Infectious Cell Entry Mechanism of Influenza Virus

Abstract

Interaction between influenza virus WSN strain and MDCK cells was studied by using spin-labeled phospholipids and electron microscopy. Envelope fusion was negligibly small at neutral pH but greatly activated in acidic media in a narrow pH range around 5.0. The half-time was less than 1 min at 37°C at pH 5.0. Virus binding was almost independent of the pH. Endocytosis occurred with a half-time of about 7 min at 37°C at neutral pH, and about 50% of the initially bound virus was internalized after 1 h. Electron micrographs showed binding of virus particles in coated pits in the microvillous surface of plasma membrane and endocytosis into coated vesicles. Chloroquine inhibited virus replication. The inhibition occurred when the drug was added not later than 10 min after inoculation. Chloroquine caused an increase in the lysosomal pH 4.9 to 6.1. The drug did not affect virus binding, endocytosis, or envelope fusion at pH 5.0. Electron micrographs showed many virus particles remaining trapped inside vacuoles even after 30 min at 37°C in the presence of drug, in contrast to only a few particles after 10 min in vacuoles and secondary lysosomes in its absence. Virus replication in an artificial condition, i.e., brief exposure of the inoculum to acidic medium followed by incubation in neutral pH in the presence of chloroquine, was also observed. These results are discussed to provide a strong support for the infection mechanism of influenza virus proposed previously: virus uptake by endocytosis, fusion of the endocytosed vesicles with lysosome, and fusion of the virus envelope with the surrounding vesicle membrane in the secondary lysosome because of the low pH. This allows the viral genome to enter the target cell cytoplasm.

Recycling of chloroquine and its hydroxyl analogue to face bacterial, fungal and viral infections in the 21st century

“…The activity of CQ against Orthomyxoviridae (influenza A and B viruses) has been described for several decades [35,37,122] and in vitro assays on avian influenza virus strains are ongoing [36]. Shibata et al's [35] results suggested that CQ prevents the uncoating of influenza B virus.…”

Virus Entry into Animal Cells

“…Considerable information exists regarding the structure, receptor specificity, endocytosis, and low-pH-dependent fusion of these viruses. Penetration occurs from late endosomes (Yoshimura et al, 1982) following uptake in coated and noncoated vesicles (Dourmashkin and Tyrell, 1974;Matlin et al, 1982a). The low endosomal pH triggers the fusion reaction (Stegmann et al, 1985;White et al, 1982b) and infection is blocked by acidotropic weak bases (Matlin et al, 1982a;Yoshimura et al, 1982).…”

Mechanisms of viral entry: sneaking in the front door

“…Lysosomotropic agents such as ammonium chloride (Matlin et al 1981) and chloroquine (Yoshimura et al 1982) inhibited influenza infection, suggesting an endocytic entry pathway. Many studies have demonstrated the pHdependence of the Influenza entry mechanism (Guinea and Cerrasco 1994;Guinea and Cerrasco 1995), where fusion with the plasma membrane occurs at pH 5.5 or below but not in less acidic environments (Matlin et al 1981).…”