Virion morphology and on-virus spike protein structures of diverse SARS-CoV-2 variants.

The evolution of SARS-CoV-2 variants with increased fitness has been accompanied by structural changes in the spike (S) proteins, which are the major target for the adaptive immune response. Single-particle cryo-EM analysis of soluble S protein from SARS-CoV-2 variants has revealed this structural adaptation at high resolution. The analysis of S trimers in situ on intact virions has the potential to provide more functionally relevant insights into S structure and virion morphology.

Structures of H5N1 influenza polymerase with ANP32B reveal mechanisms of genome replication and host adaptation.

Avian influenza A viruses (IAVs) pose a public health threat, as they are capable of triggering pandemics by crossing species barriers. Replication of avian IAVs in mammalian cells is hindered by species-specific variation in acidic nuclear phosphoprotein 32 (ANP32) proteins, which are essential for viral RNA genome replication. Adaptive mutations enable the IAV RNA polymerase (FluPolA) to surmount this barrier.

An Influenza A virus can evolve to use human ANP32E through altering polymerase dimerization.

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.

Mammalian ANP32A and ANP32B Proteins Drive Differential Polymerase Adaptations in Avian Influenza Virus.

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.

Avian Influenza A Virus Polymerase Can Utilize Human ANP32 Proteins To Support cRNA but Not vRNA Synthesis.

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.

The C-terminal LCAR of host ANP32 proteins interacts with the influenza A virus nucleoprotein to promote the replication of the viral RNA genome.

The segmented negative-sense RNA genome of influenza A virus is assembled into ribonucleoprotein complexes (RNP) with viral RNA-dependent RNA polymerase and nucleoprotein (NP). It is in the context of these RNPs that the polymerase transcribes and replicates viral RNA (vRNA). Host acidic nuclear phosphoprotein 32 (ANP32) family proteins play an essential role in vRNA replication by mediating the dimerization of the viral polymerase via their N-terminal leucine-rich repeat (LRR) domain.

Structural basis of RNA polymerase inhibition by viral and host factors.

RNA polymerase inhibition 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 bases underlying RNAP inhibition in archaea. The Acidianus two-tailed virus encodes the RIP factor that binds inside the DNA-binding channel of RNAP, inhibiting transcription by occlusion of binding sites for nucleic acid and the transcription initiation factor TFB.

A natural variant in ANP32B impairs influenza virus replication in human cells.

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 and genes.

Host Determinants of Influenza RNA Synthesis.

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.

ANP32 Proteins Are Essential for Influenza Virus Replication in Human Cells.

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 the human cidic uclear hosphoproteins (ANPs) 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.

Conformational heterogeneity and bubble dynamics in single bacterial transcription initiation complexes.

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.

Structure and mechanisms of viral transcription factors in archaea.

Virus-encoded transcription factors have been pivotal in exploring the molecular mechanisms and regulation of gene expression in bacteria and eukaryotes since the birth of molecular biology, while our understanding of viral transcription in archaea is still in its infancy. Archaeal viruses do not encode their own RNA polymerases (RNAPs) and are consequently entirely dependent on their hosts for gene expression; this is fundamentally different from many bacteriophages and requires alternative regulatory strategies. Archaeal viruses wield a repertoire of proteins to expropriate the host transcription machinery to their own benefit.

Repression of RNA polymerase by the archaeo-viral regulator ORF145/RIP.

Little is known about how archaeal viruses perturb the transcription machinery of their hosts. Here we provide the first example of an archaeo-viral transcription factor that directly targets the host RNA polymerase (RNAP) and efficiently represses its activity. ORF145 from the temperate Acidianus two-tailed virus (ATV) forms a high-affinity complex with RNAP by binding inside the DNA-binding channel where it locks the flexible RNAP clamp in one position.

Archaeal TFEα/β is a hybrid of TFIIE and the RNA polymerase III subcomplex hRPC62/39.

Transcription initiation of archaeal RNA polymerase (RNAP) and eukaryotic RNAPII is assisted by conserved basal transcription factors. The eukaryotic transcription factor TFIIE consists of α and β subunits. Here we have identified and characterised the function of the TFIIEβ homologue in archaea that on the primary sequence level is related to the RNAPIII subunit hRPC39.

