A point mutation in human parainfluenza virus type 2 nucleoprotein leads to two separate effects on virus replication

Paramyxovirus genomes, like that of human parainfluenza virus type 2 (hPIV2), are precisely a multiple of six nucleotides long (“rule of six”), in which each nucleoprotein subunit (NP) binds precisely 6 nucleotides. Ten residues of its RNA binding groove contact the genome RNA; but only one, Q202, directly contacts a nucleotide base. Mutation of NP Q202 leads to two phenotypes; the ability of the viral polymerase to replicate minigenomes with defective bipartite promoters where NP wt is inactive, and the inability to rescue rPIV2 carrying this point mutation by standard means. The absence a rPIV2 NP Q202A prevented further study of this latter phenotype. By extensive and repeated co-cultivation of transfected cells, a rPIV2 carrying this mutation was finally recovered, and this virus was apparently viable due to the presence of an additional NP mutation (I35L). Our results suggest that these two phenotypes are due to separate effects of the Q202 mutation, and that of the problematic rescue phenotype may be due to the inability of the transfected cell to incorporate viral nucleocapsids during virus budding. Importance Paramyxovirus genomes are contained within a non-covalent homopolymer of its nucleoprotein (NP) and form helical nucleocapsids (NC) whose 3’ ends contain the promoters for the initiation of viral RNA synthesis. This work suggests that these NC 3’ ends may play another role in the virus life cycle, namely via their specific interaction with virus modified cell membranes needed for the incorporation of viral NCs into budding virions.

Structural Basis of Human Parainfluenza Virus 3 Unassembled Nucleoprotein in Complex with Its Viral Chaperone

Human parainfluenza virus 3 (HPIV3) belongs to the Paramyxoviridae , causing annual worldwide epidemics of respiratory diseases, especially in newborns and infants. The core components consist of just three viral proteins: nucleoprotein (N), phosphoprotein (P), and RNA polymerase (L), playing essential roles in replication and transcription of HPIV3 as well as other paramyxoviruses. Viral genome encapsidated by N is as a template and recognized by RNA-dependent RNA polymerase complex composed of L and P. The offspring RNA also needs to assemble with N to form nucleocapsids. The N is one of the most abundant viral proteins in infected cells and chaperoned in the RNA-free form (N 0 ) by P before encapsidation. In this study, we presented the structure of unassembled HPIV3 N 0 in complex with the N-terminal portion of the P, revealing the molecular details of the N 0 and the conserved N 0 -P interaction. Combined with biological experiments, we showed that the P binds to the C-terminal domain of N 0 mainly by hydrophobic interaction and maintains the unassembled conformation of N by interfering with the formation of N-RNA oligomers, which might be a target for drug development. Based on the complex structure, we developed a method to obtain the monomeric N 0 . Furthermore, we designed a P-derived fusion peptide with 10-times higher affinity, which hijacked the N and interfered with the binding of the N to RNA significantly. Finally, we proposed a model of conformational transition of N from the unassembled state to the assembled state, which helped to further understand viral replication. IMPORTANCE Human parainfluenza virus 3 causes annual epidemics of respiratory diseases, especially in newborns and infants. For the replication of HPIV3 and other paramyxoviruses, only three viral proteins are required: phosphoprotein (P), RNA polymerase (L), and nucleoprotein (N). Here, we reported the crystal structure of the complex of N and its chaperone P. We described in details how P acts as a chaperone to maintain the unassembled conformation of N. Our analysis indicated that the interaction between P and N is conserved and mediated by hydrophobicity, which can be used as a target for drug development. We obtained a high-affinity P-derived peptide inhibitor, specifically targeted N and greatly interfered with the binding of the N to RNA, thereby inhibiting viral encapsidation and replication. In summary, our results provide new insights into the paramyxovirus genome replication and nucleocapsid assembly, and lay the basis for drug development.

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

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.

Human parainfluenza virus type 1 regulates cholesterol biosynthesis and establishes quiescent infection in human airway cells

Human parainfluenza virus type 1 (hPIV1) and 3 (hPIV3) cause seasonal epidemics, but little is known about their interaction with human airway cells. In this study, we determined cytopathology, replication, and progeny virion release from human airway cells during long-term infection in vitro. Both viruses readily established persistent infection without causing significant cytopathic effects. However, assembly and release of hPIV1 rapidly declined in sharp contrast to hPIV3 due to impaired viral ribonucleocapsid (vRNP) trafficking and virus assembly. Transcriptomic analysis revealed that both viruses induced similar levels of type I and III IFNs. However, hPIV1 induced specific ISGs stronger than hPIV3, such as MX2, which bound to hPIV1 vRNPs in infected cells. In addition, hPIV1 but not hPIV3 suppressed genes involved in lipid biogenesis and hPIV1 infection resulted in ubiquitination and degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, a rate limiting enzyme in cholesterol biosynthesis. Consequently, formation of cholesterol-rich lipid rafts was impaired in hPIV1 infected cells. These results indicate that hPIV1 is capable of regulating cholesterol biogenesis, which likely together with ISGs contributes to establishment of a quiescent infection.

Structural architecture of a dimeric paramyxovirus polymerase complex

Human parainfluenza virus type 3 (hPIV3), a member of non-segmented, negative-strand RNA viruses (nsNSVs), is the second most common cause of severe respiratory disease in pediatrics. The transcription and replication processes of nsNSVs are catalyzed by a multi-functional RNA-dependent RNA polymerase (RdRp) complex composed of the large protein (L) and the phosphoprotein (P). Previous studies have shown that the polymerase can adopt a dimeric form, however, the structure of the dimer and how it functions are not understood. Here we determined the cryo-EM structure of hPIV3 L-P complex at 2.7 Å with substantial structural details. A putative catalytic magnesium ion could be built in our structure, and structural comparison revealed atomic features conserved with other RNA viruses. Interactions identified between the two priming and intrusion loops and the connector domain potentially trigger the spatial movement of three C-terminal L domains for different steps of transcription and replication. Structural comparison with other nsNSV RdRps suggests common features of L-P binding. Furthermore, we report for the first time the structural basis of the L-L interaction in the partially modelled dimeric L-P structure, in which the connector domain of one L is positioned at the putative RNA template entry of the other L. Based on these findings, we propose a model by which L dimerization promotes efficient conversion of nascent RNA into a template.