E627K strongly favors use of mammalian ANP32B proteins, whereas D701N shows no such bias. Accordingly, PB2-E627K adaptation emerges in species with strong pro-viral ANP32B proteins, such as humans and mice, while D701N is more commonly seen in isolates from swine, dogs, and horses, where ANP32A proteins are the preferred cofactor. Using an experimental evolution approach, we show that the passage of viruses containing avian polymerases in human cells drove acquisition of PB2-E627K, but not in the absence of ANP32B. Finally, we show that the strong pro-viral support of ANP32B for PB2-E627K maps to the low-complexity acidic region (LCAR) tail of ANP32B.IMPORTANCE Influenza viruses naturally reside in wild aquatic birds. However, the high mutation rate of influenza viruses allows them to rapidly and frequently adapt to new hosts, including mammals. Viruses that succeed in these zoonotic jumps pose a pandemic threat whereby the virus adapts sufficiently to efficiently transmit human-to-human. The influenza virus polymerase is central to viral replication and restriction of polymerase activity is a major barrier to species jumps. ANP32 proteins are essential for influenza polymerase activity. In this study, we describe how avian influenza viruses can adapt in several different ways to use ma
Swann O, Rasmussen A, Peacock T, et al., 2023, Avian Influenza A Virus polymerase can utilise human ANP32 proteins to support cRNA but not vRNA synthesis, mBio, Vol: 14, Pages: 1-14, ISSN: 2150-7511
Host restriction limits the emergence of novel pandemic strains from the influenza A virus avian reservoir. For efficient replication in mammalian cells, the avian influenza RNA-dependent RNA polymerase must adapt to use human orthologues of the host factor ANP32, which lack a 33-amino-acid insertion relative to avian ANP32A. Here, we find that influenza polymerase requires ANP32 proteins to support both steps of genome replication: cRNA and vRNA synthesis. However, avian strains are only restricted in vRNA synthesis in human cells. Therefore, avian influenza polymerase can use human ANP32 orthologues to support cRNA synthesis, without acquiring mammalian adaptations. This implies a fundamental difference in the mechanism by which ANP32 proteins support cRNA versus vRNA synthesis.IMPORTANCE To infect humans and cause a pandemic, avian influenza must first adapt to use human versions of the proteins the virus hijacks for replication, instead of the avian orthologues found in bird cells. One critical host protein is ANP32. Understanding the details of how host proteins such as ANP32 support viral activity may allow the design of new antiviral strategies that disrupt these interactions. Here, we use cells that lack ANP32 to unambiguously demonstrate ANP32 is needed for both steps of influenza genome replication. Unexpectedly, however, we found that avian influenza can use human ANP32 proteins for the first step of replication, to copy a complementary strand, without adaptation but can only utilize avian ANP32 for the second step of replication that generates new genomes. This suggests ANP32 may have a distinct role in supporting the second step of replication, and it is this activity that is specifically blocked when avian influenza infects human cells.
