Intracellular Cleavage of Human Influenza A Virus Hemagglutinin and Its Inhibition

O. P. Zhirnov^1*, I. V. Vorobjeva¹, A. V. Ovcharenko¹, and H. D. Klenk²

¹Ivanovsky Institute of Virology, Russian Academy of Medical Sciences, ul. Gamalei 16, Moscow 123098, Russia; fax: (7-095) 190-3058; E-mail: zhirnov@virology.ru

²Institute of Virology, Philipps University, Marburg 35037, Germany; fax: +49 (6421) 286-8962; E-mail: klenk@staff.uni-marburg.de

^* To whom correspondence should be addressed.

Received April 5, 2002; Revision received December 10, 2002

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Replication of human influenza A viruses and proteolytic cleavage of the viral glycoprotein HA0 --> HA1/2 were studied in passaged cultures of epithelial cells of the mucosal membrane of human large intestine (CACO-2 line), dog kidney cells (MDCK), and monkey kidney cells (CV-1). Cleavage of the viral glycoprotein HA0, synthesis of activated virions, multicycle virus infection, and effective production of viral foci under an agarose overlayer were found in CACO-2 cells. By pulse-chase labeling of viral glycoproteins, testing the sensitivity to endoglycosidase-H of the viral glycoproteins HA0 and HA1/2 synthesized, and inhibiting the HA0 proteolysis with brefeldin A, the HA0 --> HA1/2 proteolysis was established to occur in the late stages of intracellular transport in the trans-Golgi and plasma membrane areas of the cells. Proteolysis of the viral glycoprotein HA0 in CACO-2 cells was suppressed by aprotinin, a natural inhibitor of serine proteinases. Unlike MDCK and CV-1 cells resistant to apoptosis induced by influenza virus, CACO-2 cells retained their viability for 2-3 days after infection with human influenza A virus.

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KEY WORDS: influenza virus, replication, hemagglutinin cleavage, aprotinin

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