Deep mutational scanning identifies sites in influenza nucleoprotein that affect viral inhibition by MxA

Ashenberg, Orr; Jai, Padmakumar; Doud, Michael B.; Bloom, Jesse D.

doi:10.1371/journal.ppat.1006288

Cited by 65 publications

(73 citation statements)

References 69 publications

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“…The passaged HA had two mutations, G78D and T212I, which enhanced viral growth as shown in Figure S1. The HA with these two mutations was cloned into pHW2000 (81) and pICR2 (82) to create pHW-Perth09-HA-G78D-T212I and pICR2-Perth09-HA-G78D-T212I. For all subsequent experiments, we used viruses with the HA containing these two mutations to improve titers and viral genetic stability, and this is the HA that we refer to as Perth/2009 in the manuscript text.…”

Section: Creation Of Mdck-siat1-tmprss2 Cells When Growing Influenzamentioning

confidence: 99%

Deep mutational scanning of hemagglutinin helps predict evolutionary fates of human H3N2 influenza variants

Lee

Huddleston

Doud

et al. 2018

Preprint

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Human influenza virus rapidly accumulates mutations in its major surface protein hemagglutinin (HA). The evolutionary success of influenza virus lineages depends on how these mutations affect HA's functionality and antigenicity. Here we experimentally measure the effects on viral growth in cell culture of all single amino-acid mutations to the HA from a recent human H3N2 influenza virus strain. We show that mutations that are measured to be more favorable for viral growth are enriched in evolutionarily successful H3N2 viral lineages relative to mutations that are measured to be less favorable for viral growth. Therefore, despite the well-known caveats about cell-culture measurements of viral fitness, such measurements can still be informative for understanding evolution in nature. We also compare our measurements for H3 HA to similar data previously generated for a distantly related H1 HA, and find substantial differences in which amino acids are preferred at many sites. For instance, the H3 HA has less disparity in mutational tolerance between the head and stalk domains than the H1 HA. Overall, our work suggests that experimental measurements of mutational effects can be leveraged to help understand the evolutionary fates of viral lineages in nature -but only when the measurements are made on a viral strain similar to the ones being studied in nature.influenza virus | hemagglutinin | deep mutational scanning | antigenic drift | epistasis S easonal H3N2 influenza virus evolves rapidly, fixing 3 to 4 amino-acid mutations per year in its hemagglutinin (HA) surface protein (1, 2). Many of these mutations contribute to the rapid antigenic drift that necessitates frequent updates to the annual influenza vaccine (3). This evolution is further characterized by competition and turnover among groups of strains called clades bearing different complements of mutations (4-8). Clades vary widely in their evolutionary success, with some dying out soon after emergence and others going on to take over the virus population. Several lines of evidence indicate that successful clades have higher fitness than clades that remain at low frequency (4-6, 9). A key goal in the study of H3N2 evolution is to identify the features that enable certain clades to succeed as others die out.Two main characteristics distinguish evolutionarily successful clades from their competitors: greater antigenic change, and efficient viral growth and transmission. In principle, experiments could be informative for identifying how mutations affect these features. Most work on influenza evolution to date has utilized experimental data to assess the antigenicity of circulating strains (11)(12)(13)(14)(15)(16). However, the non-antigenic effects of mutations also play an important role (5,7,9,17). Specifically, due to influenza virus's high mutation rate (18)(19)(20) and lack of intra-segment recombination (21), deleterious mutations become linked to beneficial ones. The resulting accumulation of deleterious mutations can affect non-antigenic properties central...