The sabotage of the bacterial transcription machinery by a small bacteriophage protein.

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.

A bacteriophage transcription regulator inhibits bacterial transcription initiation by σ-factor displacement.

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.

A non-bacterial transcription factor inhibits bacterial transcription by a multipronged mechanism.

The process of transcription initiation is the major target for regulation of gene expression in bacteria and is performed by a multi-subunit RNA polymerase enzyme (RNAp). A complex network of regulatory elements controls the activity of the RNAp to fine-tune transcriptional output. Thus, RNAp is a nexus for controlling bacterial gene expression at the transcription level.

Overexpression of Escherichia coli udk mimics the absence of T7 Gp2 function and thereby abrogates successful infection by T7 phage.

Successful infection of Escherichia coli by bacteriophage T7 relies upon the transcription of the T7 genome by two different RNA polymerases (RNAps). The bacterial RNAp transcribes early T7 promoters, whereas middle and late T7 genes are transcribed by the T7 RNAp. Gp2, a T7-encoded transcription factor, is a 7 kDa product of an essential middle T7 gene 2, and is a potent inhibitor of the host RNAp.

Key roles of the downstream mobile jaw of Escherichia coli RNA polymerase in transcription initiation.

Differences in kinetics of transcription initiation by RNA polymerase (RNAP) at different promoters tailor the pattern of gene expression to cellular needs. After initial binding, large conformational changes occur in promoter DNA and RNAP to form initiation-capable complexes. To understand the mechanism and regulation of transcription initiation, the nature and sequence of these conformational changes must be determined.

Substitutions in the Escherichia coli RNA polymerase inhibitor T7 Gp2 that allow inhibition of transcription when the primary interaction interface between Gp2 and RNA polymerase becomes compromised.

The Escherichia coli-infecting bacteriophage T7 encodes a 7 kDa protein, called Gp2, which is a potent inhibitor of the host RNA polymerase (RNAp). Gp2 is essential for T7 phage development. The interaction site for Gp2 on the E.

Structural and mechanistic basis for the inhibition of Escherichia coli RNA polymerase by T7 Gp2.

The T7 phage-encoded small protein Gp2 is a non-DNA-binding transcription factor that interacts with the jaw domain of the Escherichia coli (Ec) RNA polymerase (RNAp) β' subunit and inhibits transcriptionally proficient promoter-complex (RPo) formation. Here, we describe the high-resolution solution structure of the Gp2-Ec β' jaw domain complex and show that Gp2 and DNA compete for binding to the β' jaw domain. We reveal that efficient inhibition of RPo formation by Gp2 requires the amino-terminal σ(70) domain region 1.

Molecular mechanism of transcription inhibition by phage T7 gp2 protein.

Escherichia coli T7 bacteriophage gp2 protein is a potent inhibitor of host RNA polymerase (RNAP). gp2 inhibits formation of open promoter complex by binding to the β' jaw, an RNAP domain that interacts with downstream promoter DNA. Here, we used an engineered promoter with an optimized sequence to obtain and characterize a specific promoter complex containing RNAP and gp2.

Reprint of: inhibition of Escherichia coli RNAp by T7 Gp2 protein: role of negatively charged strip of amino acid residues in Gp2.

Gp2, a 7 kDa protein encoded by T7 bacteriophage, is a potent inhibitor of Escherichia coli RNA polymerase (RNAp), the enzyme responsible for transcription of all bacterial genes and early viral genes. A prominent feature in the structure of Gp2 is a contiguous strip of seven negatively charged amino acid residues (negatively charged strip or NCS), located along one side of the molecule. The role of the NCS in Gp2 function is not known.

Inhibition of Escherichia coli RNAp by T7 Gp2 protein: role of negatively charged strip of amino acid residues in Gp2.

Gp2, a 7 kDa protein encoded by T7 bacteriophage, is a potent inhibitor of Escherichia coli RNA polymerase (RNAp), the enzyme responsible for transcription of all bacterial genes and early viral genes. A prominent feature in the structure of Gp2 is a contiguous strip of seven negatively charged amino acid residues (negatively charged strip or NCS), located along one side of the molecule. The role of the NCS in Gp2 function is not known.