Swann OC, Rasmussen AB, Peacock TP, et al., 2022, Avian Influenza A Virus polymerase can utilise human ANP32 proteins to support cRNA but not vRNA synthesis
<jats:title>Abstract</jats:title><jats:p>Host restriction limits the emergence of novel pandemic strains from the Influenza A Virus avian reservoir. For efficient replication in mammalian cells, the avian influenza RNA-dependent RNA polymerase must adapt to use human orthologues of the host factor ANP32, which lack a 33 amino acid insertion relative to avian ANP32A. Here we find that influenza polymerase requires ANP32 proteins to support both steps of replication: cRNA and vRNA synthesis. Nevertheless, avian strains are only restricted in vRNA synthesis in human cells. Therefore, avian polymerase can use human ANP32 orthologues to support cRNA synthesis, without acquiring mammalian adaptations. This implies a fundamental difference in the mechanism by which ANP32 proteins support cRNA vs vRNA synthesis.</jats:p><jats:sec><jats:title>Importance</jats:title><jats:p>In order to infect humans and cause a pandemic, avian influenza must first learn how to use human versions of the proteins the virus hijacks for replication – instead of the avian versions found in bird cells. One such protein is ANP32. Understanding the details of how host proteins such as ANP32 support viral activity may allow the design of new antiviral treatments that disrupt these interactions. In this work, we use cells that lack ANP32 to unambiguously demonstrate ANP32 is needed for both steps of influenza genome replication. Surprisingly however, we find that avian influenza can use human ANP32 proteins for the first step of replication without any adaptation, but only avian ANP32 for the second step of replication. This suggests ANP32 may have an additional role in supporting the second step of replication, and it is this activity that is specifically blocked when avian influenza infects human cells.</jats:p></jats:sec>
Wang F, Sheppard CM, Mistry B, et al., 2022, The C-terminal LCAR of host ANP32 proteins interacts with the influenza A virus nucleoprotein to promote the replication of the viral RNA genome, NUCLEIC ACIDS RESEARCH, Vol: 50, Pages: 5713-5725, ISSN: 0305-1048
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- Citations: 5
Pilotto S, Fouqueau T, Lukoyanova N, et al., 2021, Structural basis of RNA polymerase inhibition by viral and host factors, NATURE COMMUNICATIONS, Vol: 12
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- Citations: 3
Werner F, Pilotto S, Fouqueau T, et al., 2021, Structural basis of RNA polymerase inhibition by viral and host factors
<jats:title>Abstract</jats:title> <jats:p>The inhibition of RNA polymerases activity plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. Here we present the high-resolution structures of two such RNAP-inhibitor complexes that provide the structural basis underlying RNAP inhibition in archaea. The Acidianus two-tailed virus (ATV) encodes the RIP factor that binds to the inside the DNA-binding channel of RNAP, inhibiting transcription by occlusion of binding sites for nucleic acid and the transcription initiation factor TFB. Infection with the Sulfolobus Turreted Icosahedral Virus (STIV) induces the expression of the host factor TFS4, which binds in the RNAP secondary channel similarly to eukaryotic transcript cleavage factors. In contrast to RIP, TFS4 binding allosterically induces a widening of the DNA binding channel which disrupts trigger loop and bridge helix motifs. Importantly, the conformational changes induced by TFS4 are closely related to inactivated states of RNAP in other domains of life indicating a deep evolutionary conservation of allosteric RNAP inhibition.</jats:p>
Peacock TP, Sheppard CM, Brown JC, et al., 2021, The SARS-CoV-2 variants associated with infections in India, B.1.617, show enhanced spike cleavage by furin
<jats:title>Abstract</jats:title><jats:p>The spike (S) glycoprotein of the SARS-CoV-2 virus that emerged in 2019 contained a suboptimal furin cleavage site at the S1/S2 junction with the sequence <jats:sub>681</jats:sub>P<jats:bold>RR</jats:bold>A<jats:bold>R</jats:bold>/S<jats:sub>686</jats:sub>. This cleavage site is required for efficient airway replication, transmission, and pathogenicity of the virus. The B.1.617 lineage has recently emerged in India, coinciding with substantial disease burden across the country. Early evidence suggests that B.1.617.2 (a sublineage of B.1.617) is more highly transmissible than contemporary lineages. B.1.617 and its sublineages contain a constellation of S mutations including the substitution P681R predicted to further optimise this furin cleavage site. We provide experimental evidence that virus of the B.1.617 lineage has enhanced S cleavage, that enhanced processing of an expressed B.1.617 S protein in cells is due to P681R, and that this mutation enables more efficient cleavage of a peptide mimetic of the B.1.617 S1/S2 cleavage site by recombinant furin. Together, these data demonstrate viruses in this emerging lineage have enhanced S cleavage by furin which we hypothesise could be enhancing transmissibility and pathogenicity.</jats:p>
Staller E, Sheppard CM, Baillon L, et al., 2021, A natural variant in ANP32B impairs influenza virus replication in human cells, JOURNAL OF GENERAL VIROLOGY, Vol: 102, ISSN: 0022-1317
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- Citations: 4
