Biology subjects

Staller, E.

Publications and source records attributed to Staller, E..

7 recordsLinked to original sources

bioRxiv2026

Influenza A virus initiates genome selection through promoter-dependent nuclear export

Influenza A virus replicates its segmented RNA genome in the nucleus in the context of viral ribonucleoprotein (vRNP) complexes. Newly synthesised vRNPs are exported to the cytoplasm for virion assembly, whereas structurally similar complementary RNPs (cRNPs), which function as replication intermediates, are not incorporated into virions. The molecular basis for this selectivity remains unclear. Here, combining promoter mutagenesis, single-molecule fluorescence in situ hybridisation (smFISH), virus-like particle (VLP) assays and cryo-electron microscopy (cryo-EM), we find that cRNPs are retained in the nucleus and that export competence is encoded by promoter architecture, with the length of the single-stranded 3' promoter region as the principal contributing feature and promoter sequence and duplex architecture further modulating export efficiency. cRNPs carrying export-competent promoters are incorporated into virus-like particles, demonstrating a functional link between nuclear export and packaging competence. Cryo-EM analyses reveal that both 3' vRNA and cRNA promoters bind a common polymerase surface site but are associated with distinct polymerase conformational landscapes, with only the vRNA promoter being compatible with an encapsidase conformation of the polymerase. Together, our findings identify a promoter-dependent checkpoint that links RNA promoter architecture and polymerase conformation to RNP export and packaging. These results demonstrate that influenza virus initiates genome selection in the nucleus and reveal nuclear export as an early step in genome selection.

microbiology↗

bioRxiv2025

Polymerase mutations underlie adaptation of H5N1 influenza virus to dairy cattle and other mammals.

In early 2024, an unprecedented outbreak of H5N1 high pathogenicity avian influenza was detected in dairy cattle in the USA1. As of mid-2025 the epidemic is ongoing, resulting in spillbacks into poultry, wild birds and other mammals including humans2. Here, we present molecular and virological evidence that the cattle B3.13 genotype H5N1 viruses rapidly accumulated adaptations in polymerase genes that enabled better replication in bovine cells and tissues, as well as cells of other mammalian species including humans and pigs. We find evidence of several mammalian adaptations gained early in the evolution of these viruses in cattle including PB2 M631L, which is found in all cattle sequences, and PA K497R, which is found in the majority. Structurally, PB2 M631L maps to the polymerase-ANP32 interface, an essential host factor for viral genome replication. We show that this mutation adapts the polymerase to better interact with bovine ANP32 proteins, particularly ANP32A, and thereby enhances virus replication in bovine mammary systems and primary human airway cultures. Importantly, we show that ongoing evolution during 2024 and 2025 in the PB2 gene, including E627K and a convergently arising D740N substitution, further increase polymerase activity and virus replication in a range of mammalian cells. Thus, the continued circulation of H5N1 in dairy cattle not only allows virus adaption improving replicative ability in cattle, but also increases the risk of zoonotic spillover.

microbiology↗

bioRxiv2020

The furin cleavage site of SARS-CoV-2 spike protein is a key determinant for transmission due to enhanced replication in airway cells.

SARS-CoV-2 enters cells via its spike glycoprotein which must be cleaved sequentially at the S1/S2, then the S2 cleavage sites (CS) to mediate membrane fusion. SARS-CoV-2 has a unique polybasic insertion at the S1/S2 CS, which we demonstrate can be cleaved by furin. Using lentiviral pseudotypes and a cell-culture adapted SARS-CoV-2 virus with a S1/S2 deletion, we show that the polybasic insertion is selected for in lung cells and primary human airway epithelial cultures but selected against in Vero E6, a cell line used for passaging SARS-CoV-2. We find this selective advantage depends on expression of the cell surface protease, TMPRSS2, that allows virus entry independent of endosomes thus avoiding antiviral IFITM proteins. SARS-CoV-2 virus lacking the S1/S2 furin CS was shed to lower titres from infected ferrets and was not transmitted to cohoused sentinel animals. Thus, the polybasic CS is a key determinant for efficient SARS-CoV-2 transmission.

microbiology↗

bioRxiv2020

Mammalian ANP32A and ANP32B proteins drive alternative avian influenza virus polymerase adaptations

ANP32 proteins, which act as influenza polymerase co-factors, vary between birds and mammals. The well-known mammalian adaptation, PB2-E627K, enables influenza polymerase to use mammalian ANP32 proteins. However, some mammalian-adapted influenza viruses do not harbour this adaptation. Here, we show that alternative PB2 adaptations, Q591R and D701N also allow influenza polymerase to use mammalian ANP32 proteins. PB2-E627K strongly favours 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 more strongly pro-viral. In an experimental evolution approach, passage of avian viruses in human cells drives acquisition of PB2-E627K, but not when ANP32B is ablated. The strong pro-viral support of ANP32B for PB2-E627K maps to the LCAR region of ANP32B.

microbiology↗

bioRxiv2020

A rare 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 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.

microbiology↗

bioRxiv2020

Swine ANP32A supports avian influenza virus polymerase

Avian influenza viruses occasionally infect and adapt to mammals, including humans. Swine are often described as mixing vessels, being susceptible to both avian and human origin viruses, which allows the emergence of novel reassortants, such as the precursor to the 2009 H1N1 pandemic. ANP32 proteins are host factors that act as influenza virus polymerase cofactors. In this study we describe how swine ANP32A, uniquely among the mammalian ANP32 proteins tested, supports some, albeit limited, activity of avian origin influenza virus polymerases. We further show that after the swine-origin influenza virus emerged in humans and caused the 2009 pandemic it evolved polymerase gene mutations that enabled it to more efficiently use human ANP32 proteins. We map the super pro-viral activity of swine ANP32A to a pair of amino acids, 106 and 156, in the LRR and central domains and show these mutations enhance binding to influenza virus trimeric polymerase. These findings help elucidate the molecular basis for the mixing vessel trait of swine and further our understanding of the evolution and ecology of viruses in this host.

microbiology↗

bioRxiv2019

Avian ANP32B does not support influenza A virus polymerase and influenza A virus relies exclusively on ANP32A in chicken cells.

Influenza A viruses (IAV) are subject to species barriers that prevent frequent zoonotic transmission and pandemics. One of these barriers is the poor activity of avian IAV polymerases in human cells. Differences between avian and mammalian ANP32 proteins underlie this host range barrier. Human ANP32A and ANP32B homologues both support function of human-adapted influenza polymerase but do not support efficient activity of avian IAV polymerase which requires avian ANP32A. We show here that avian ANP32B is evolutionarily distinct from mammalian ANP32B, and that chicken ANP32B does not support IAV polymerase activity even of human-adapted viruses. Consequently, IAV does not replicate in chicken cells that lack ANP32A. Amino acid differences in LRR5 domain accounted for the inactivity of chicken ANP32B. Transfer of these residues to chicken ANP32A abolished support of IAV polymerase. Understanding ANP32 function will help develop antiviral strategies and aid the design of influenza virus resistant genome edited chickens.

microbiology↗