Specific Residues in the 2009 H1N1 Swine-Origin Influenza Matrix Protein Influence Virion Morphology and Efficiency of Viral Spread In Vitro

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Influenza virus strains are often pleiomorphic, a characteristic that is largely attributed to specific residues in matrix protein 1 (M1). Although the mechanism by which M1 controls virion morphology has not yet been defined, it is suggested that the M1 interaction with other viral proteins plays an important role. In this study, we rescued recombinant virus WSN-AichiM1 containing the spherical A/WSN/33 (WSN) backbone and the M1 protein from A/Aichi/2/68 (Aichi). Aichi M1 differs from WSN M1 by 7 amino acids but includes those identified to be responsible for filamentous virion formation. Interestingly, Aichi virus produced spherical virions, while WSN-AichiM1 exhibited a long filamentous morphology, as detected by immunofluorescence and electron microscopy. Additional incorporation of Aichi nucleoprotein (NP) but not the hemagglutinin (HA), neuraminidase (NA), or M2 gene to WSN-AichiM1 abrogated filamentous virion formation, suggesting that specific M1-NP interactions affect virion morphology. Further characterization of viruses containing WSN/Aichi chimeric NPs identified residues 214, 217, and 253 of Aichi NP as necessary and sufficient for the formation of spherical virions. NP residues 214 and 217 localize at the minor groove between the two opposite-polarity NP helical strands of viral ribonucleocapsids, and residue 253 also localizes near the surface of the groove. These findings indicate that NP plays a critical role in influenza virus morphology, possibly through its interaction with the M1 layer during virus budding.

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confidence: 64%

Specific Nucleoprotein Residues Affect Influenza Virus Morphology

Bialas

Bussey

Stone

et al. 2014

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“…Several previous studies have demonstrated that the gene products of the M segment affect the morphology of the influenza virion (28,29,31,32). Elleman and Barclay (29) reported that differences in morphology between (i) the laboratory-adapted strains A/PR/8/34 and A/WSN/33 and (ii) the human isolate The matrix proteins of PR8 and NL602 have 13 amino acid differences (95% identity), while the M2 proteins of the strains differ by 14 amino acids (86% identity).…”

Section: Discussionmentioning

confidence: 99%

“…M2 is a transmembrane channel protein that is involved in (i) membrane scission during viral egress (23); (ii) acidification of the virion during viral entry, which leads to release of vRNPs from the M1 protein (24)(25)(26)(27); and (iii) preventing premature conformational change of specific hemagglutinin proteins in the Golgi complex, mediated through control of the pH within this organelle (24,25,27). Both M1 and M2 have been identified as determinants of influenza virion morphology (28)(29)(30)(31)(32)(33), and point mutations in either protein can convert an exclusively spherical influenza virus (approximately 200 nm diameter) to one that produces a mixture of spheres and filaments (which can be greater than 1 m in length) and vice versa (28)(29)(30)(31)(32)(33)(34). As clinical isolates of influenza A virus overwhelmingly include some percentage of filamentous virions and laboratory adaptation frequently results in the generation of strains with an exclusively spherical morphology (29,34), an understanding of the biological relevance of filamentous influenza A virus particles has long been sought.…”

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confidence: 99%

The M Segment of the 2009 Pandemic Influenza Virus Confers Increased Neuraminidase Activity, Filamentous Morphology, and Efficient Contact Transmissibility to A/Puerto Rico/8/1934-Based Reassortant Viruses

Campbell

Danzy

Kyriakis

et al. 2014

103

117

To aid in understanding how the M segment might affect transmission, we evaluated neuraminidase activity and virion morphology of reassortant viruses. Transmission was found to correlate with higher neuraminidase activity and a more filamentous morphology. Importantly, we found that introduction of the pandemic M segment alone resulted in an increase in the neuraminidase activity of two pairs of otherwise isogenic PR8-based viruses. Thus, our data demonstrate the surprising result that functions encoded by the influenza A virus M segment impact neuraminidase activity and, perhaps through this mechanism, have a potent effect on transmissibility. IMPORTANCEOur work uncovers a previously unappreciated mechanism through which the influenza A virus M segment can alter the receptor-destroying activity of an influenza virus. Concomitant with changes to neuraminidase activity, the M segment impacts the morphology of the influenza A virion and transmissibility of the virus in the guinea pig model. We suggest that changes in NA activity underlie the ability of the influenza M segment to influence virus transmissibility. Furthermore, we show that coadapted M, NA, and HA segments are required to provide optimal transmissibility to an influenza virus. The M-NA functional interaction we describe appears to underlie the prominent role of the 2009 pandemic M segment in supporting efficient transmission and may be a highly important means by which influenza A viruses restore HA/NA balance following reassortment or transfer to new host environments.

“…Plasmids. Construction of pCAGGS and pPolI vectors containing A/chicken/Nanchang/3-120/01 (Nan) and Cal genes were described previously (3,16,17). Chimera PB2 genes were created using compatible restriction sites in Nan and Cal PB2 and then cloned into pCAGGS.…”

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confidence: 99%

Identification of Influenza A Virus PB2 Residues Involved in Enhanced Polymerase Activity and Virus Growth in Mammalian Cells at Low Temperatures

Hayashi

Wills

Bussey

et al. 2015

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Mutations in the polymerase genes are known to play a major role in avian influenza virus adaptation to mammalian hosts. Despite having avian origin PA and PB2, the 2009 pandemic H1N1 virus (pH1N1) can replicate well in mammalian respiratory tracts, suggesting that these proteins have acquired mutations for efficient growth in humans. We have previously shown that PA from the pH1N1 virus A/California/04/09 (Cal) strongly enhances activity of an otherwise avian polymerase complex derived from A/chicken/Nanchang/3-120/01 (Nan) in mammalian cells. However, this enhancement was observed at 37°C but not at the lower temperature of 34°C. An additional introduction of Cal PB2 enhanced activity at 34°C, suggesting the presence of unidentified residues in Cal PB2 that are required for efficient growth at low temperature. Here, we sought to determine the key PB2 residues which confer enhanced polymerase activity and virus growth in human cells at low temperature. Using a reporter gene assay, we identified novel mutations, PB2 V661A and V683T/A684S, which are involved in enhanced Cal polymerase activity at low temperature. The PB2 T271A mutation, which we previously reported, also contributed to enhanced activity. The growth of recombinant Cal containing PB2 with Nan residues 271T/661V/683V/684A was strongly reduced in human cells compared to wild-type virus at low temperature. Among the four residues, 271A and 684S are conserved in human and pH1N1 viruses but not in avian viruses, suggesting an important role in mammalian adaptation of pH1N1 virus. IMPORTANCEThe PB2 protein plays a key role in the host adaptation, cold sensitivity, and pathogenesis of influenza A virus. Despite containing an avian origin PB2 lacking the mammalian adaptive mutations 627K or 701N, pH1N1 influenza virus strains can replicate efficiently in the low temperature upper respiratory tract of mammals, suggesting the presence of unknown mutations in the pH1N1 PB2 protein responsible for its low temperature adaptation. Here, in addition to PB2 271A, which has been shown to increase polymerase activity, we identified novel PB2 residues 661A and 683T/684S in pH1N1 which confer enhanced polymerase activity and virus growth in mammalian cells especially at low temperature. Our findings suggest that the presence of these PB2 residues contributes to efficient replication of the pH1N1 virus in the upper respiratory tract, which resulted in efficient human-to-human transmission of this virus.