Staller E, Baillon L, Frise R, et al., 2020, A rare variant in ANP32B impairs influenza virus replication in human cells, biorxiv
Viruses require host factors to support their replication, and genetic variation in such factors can affect susceptibility to infectious disease. Influenza virus replication in human cells relies on ANP32 proteins, which are involved in assembly of replication-competent dimeric influenza virus polymerase (FluPol) complexes. Here, we investigate naturally occurring single nucleotide variants (SNV) in the human Anp32A and Anp32B genes. We note that variant rs182096718 in Anp32B is found at a higher frequency than other variants in either gene. This SNV results in a D130A substitution in ANP32B, which is less able to support FluPol activity than wildtype ANP32B and binds FluPol with lower affinity. Interestingly, ANP32B-D130A exerts a dominant negative effect over wildtype ANP32B and interferes with the functionally redundant paralogue ANP32A. FluPol activity and virus replication are attenuated in CRISPR-edited cells expressing wildtype ANP32A and mutant ANP32B-D130A. We propose a model in which the D130A mutation impairs FluPol dimer formation, thus resulting in compromised replication. We suggest that both homozygous and heterozygous carriers of rs182096718 may have some genetic protection against influenza viruses.
Staller E, Sheppard CM, Neasham PJ, et al., 2019, ANP32 proteins are essential for influenza virus replication in human cells, Journal of Virology, Vol: 93, ISSN: 0022-538X
ANP32 proteins have been implicated in supporting influenza virus replication, but most of the work to date has focused on the ability of avian Anp32 proteins to overcome restriction of avian influenza polymerases in human cells. Using a CRISPR approach we show that human ANP32A and ANP32B are functionally redundant but essential host factors for mammalian-adapted influenza A virus (IAV) and influenza B virus (IBV) replication in human cells. When both proteins are absent from human cells, influenza polymerases are unable to replicate the viral genome, and infectious virus cannot propagate. Provision of exogenous ANP32A or –B recovers polymerase activity and virus growth. We demonstrate that this redundancy is absent in the murine Anp32 orthologues: murine Anp32A is incapable of recovering IAV polymerase activity, while murine Anp32B can. Intriguingly, IBV polymerase is able to use murine Anp32A. We show using a domain swap and point mutations that the LRR 5 region comprises an important functional domain for mammalian ANP32 proteins. Our approach has identified a pair of essential host factors for influenza virus replication and can be harnessed to inform future interventions.
Peacock TP, Sheppard CM, Staller E, et al., 2019, Host Determinants of Influenza RNA Synthesis., Annu Rev Virol
Influenza viruses are a leading cause of seasonal and pandemic respiratory illness. Influenza is a negative-sense single-stranded RNA virus that encodes its own RNA-dependent RNA polymerase (RdRp) for nucleic acid synthesis. The RdRp catalyzes mRNA synthesis, as well as replication of the virus genome (viral RNA) through a complementary RNA intermediate. Virus propagation requires the generation of these RNA species in a controlled manner while competing heavily with the host cell for resources. Influenza virus appropriates host factors to enhance and regulate RdRp activity at every step of RNA synthesis. This review describes such host factors and summarizes our current understanding of the roles they play in viral synthesis of RNA. Expected final online publication date for the Annual Review of Virology Volume 6 is September 30, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Duchi D, Gryte K, Robb NC, et al., 2017, Conformational heterogeneity and bubble dynamics in single bacterial transcription initiation complexes., Nucleic Acids Research, Vol: 46, Pages: 677-688, ISSN: 0305-1048
Transcription initiation is a major step in gene regulation for all organisms. In bacteria, the promoter DNA is first recognized by RNA polymerase (RNAP) to yield an initial closed complex. This complex subsequently undergoes conformational changes resulting in DNA strand separation to form a transcription bubble and an RNAP-promoter open complex; however, the series and sequence of conformational changes, and the factors that influence them are unclear. To address the conformational landscape and transitions in transcription initiation, we applied single-molecule Förster resonance energy transfer (smFRET) on immobilized Escherichia coli transcription open complexes. Our results revealed the existence of two stable states within RNAP-DNA complexes in which the promoter DNA appears to adopt closed and partially open conformations, and we observed large-scale transitions in which the transcription bubble fluctuated between open and closed states; these transitions, which occur roughly on the 0.1 s timescale, are distinct from the millisecond-timescale dynamics previously observed within diffusing open complexes. Mutational studies indicated that the σ70 region 3.2 of the RNAP significantly affected the bubble dynamics. Our results have implications for many steps of transcription initiation, and support a bend-load-open model for the sequence of transitions leading to bubble opening during open complex formation.
