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. 2013 May;87(9):5161-9.
doi: 10.1128/JVI.00210-13. Epub 2013 Feb 28.

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

Longping V Tse  1 , Valerie C Marcano, Weishan Huang, Misty S Pocwierz, Gary R Whittaker
Affiliations

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

Longping V Tse et al. J Virol. 2013 May.

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.

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Figures

Fig 1
Fig 1
Multiple-sequence alignment of the influenza virus H1 HA cleavage site and fusion peptide. (A) A total of 4,325 H1N1 HA amino acid (aa) sequences of H1N1 from the NCBI Influenza Virus Resource (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html) were aligned using muscle v3.8.31, and the aligned sequences of representative viruses at the HA cleavage site are shown along with the consensus sequence. Dots represent identical residues, P2 to P1′ represent positions of the cleavage site, and the location of the fusion peptide is shown. The representative strains selected are the following: A/South Carolina/1/1918 (accession number AAD17229), A/Puerto Rico/8/1934 (ABO21709), A/Wilson-Smith/1933 (ABD77796), A/New Caledonia/20/1999 (ABF21272), A/California/04/2009 (ACP41105), A/WSN/33 (AAA43209), A/Beijing/718/2009 (ACZ98546), and A/Beijing/720/2009 (ACZ98560). (B) List of H1 viruses with serine (S)-to-tyrosine (Y) mutation at the P2 position of the cleavage site, along with the information on subtype, accession number, and species of isolation.
Fig 2
Fig 2
Cleavage of A/Beijing/718/2009 influenza virus HAs by human plasmin. (A) Human plasmin-dependent HA cleavage of A/Beijing/718/2009 (Bei718) HA and Bei718 HA-328S. Surface biotinylation was performed on 293T cells expressing each HA. Transfected cells were treated with human plasmin (100 mU/ml) at the indicated times, and the cleavage product(s) was detected by Western blot analysis using anti-HA antibody. (B) Quantification of the time-dependent human plasmin-mediated HA cleavage efficiency of Bei718 HA and Bei718 HA-328S. The error bars represent the standard deviations of the results of three independent experiments. Statistical analyses were performed using Student's t test (unpaired, one tail) by GraphPad Prism, comparing the individual treatment times (*, P < 0.05; **, P < 0.005; ***, P < 0.0001).
Fig 3
Fig 3
Cleavage of the A/California/04/2009 influenza virus HAs by human plasmin. (A) Human plasmin-dependent HA cleavage of A/California/04/09 (CA0409) HA and CA0409 HA-328Y. Surface biotinylation was performed on 293T cells expressing each HA. Transfected cells were treated with human plasmin (100 mU/ml) at the indicated times, and the cleavage product(s) was detected by Western blot analysis using anti-HA antibody. (B) Quantification of the time-dependent human plasmin-mediated HA cleavage efficiency of CA0409 HA and CA0409 HA-328Y. The error bars represent the standard deviations of the results of three independent experiments. Statistical analyses were performed using Student's t test (unpaired, one tail) by GraphPad Prism, comparing the individual treatment times (*, P < 0.05; **, P < 0.005).
Fig 4
Fig 4
Plaque-forming properties of recombinant virus on MDCK cells. Representative pictures of immunoplaque assays of A/California/04/2009 (rCA0409) (A) and rCA0409 HA-328Y (B) virus on MDCK cells are shown. Plaque assays were performed by inoculating 30 to 40 PFU virus on MDCK cells in the presence of TPCK-trypsin (1 μg/ml), human plasmin (h-Pm) (3 mU/ml), or 5% FBS or were mock treated with or without addition of 6-AHA (5 mg/ml) for 3 days. Cells were fixed, and viral NP were stained with mouse α-NP antibody (Ab). Secondary α-mouse Ab conjugated with alkaline phosphatase (AP) was used to detect virally infected cell clumps visually. Images were taken using an Alpha DigiDoc AD-1200 imager. At least three individual experiments were performed for each condition.
Fig 5
Fig 5
