The influenza A virus continues to be a major cause of morbidity and mortality worldwide despite implementation of vaccines and a few antiviral drugs. Our long-term goal is to determine the molecular mechanisms regulating assembly and spread of influenza A virus, which potentially serve as new targets for antivirals. In the current proposal, our objective is to determine the mechanism that gives rise to the cell type- specific differences in infectious virus assembly. The Influenza A virus can infect macrophages, but macrophages are much less permissive than airway epithelial cells. Our preliminary studies showed that infectious titer release from human primary monocyte-derived macrophages (MDMs) is severely impaired relative to differentiated THP-1, a human monocytic cell line. Using correlative fluorescence and scanning microscopy further revealed that efficient assembly of virus particles at the plasma membrane is detectable in differentiated THP-1 but not in MDMs even though both differentiated THP-1 and primary MDM express viral structural proteins at the cell surface to the same extent. The expression levels of total viral RNA and proteins are also similar. These results indicate that assembly of progeny virus particles is highly inefficient in human primary macrophages. Interestingly, we observed that influenza transmembrane proteins HA and M2, both of which plays key roles in virus assembly and release, fail to come in close proximity of each other in MDMs but not in differentiated THP-1 or epithelial cells and that this restriction in MDMs is reversed upon disruption of actin cytoskeleton. Based on these and other preliminary results, in this proposal we plan to test our central hypothesis that actin cytoskeletons restrict influenza A virus assembly via modulation of viral protein distribution at the plasma membrane in a cell-dependent manner. We will first identify viral structural proteins that render the virus susceptible to this inhibition of HA-M2 association (Aim 1). Then, we will determine the cellular mechanism that contributes to this inhibition (Aim 2). The rationale behind focusing on cell-specific differences in influenza virus assembly and release is to facilitate identification of cellular mechanisms that support or inhibit influenza virus propagation. Once identified, such mechanisms may serve as potential targets for therapeutic modulation designed to block virus spread in the respiratory epithelial cells.
Influenza A viruses, which cause seasonal epidemics each year and severe pandemics occasionally, continue to impose a major threat upon human health. Although it has been known that influenza infection of human macrophages result in very low virion production, little is known about the nature of this defect. The goals of this study are to fill this knowledge gap and to identify the cellular mechanism contributing to the deficiency of progeny virus production. The outcomes of this study may inform development of novel therapeutic strategies against influenza virus spread in the respiratory tract.