Sheppard C, Werner F, 2017, Structure and mechanisms of viral transcription factors in archaea, EXTREMOPHILES, Vol: 21, Pages: 829-838, ISSN: 1431-0651
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- Citations: 9
Sheppard C, Blombach F, Belsom A, et al., 2016, Repression of RNA polymerase by the archaeo-viral regulator ORF145/RIP, NATURE COMMUNICATIONS, Vol: 7, ISSN: 2041-1723
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- Citations: 18
Brown DR, Sheppard CM, Matthews S, et al., 2016, The Xp10 bacteriophage protein P7 inhibits transcription by the major and major variant forms of the host RNA polymerase via a common mechanism, Journal of Molecular Biology, Vol: 428, Pages: 3911-3919, ISSN: 1089-8638
The σ factor is a functionally obligatory subunit of the bacterial transcription machinery, the RNA polymerase. Bacteriophage-encoded small proteins that either modulate or inhibit the bacterial RNAP to allow the temporal regulation of bacteriophage gene expression often target the activity of the major bacterial σ factor, σ70. Previously, we showed that during Xanthomonas oryzae phage Xp10 infection, the phage protein P7 inhibits the host RNAP by preventing the productive engagement with the promoter and simultaneously displaces the σ70 factor from the RNAP. In this study, we demonstrate that P7 also inhibits the productive engagement of the bacterial RNAP containing the major variant bacterial σ factor, σ54, with its cognate promoter. The results suggest for the first time that the major variant form of the host RNAP can also be targeted by bacteriophage-encoded transcription regulatory proteins. Since the major and major variant σ factor interacting surfaces in the RNAP substantially overlap, but different regions of σ70 and σ54 are used for binding to the RNAP, our results further underscore the importance of the σ–RNAP interface in bacterial RNAP function and regulation and potentially for intervention by antibacterials.
Blombach F, Salvadori E, Fouqueau T, et al., 2015, Archaeal TFEα/β is a hybrid of TFIIE and the RNA polymerase III subcomplex hRPC62/39, ELIFE, Vol: 4, ISSN: 2050-084X
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- Citations: 36
Liu B, Shadrin A, Sheppard C, et al., 2014, A bacteriophage transcription regulator inhibits bacterial transcription initiation by Sigma-factor displacement, Nucleic Acids Research, Vol: 42, Pages: 4294-4305, ISSN: 0305-1048
Bacteriophages (phages) appropriate essential processes of bacterial hosts to benefit their own development. The multisubunit bacterial RNA polymerase (RNAp) enzyme, which catalyses DNA transcription, is targeted by phage-encoded transcription regulators that selectively modulate its activity. Here, we describe the structural and mechanistic basis for the inhibition of bacterial RNAp by the transcription regulator P7 encoded by Xanthomonas oryzae phage Xp10. We reveal that P7 uses a two-step mechanism to simultaneously interact with the catalytic β and β’ subunits of the bacterial RNAp and inhibits transcription initiation by inducing the displacement of the σ70-factor on initial engagement of RNAp with promoter DNA. The new mode of interaction with and inhibition mechanism of bacterial RNAp by P7 underscore the remarkable variety of mechanisms evolved by phages to interfere with host transcription.
