Apoptosis by influenza viruses correlates with efficiency of viral mRNA synthesis.

A mutant influenza virus, A/NWS-Mvi, grows well in the presence of exogenous sialidase activity sufficient to remove all cell surface sialic acids. Related wild-type viruses grow very poorly under these conditions, although mutant and wild-type viruses bind to desialylated cells with similar efficiency and show similar reduction of binding to sialidase-treated cells compared to native cells. Here we examine entry, transcription, translation, and RNA replication and find that, although the viruses appear to utilize the same entry pathway, the mutant NWS-Mvi transcribes and replicates RNA to higher levels than the wild-type strains. The kinetics of replication in multi-cycle infection show that this enhancement of RNA synthesis facilitates growth where entry is restricted. The hemagglutinin (HA) protein of NWS-Mvi lyses red blood cells 0.1 pH unit higher than wild-type viruses. This higher fusion pH may allow more efficient release of nucleocapsids from endosomes and contribute to the enhanced RNA synthesis. The efficient RNA synthesis assists virus survival at low inocula or under stringent growth conditions, such as the presence of antiviral agents. NWS-Mvi induces apoptosis in infected cells more readily than wild-type viruses, apparently as a consequence of enhanced production of viral mRNA. Since growth of NWS-Mvi is more efficient, apoptosis may play a positive role in viral replication by removing cells that have already been infected from those capable of making more virus.

Influenza virus infection of desialylated cells.

Sialic acid has long been considered to be the sole receptor for influenza virus. The viral hemagglutinin (HA) is known to bind cell surface sialic acid, and sialic acids on viral glyco-proteins are cleaved by the viral neuraminidase (NA) to promote efficient release of progeny virus particles. However, NWS-Mvi, a mutant virus completely lacking NA, grows well in MDCK cells continuously treated with exogenous neuraminidase (sialidase). Exogenous sialidase quantitatively releases all sialic acids from purified glycoproteins and glycolipids of MDCK cells and efficiently removes surface sialic acid from intact cells. Binding of NWS-Mvi and parent influenza viruses to MDCK cells is indistinguishable, and is only partially reduced by sialidase treatment of the cells. Both mutant and wild-type viruses enter enzymatically desialylated cells and initiate transcription. The ability of influenza A reassortant viruses to infect desialylated cells is shared by recent H3N2 clinical isolates, suggesting that this may be a general property of influenza A viruses. We propose that influenza virus infection can result from sialic acid-independent receptors, either directly or in a multistage process. When sialic acid is present, it may act to enhance virus binding to the cell surface to increase interaction with secondary receptors to mediate entry. Understanding virus entry will be critical to further efforts in infection control and prevention.

Antigenic, sequence, and crystal variation in influenza B neuraminidase.

The neuraminidase (NA) genes of influenza B viruses B/Maryland/59, B/Hong Kong/8/73, B/Singapore/222/79, B/Oregon/5/80, B/USSR/100/83, B/Victoria/3/85, B/Leningrad/179/86, B/Memphis/6/86, and B/Memphis/3/89 have been sequenced. The deduced amino acid sequences show high variability in the stalk domain of the NA, but a surprising degree of sequence conservation in the head region which carries all the antigenic and enzyme activity. The variable region coding for the neuraminidase stalk also translates into a variable section in the overlapping NB polypeptide, which is coded from a second reading frame that overlaps the first 100 amino acids of NA. The influenza B NAs are antigenically distinguishable with monoclonal antibodies in neuraminidase-inhibition tests, even when there is only one amino acid sequence difference. However, seven of nine escape mutants selected with monoclonal antibodies were distinguished only by the antibody used for selection. When NA heads of influenza B viruses are crystallized, there are remarkable differences in crystal morphology between neuraminidases which have very few sequence changes.