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The Proteolytic Activation of (H3N2) Influenza A Virus Hemagglutinin Is Facilitated by Different Type II Transmembrane Serine Proteases.

Kühn N, Bergmann S, Kösterke N, Lambertz RL, Keppner A, van den Brand JM, Pöhlmann S, Weiß S, Hummler E, Hatesuer B, Schughart K - J. Virol. (2016)

Bottom Line: In this study, we investigated an additional trypsin-like protease, TMPRSS4.Thus, our results identified TMPRSS4 as a second host cell protease that, in addition to TMPRSS2, is able to activate the HA of H3N2 influenza virus in vivo Influenza epidemics and recurring pandemics are responsible for significant global morbidity and mortality.Here we show that deletion of two host protease genes,Tmprss2 and Tmprss4, strongly reduced viral spread as well as lung pathology and resulted in increased survival after H3N2 virus infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Infection Genetics, Helmholtz Centre for Infection Research, and University of Veterinary Medicine Hannover, Braunschweig, Germany.

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Virus isolated from Tmprss2−/−Tmprss4−/− lungs exhibits reduced infectivity in the absence of trypsin. Eight- to 11-week-old female mice were infected with 2 × 103 FFU of mouse-adapted H3N2 influenza virus by intranasal application. On day 2 p.i., the ratio of processed to nonprocessed virus (HA cleavage or not) in lung homogenates was determined using a modified FFU assay without exogenous trypsin. The total number of virus particles (mature or not) was determined in the presence of trypsin. Samples from mutant mice showed significantly lower levels of matured viruses than the total number of virus particles (P < 0.05). No difference was detectable in samples from infected wild-type mice. Data shown are means ± SEM for 7 replicates from two independent experiments.
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Figure 7: Virus isolated from Tmprss2−/−Tmprss4−/− lungs exhibits reduced infectivity in the absence of trypsin. Eight- to 11-week-old female mice were infected with 2 × 103 FFU of mouse-adapted H3N2 influenza virus by intranasal application. On day 2 p.i., the ratio of processed to nonprocessed virus (HA cleavage or not) in lung homogenates was determined using a modified FFU assay without exogenous trypsin. The total number of virus particles (mature or not) was determined in the presence of trypsin. Samples from mutant mice showed significantly lower levels of matured viruses than the total number of virus particles (P < 0.05). No difference was detectable in samples from infected wild-type mice. Data shown are means ± SEM for 7 replicates from two independent experiments.

Mentions: Finally, to examine whether the reduced viral replication in the lungs of mutant mice is also reflected in reduced HA activation, we performed a modified FFU assay of lung homogenates. To prevent artificial HA cleavage and following viral entry into MDCK II cells by addition of exogenous trypsin, the homogenates were incubated on the cells without additional proteinase in the medium. Under these conditions, only completely matured virus particles will enter cells and form foci. We were able to detect a significant smaller amount of matured virus than the total amount of virus in samples from double-knockout mice (Fig. 7). In contrast, no difference was observed in wild-type samples with and without trypsin treatment. Taken together, these data show that TMPRSS2 and TMPRSS4 mediate activation of H3N2 influenza A viruses by cleavage maturation of viral hemagglutinin progeny and that deletion of these host factors results in reduced viral replication, spreading, and pathogenesis.


The Proteolytic Activation of (H3N2) Influenza A Virus Hemagglutinin Is Facilitated by Different Type II Transmembrane Serine Proteases.

Kühn N, Bergmann S, Kösterke N, Lambertz RL, Keppner A, van den Brand JM, Pöhlmann S, Weiß S, Hummler E, Hatesuer B, Schughart K - J. Virol. (2016)

Virus isolated from Tmprss2−/−Tmprss4−/− lungs exhibits reduced infectivity in the absence of trypsin. Eight- to 11-week-old female mice were infected with 2 × 103 FFU of mouse-adapted H3N2 influenza virus by intranasal application. On day 2 p.i., the ratio of processed to nonprocessed virus (HA cleavage or not) in lung homogenates was determined using a modified FFU assay without exogenous trypsin. The total number of virus particles (mature or not) was determined in the presence of trypsin. Samples from mutant mice showed significantly lower levels of matured viruses than the total number of virus particles (P < 0.05). No difference was detectable in samples from infected wild-type mice. Data shown are means ± SEM for 7 replicates from two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836353&req=5

Figure 7: Virus isolated from Tmprss2−/−Tmprss4−/− lungs exhibits reduced infectivity in the absence of trypsin. Eight- to 11-week-old female mice were infected with 2 × 103 FFU of mouse-adapted H3N2 influenza virus by intranasal application. On day 2 p.i., the ratio of processed to nonprocessed virus (HA cleavage or not) in lung homogenates was determined using a modified FFU assay without exogenous trypsin. The total number of virus particles (mature or not) was determined in the presence of trypsin. Samples from mutant mice showed significantly lower levels of matured viruses than the total number of virus particles (P < 0.05). No difference was detectable in samples from infected wild-type mice. Data shown are means ± SEM for 7 replicates from two independent experiments.
Mentions: Finally, to examine whether the reduced viral replication in the lungs of mutant mice is also reflected in reduced HA activation, we performed a modified FFU assay of lung homogenates. To prevent artificial HA cleavage and following viral entry into MDCK II cells by addition of exogenous trypsin, the homogenates were incubated on the cells without additional proteinase in the medium. Under these conditions, only completely matured virus particles will enter cells and form foci. We were able to detect a significant smaller amount of matured virus than the total amount of virus in samples from double-knockout mice (Fig. 7). In contrast, no difference was observed in wild-type samples with and without trypsin treatment. Taken together, these data show that TMPRSS2 and TMPRSS4 mediate activation of H3N2 influenza A viruses by cleavage maturation of viral hemagglutinin progeny and that deletion of these host factors results in reduced viral replication, spreading, and pathogenesis.

Bottom Line: In this study, we investigated an additional trypsin-like protease, TMPRSS4.Thus, our results identified TMPRSS4 as a second host cell protease that, in addition to TMPRSS2, is able to activate the HA of H3N2 influenza virus in vivo Influenza epidemics and recurring pandemics are responsible for significant global morbidity and mortality.Here we show that deletion of two host protease genes,Tmprss2 and Tmprss4, strongly reduced viral spread as well as lung pathology and resulted in increased survival after H3N2 virus infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Infection Genetics, Helmholtz Centre for Infection Research, and University of Veterinary Medicine Hannover, Braunschweig, Germany.

Show MeSH
Related in: MedlinePlus