Liu B, Shadrin A, Sheppard C, et al., 2014, The sabotage of the bacterial transcription machinery by a small bacteriophage protein., Bacteriophage, Vol: 4, ISSN: 2159-7073
Many bacteriophages produce small proteins that specifically interfere with the bacterial host transcription machinery and thus contribute to the acquisition of the bacterial cell by the bacteriophage. We recently described how a small protein, called P7, produced by the Xp10 bacteriophage inhibits bacterial transcription initiation by causing the dissociation of the promoter specificity sigma factor subunit from the host RNA polymerase holoenzyme. In this addendum to the original publication, we present the highlights of that research.
Sheppard C, James E, Barton G, et al., 2013, A non-bacterial transcription factor inhibits bacterial transcription by a multipronged mechanism, RNA BIOLOGY, Vol: 10, Pages: 495-501, ISSN: 1547-6286
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- Citations: 9
Shadrin A, Sheppard C, Savalia D, et al., 2013, Overexpression of <i>Escherichia coli udk</i> mimics the absence of T7 Gp2 function and thereby abrogates successful infection by T7 phage, MICROBIOLOGY-SGM, Vol: 159, Pages: 269-274, ISSN: 1350-0872
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- Citations: 7
Drennan A, Kraemer M, Capp M, et al., 2012, Key Roles of the Downstream Mobile Jaw of <i>Escherichia coli</i> RNA Polymerase in Transcription Initiation, BIOCHEMISTRY, Vol: 51, Pages: 9447-9459, ISSN: 0006-2960
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- Citations: 18
Shadrin A, Sheppard C, Severinov K, et al., 2012, Substitutions in the <i>Escherichia coli</i> RNA polymerase inhibitor T7 Gp2 that allow inhibition of transcription when the primary interaction interface between Gp2 and RNA polymerase becomes compromised, MICROBIOLOGY-SGM, Vol: 158, Pages: 2753-2764, ISSN: 1350-0872
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- Citations: 11
James E, Liu M, Sheppard C, et al., 2012, Structural and Mechanistic Basis for the Inhibition of <i>Escherichia coli</i> RNA Polymerase by T7 Gp2, MOLECULAR CELL, Vol: 47, Pages: 755-766, ISSN: 1097-2765
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- Citations: 34
Drennan A, Saecker R, Heitkamp S, et al., 2012, E. Coli RNA Polymerase: A Molecular DNA Opening Machine, BIOPHYSICAL JOURNAL, Vol: 102, Pages: 286A-286A, ISSN: 0006-3495
Mekler V, Minakhin L, Sheppard C, et al., 2011, Molecular Mechanism of Transcription Inhibition by Phage T7 gp2 Protein, JOURNAL OF MOLECULAR BIOLOGY, Vol: 413, Pages: 1016-1027, ISSN: 0022-2836
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- Citations: 26
Sheppard C, Camara B, Shadrin A, et al., 2011, Reprint of: Inhibition of Escherichia coli RNAp by T7 Gp2 protein: Role of Negatively Charged Strip of Amino Acid Residues in Gp2, JOURNAL OF MOLECULAR BIOLOGY, Vol: 412, Pages: 832-841, ISSN: 0022-2836
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- Citations: 3
Sheppard C, Camara B, Shadrin A, et al., 2011, Inhibition of <i>Escherichia coli</i> RNAp by T7 Gp2 Protein: Role of Negatively Charged Strip of Amino Acid Residues in Gp2, JOURNAL OF MOLECULAR BIOLOGY, Vol: 407, Pages: 623-632, ISSN: 0022-2836
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- Citations: 9
Sherwood V, Manbodh R, Sheppard C, et al., 2008, RASSF7 is a member of a new family of RAS association domain-containing proteins and is required for completing mitosis, MOLECULAR BIOLOGY OF THE CELL, Vol: 19, Pages: 1772-1782, ISSN: 1059-1524
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- Citations: 48
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