| The influenza A viral genome is composed of eight negative-sense RNA chromosomes (called vRNAs), all of which must be packaged to produce an infectious virion. It is not clear whether individual vRNAs are packaged specifically or at random, however, and the total vRNA capacity of the virion is unknown. We have created modified forms of the viral nucleoprotein (NP), neuraminidase (NA), and nonstructural (NS) vRNAs that encode green or yellow fluorescent proteins, and have studied the efficiency with which these are packaged using a plasmid-based influenza assembly system. We found that, under conditions where virions are limiting, pairs of alternatively-tagged vRNAs compete for packaging, but do so in a nonspecific manner. Moreover, 3–5% of transduction-competent viruses were found to incorporate two alternative reporters, regardless of whether those reporters represented the same or different vRNAs—a finding compatible with random, but not with specific packaging. Probabilistic estimates suggest that, in order to achieve this level of dual-transduction by chance alone, each influenza A virion must package an average of 9–11 vRNAS.; Influenza A and C viruses each utilize specific forms of sialic acid on cell surfaces as receptors to bind and infect their host cells. The extent to which various specific types of sialoglycoproteins function as influenza receptors, however, is largely unknown. Sialomucins are a distinct class of surface proteins that contain regions that are densely substituted with O-linked sialylated oligosaccharides, which could provide an ideal substrate for polyvalent interactions between influenza virus and the cell. Here we demonstrate that pretreatment with an enzyme, which selectively degrades sialomucins as a class from cell surfaces, profoundly inhibits the attachment of influenza A or C viruses as measured by hemagglutination. Surprisingly, however, neither enzymatically depleting surface sialomucins from CHO or MDCK cells nor the overexpression of a specific surface sialomucin (CD34) in CHO cells had any discernible effect on influenza infectivity, as measured by the levels or kinetics of viral RNA expression. Despite their predominant role in influenza attachment to erythrocytes, we find no evidence that sialomucins function as receptors for influenza A or C viruses on mammalian cells. |
