Human Parainfluenza Virus 3 Phosphoprotein Is a Tetramer and Shares Structural and Interaction Features with Ebola Phosphoprotein VP35.

Joaquin Rodriguez Galvan,Gretchen Fujimura,Elisar Barbar,Cat Hoang Veseley,Patrick Reardon,Jesse Howe,Brianna Donner,Heather M Forsythe

doi:10.3390/biom11111603

Joaquin Rodriguez Galvan, Gretchen Fujimura + Show 6 more

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https://doi.org/10.3390/biom11111603

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Abstract

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.

Highlights

The human parainfluenza virus (HPIV), in the Paramyxoviridae family, is a cluster of seasonal respiratory viruses (RVs) that generally cause mild to moderate respiratory impairments [1]
In addition to a long, disordered N-terminal domain (PTAIL ), there was a short stretch of residues (472–488, for human parainfluenza virus 3 (HPIV3) P) that fell above the disorder threshold
WaggaWagga coiled-coil prediction outputs implied a similar trend for the three phosphoproteins, with a predicted coiled-coil starting at similar regions for MeV P, SeV P, and HPIV3 phosphoprotein (HPIV3 P), respectively (Figure 1C)

Summary

Introduction

The human parainfluenza virus (HPIV), in the Paramyxoviridae family, is a cluster of seasonal respiratory viruses (RVs) that generally cause mild to moderate respiratory impairments [1]. HPIV infections typically follow a common cold-like trajectory in healthy adults but elevate the risk of pneumonia in children and immunocompromised patients [2]. HPIV variants HPIV3 and HPIV4 are associated with the poorest clinical outcomes; both are highly transmissible, in hospitals, and accountable for increased ICU admission rates in children [2,3]. HPIV3, in particular, causes high morbidity yet very low mortality among vulnerable patients [4]. The underlying reasons for these observations as well as the molecular pathogenesis of HPIV3 infections are unclear. There is an urgent need to identify novel mechanisms at the host–pathogen interface; unveiling these processes

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