Mouse model of viral pathogenesis following intranasal and intracranial inoculation. (A) Summary of viral titer results at 3 days or 6 days postintranasal inoculation and 3 days postintracranial inoculation. Values inside the parentheses indicate the portion of positive samples for viral detection in each group. Average titers were calculated based on positive samples and were represented as log10 PFU/g of tissue. <, viral titer below the detection limit (i.e., 20 PFU/ml). (B and C) Percentage of initial weight loss of mice. Mouse body weights were measured every 24 h for intranasal inoculation (B) or every 12 h for intracranial inoculation (C). Body weights were normalized to the initial body weight prior to viral inoculation.
Fig 6
Fig 6
HA cleavage by human plasminogen independent from its cognate neuraminidase. (A) Time-dependent human plasminogen-mediated cleavage of A/Beijing/718/2009 (Bei718) HA and Bei718 HA-328S. Surface biotinylation was performed as described for Fig. 2 by cotransfecting Bei718 HA with vector control, WSN NA, or CA0409 NA and treated with human plasminogen (6 μg/ml) for the indicated duration. (B) Quantification of three individual experiments. The error bars represent the standard deviations of the results of three independent experiments, and statistical analyses were performed as described for Fig. 2.
Fig 7
Fig 7
HA cleavage by the use of conditioned medium from MDCK cells. (A) Cleavage of A/Beijing/718/2009 HA. Plasmin (Plg)-MDCK-conditioned medium was prepared by incubating human plasmin (h-Plg) (5 μg/ml) on MDCK cells for 18 h. The conditioned medium was used to treat HA-transfected cells for 30 min. Human plasmin (100 mU/ml) was used as a positive control and MDCK medium alone as a negative control. (B) Quantification of plasminogen (Plg)-MDCK-conditioned medium-mediated HA cleavage. Relative cleavage efficiencies were calculated as described for Fig. 2. The error bars represent the standard deviations of the results of three independent experiments, and statistical analyses were performed as described for Fig. 2.
Fig 8
Fig 8
Plasminogen binding on HA- and NA-transfected cells by flow cytometry. (A) Percentage of human plasminogen-binding cells. A/WSN/33 (WSN) NA was used as a positive control, pEF4 was used for background binding, and WSN NA R146N ΔK470 was used as a negative control. CA0409 NA, eight different H1 HAs (WSN, A/South Carolina/1/1918 [1918], CA0409, Bei178, A/NWS/1934 [NWS], A/Wilson-Smith/1933 [WS], A/Puerto Rico/8/1934 [PR8], and A/New Caledonia/20/1999 [New Caledonia]), and one H9 HA (not shown) were tested. The error bars represent the standard deviations of the results of three independent experiments. Statistical analyses were performed as described for Fig. 2 by comparing each sample to the negative control. (B) Representative FACS traces are shown. All plots were overlaid with the background control (pEF4 empty vector) to illustrate the peak shift on the x axis (allophycocyanin [APC] staining for plasminogen). The y axis data represent the percentage of cells normalized to the maximum (Max) cell number in a single channel within the plot.
Fig 9
Fig 9
HA cleavage by bacterial streptokinase-activated human plasminogen. (A) Surface biotinylation analysis was performed as mentioned above. 293T cells transfected with A/Beijing/718/2009 (Bei718) HA or HA-328S mutant were treated with different proteases as indicated for 1 h. Cleavage products were detected by Western blot analysis using anti-HA antibody. (B) Quantification of the streptokinase/plasminogen (Skc-Plg)-dependent HA cleavage efficiency. Cleavage efficiencies were calculated. The error bars represent the standard deviations of the results of three independent experiments, and statistical analyses were performed as described for Fig. 2.
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References

    1. Cox NJ, Subbarao K. 2000. Global epidemiology of influenza: past and present. Annu. Rev. Med. 51:407–421 - PubMed
    1. Wiley DC, Skehel JJ. 1987. The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. Annu. Rev. Biochem. 56:365–394 - PubMed
    1. Klenk H-D, Garten W. 1994. Activation cleavage of viral spike proteins by host proteases, p 241–280 In Wimmer E. (ed), Cellular receptors for animal viruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
    1. Klenk HD, Garten W. 1994. Host cell proteases controlling virus pathogenicity. Trends Microbiol. 2:39–43 - PubMed
    1. Choi SY, Bertram S, Glowacka I, Park YW, Pohlmann S. 2009. Type II transmembrane serine proteases in cancer and viral infections. Trends Mol. Med. 15:303–312 - PubMed

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