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Section: Creation Of Mdck-siat1-tmprss2 Cells When Growing Influenzamentioning

confidence: 99%

Deep mutational scanning of hemagglutinin helps predict evolutionary fates of human H3N2 influenza variants

Lee

Huddleston

Doud

et al. 2018

Preprint

Self Cite

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“…5F suggest that NA43 does not substantially contribute to viral 452 growth in cell culture, but simply titering viral supernatants generated by reverse 453 genetics is a relatively insensitive way to quantify how mutations affect growth. We 454 therefore performed competition assays between viruses with wildtype NA and NA 455 lacking the start site at position 43, since such assays are a more sensitive way to detect 456 small differences in viral fitness [104]. We performed these assays by mixing virus with 457 wildtype NA with either the 1 wt 43 GUA or 1 wt 43 UUA mutant in roughly equal 458 proportion, and then allowing the viruses to compete in low-MOI infections in cell 459 culture.…”

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confidence: 99%

Comprehensive profiling of translation initiation in influenza virus infected cells

Machkovech

Bloom

Subramaniam

2018

Preprint

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Translation can initiate at alternate, non-canonical start codons in response to stressful stimuli in mammalian cells. Recent studies suggest that viral infection and anti-viral responses alter sites of translation initiation, and in some cases, lead to production of novel immune epitopes. Here we systematically investigate the extent and impact of alternate translation initiation in cells infected with influenza virus. We perform evolutionary analyses that suggest selection against non-canonical initiation at CUG codons in influenza virus lineages that have adapted to mammalian hosts. We then use ribosome profiling with the initiation inhibitor lactidomycin to experimentally delineate translation initiation sites in a human lung epithelial cell line infected with influenza virus. We identify several candidate sites of alternate initiation in influenza mRNAs, all of which occur at AUG codons that are downstream of canonical initiation codons. One of these candidate downstream start sites truncates 14 amino acids from the N-terminus of the N1 neuraminidase protein, resulting in loss of its cytoplasmic tail and a portion of the transmembrane domain. This truncated neuraminidase protein is expressed on the cell surface during influenza virus infection, is enzymatically active, and is conserved in most N1 viral lineages. Host transcripts induced by the anti-viral response are enriched for translation initiation at non-canonical start sites and non-AUG start codons. Together, our results systematically map the landscape of translation initiation during influenza virus infection, and shed light on the evolutionary forces shaping this landscape.

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“…In particular, it is possible that intergenic correlations reveal the nature of tradeoffs shaping the life history strategies in viruses (De Paepe and Taddei, 2006). Future modeling (Neverov et al ., 2015) and empirical (Ashenberg et al ., 2017) work should probably focus on elucidating not only these constraints, but also the theoretical basis connecting correlated evolution, if not epistasis, to selection in shaping the evolutionary strategies of biological replicators.…”

Section: Resultsmentioning

confidence: 99%

Episodic diversifying selection and intragenomic interactions shape the evolution of DNA and RNA viruses

Aris‐Brosou

Parent

Ibeh

2019

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11Viruses are known to have some of the highest and most diverse mutation rates found 12 in any biological replicator, with single-stranded (ss) RNA viruses evolving the fastest, 13 and double-stranded (ds) DNA viruses having rates approaching those of bacteria. As 14 mutation rates are tightly and negatively correlated with genome size, selection is a clear 15 driver of viral evolution. However, the role of intragenomic interactions as drivers of 16 viral evolution is still unclear. To understand how these two processes affect the long-17 term evolution of viruses infecting humans, we comprehensively analyzed ssRNA, ssDNA, 18 dsRNA, and dsDNA viruses, to find which virus types and which functions show evidence 19 for episodic diversifying selection and correlated evolution. We show that selection mostly 20 affects single stranded viruses, that correlated evolution is more prevalent in DNA viruses, 21 and that both processes, taken independently, mostly affect viral replication. However, 22 the genes that are jointly affected by both processes are involved in key aspects of their life 23 cycle, favoring viral stability over proliferation. We further show that both evolutionary 24 processes are intimately linked at the amino acid level, which suggests that it is the 25 joint action of selection and correlated evolution, and not just selection, that shapes the 26 evolutionary trajectories of viruses -and possibly of their epidemiological potential.27

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Deep mutational scanning identifies sites in influenza nucleoprotein that affect viral inhibition by MxA

Cited by 65 publications

References 69 publications

Deep mutational scanning of hemagglutinin helps predict evolutionary fates of human H3N2 influenza variants

Deep mutational scanning of hemagglutinin helps predict evolutionary fates of human H3N2 influenza variants

Comprehensive profiling of translation initiation in influenza virus infected cells

Episodic diversifying selection and intragenomic interactions shape the evolution of DNA and RNA viruses

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