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Plasmin-mediated activation of pandemic H1N1 influenza virus hemagglutinin is independent of the viral neuraminidase.

ABSTRACT: Influenza virus is well recognized to modulate host tropism and pathogenesis based on mutations in the proteolytic cleavage site of the viral hemagglutinin (HA), which activates HA and exposes the fusion peptide for membrane fusion. Instead of the conventional trypsin-mediated cleavage event, modification of the cleavage site allows extended use of host cell proteases and enhanced spread in vivo. For H1N1 influenza viruses, the mouse-adapted A/WSN/33 strain is known to replicate in the brain based on recruitment of plasminogen by the viral neuraminidase (NA), as well as a Ser-Tyr substitution at the P2 position of the HA cleavage site. Here, we show that an equivalent Ser-Tyr substitution has occurred in the HA of naturally occurring human H1N1 influenza viruses. We characterize one of these viruses (A/Beijing/718/2009), as well as the prototype A/California/04/2009 with a Ser-Tyr substitution in the cleavage site, and show that these HAs are preferentially cleaved by plasmin. Importantly, cleavage activation by plasmin/plasminogen was independent of the viral NA, suggesting a novel mechanism for HA cleavage activation. We show that the viral HA itself can recruit plasminogen for HA cleavage. We further show that cellular factors, as well as streptokinase from bacteria commonly coinfecting the respiratory tract of influenza patients, can be a source of activated plasminogen for plasmin-mediated cleavage of influenza virus HAs that contain a Ser-Tyr substitution in the cleavage site.

SUBMITTER: Tse LV

PROVIDER: S-EPMC3624321 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Plasmin-mediated activation of pandemic H1N1 influenza virus hemagglutinin is independent of the viral neuraminidase.

Tse Longping V LV Marcano Valerie C VC Huang Weishan W Pocwierz Misty S MS Whittaker Gary R GR

Journal of virology 20130228 9

Influenza virus is well recognized to modulate host tropism and pathogenesis based on mutations in the proteolytic cleavage site of the viral hemagglutinin (HA), which activates HA and exposes the fusion peptide for membrane fusion. Instead of the conventional trypsin-mediated cleavage event, modification of the cleavage site allows extended use of host cell proteases and enhanced spread in vivo. For H1N1 influenza viruses, the mouse-adapted A/WSN/33 strain is known to replicate in the brain bas ...[more]

PMID: 23449787

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Project description:Analyzing the evolutionary pattern of the influenza A(H1N1)pdm09 strain in different regions is important for understanding its diversification. We therefore conducted this study to elucidate the genetic variability and molecular evolution of the influenza A(H1N1)pdm09 strains that circulated during the 2009-2010 and 2010-2011 influenza seasons in Sendai, Japan. Nasopharyngeal swab specimens were collected from patients with influenza-like illnesses who visited outpatient clinics in Sendai City, Japan, from September 2009 to April 2011. A total of 75 isolates were selected from September 2009 to April 2011 to analyze the genetic changes in the entire hemagglutinin 1 (HA1) segment of the HA gene and the neuraminidase (NA) gene based on sequence analysis. Bayesian coalescent Markov chain Monte Carlo analyses of HA1 and NA gene sequences were performed for further analysis. High sequence identities were observed for HA1 and NA in influenza A(H1N1)pdm09, displaying 99.06 and 99.33 % nucleotide identities, respectively, with the A(H1N1)pdm09 vaccine strain A/California/07/2009. The substitution rates of nucleotides for HA1 in the 2009-2010 and 2010-2011 were 1.5 × 10-3 and 1.6 × 10-3 substitutions per site per year, respectively. Phylogenetic tree analysis demonstrated that Sendai isolates were clustered into global clade 7, which is characterized by an S203T mutation in the HA1 gene. Moreover, two distinct circulation clusters were present in the 2010-2011 season. Mutations were present in antigenic or receptor-binding domains of the HA1 segment, including A141V, S143G, S183P, S185T, and S203T. The Bayesian skyline plot model illustrated a steady rate for the maintenance of genetic diversity, followed by a slight increase in the later part of the 2010-2011 season. Selection analysis revealed that the HA1 (position 197) and NA (position 46) sites were under positive selection; however, no known mutation conferring resistance to NA inhibitors such as H275Y was observed. The effect on control of the influenza A(H1N1)pdm09 virus, including vaccine strain selection, requires continuous monitoring of the strain by genetic surveillance.

Project description:D222G/N substitutions in A(H1N1)pdm09 hemagglutinin may be associated with increased binding of viruses causing low respiratory tract infections and human pathogenesis. We assessed the impact of such substitutions on the balance between hemagglutinin binding and neuraminidase cleavage, viral growth and in vivo virulence.Seven viruses with differing polymorphisms at codon 222 (2 with D, 3 G, 1 N and 1 E) were isolated from patients and characterized with regards hemagglutinin binding affinity (Kd) to α-2,6 sialic acid (SAα-2,6) and SAα-2,3 and neuraminidase enzymatic properties (Km, Ki and Vmax). The hemagglutination assay was used to quantitatively assess the balance between hemagglutinin binding and neuraminidase cleavage. Viral growth properties were compared in vitro in MDCK-SIAT1 cells and in vivo in BALB/c mice. Compared with D222 variants, the binding affinity of G222 variants was greater for SAα-2,3 and lower for SAα-2,6, whereas that of both E222 and N222 variants was greater for both SAα-2,3 and SAα-2,6. Mean neuraminidase activity of D222 variants (16.0 nmol/h/10(6)) was higher than that of G222 (1.7 nmol/h/10(6) viruses) and E/N222 variants (4.4 nmol/h/10(6) viruses). The hemagglutination assay demonstrated a deviation from functional balance by E222 and N222 variants that displayed strong hemagglutinin binding but weak neuraminidase activity. This deviation impaired viral growth in MDCK-SIAT1 cells but not infectivity in mice. All strains but one exhibited low infectious dose in mice (MID50) and replicated to high titers in the lung; this D222 strain exhibited a ten-fold higher MID50 and replicated to low titers. Hemagglutinin-neuraminidase balance status had a greater impact on viral replication than hemagglutinin affinity strength, at least in vitro, thus emphasizing the importance of an optimal balance for influenza virus fitness. The mouse model is effective in assessing binding to SAα-2,3 but cannot differentiate SAα-2,3- from SAα-2,6- preference, nor estimate the hemagglutinin-neuraminidase balance in A(H1N1)pdm09 strains.

Dataset Information

Plasmin-mediated activation of pandemic H1N1 influenza virus hemagglutinin is independent of the viral neuraminidase.

Publications

Plasmin-mediated activation of pandemic H1N1 influenza virus hemagglutinin is independent of the viral neuraminidase.

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