The human parainfluenza virus 3 (HPIV3) poses a risk for pneumonia development in young children and immunocompromised patients. To investigate mechanisms of HPIV3 pathogenesis, we characterized the association state and host protein interactions of HPIV3 phosphoprotein (HPIV3 P), an indispensable viral polymerase cofactor. Sequence analysis and homology modeling predict that HPIV3 P possesses a long, disordered N-terminal tail (PTAIL) a coiled-coil multimerization domain (PMD), similar to the well-characterized paramyxovirus phosphoproteins from measles and Sendai viruses. Using a recombinantly expressed and purified construct of PMD and PTAIL, we show that HPIV3 P in solution is primarily an alpha-helical tetramer that is stable up to 60 °C. Pulldown and isothermal titration calorimetry experiments revealed that HPIV3 P binds the host hub protein LC8, and turbidity experiments demonstrated a new role for LC8 in increasing the solubility of HPIV3 P in the presence of crowding agents such as RNA. For comparison, we show that the multimerization domain of the Zaire Ebola virus phosphoprotein VP35 is also a tetramer and binds LC8 but with significantly higher affinity. Comparative analysis of the domain architecture of various virus phosphoproteins in the order Mononegavirales show multiple predicted and verified LC8 binding motifs, suggesting its prevalence and importance in regulating viral phosphoprotein structures. Our work provides evidence for LC8 binding to phosphoproteins with multiple association states, either tetrameric, as in the HPIV3 and Ebola phosphoproteins shown here, or dimeric as in rabies virus phosphoprotein. Taken together the data suggest that the association states of a virus-specific phosphoprotein and the complex formed by binding of the phosphoprotein to host LC8 are important regulators of viral function.
Chimeric human parainfluenza virus bearing the Ebola virus glycoprotein as the sole surface protein is immunogenic and highly protective against Ebola virus challenge
