The Viral Polymerase Complex Mediates the Interaction of vRNPs with Recycling Endosomes During SeV Assembly

Abstract

31Paramyxoviruses are negative sense single-stranded RNA viruses that comprise many important 32 human and animal pathogens, including human parainfluenza viruses. These viruses bud from 33 the plasma membrane of infected cells after the viral ribonucleoprotein complex (vRNP) is 34 transported from the cytoplasm to the cell membrane via The viral proteins that are critical for mediating this important initial step in viral assembly are 36 unknown. Here we use the model paramyxovirus, murine parainfluenza virus 1, or Sendai virus 37 (SeV), to investigate the roles of viral proteins in Rab11a-driven virion assembly. We previously 38 reported that infection with SeV containing high levels of copy-back defective viral genomes 39 (DVGs) generates heterogenous populations of cells. Cells enriched in full-length virus produce 40 viral particles containing standard or defective viral genomes, while cells enriched in DVGs do 41 not, despite high levels of defective viral genome replication. Here we take advantage of this 42 heterogenous cell phenotype to identify proteins that mediate interaction of vRNPs with Rab11a. 43We examine the role of matrix protein and nucleoprotein and determine that they are not 44 sufficient to drive interaction of vRNPs with recycling endosomes. Using a combination of mass 45 spectrometry and comparative protein abundance and localization in DVG-and FL-high cells, 46we identify viral polymerase complex components L and, specifically, its cofactor C proteins as 47Paramyxoviruses are single-stranded negative sense RNA viruses that include viruses of 65 clinical and global health significance, such as human parainfluenza viruses (HPIV). HPIV1 and 66 2 are the leading cause of croup in young children and HPIV3 is associated with bronchiolitis, 67 bronchitis, and pneumonia (1). These infections have also been associated with the development 68 or exacerbation of asthma and chronic airway disorders (2). Additionally, HPIVs are the 69 etiological agent of 7% of hospitalized pneumonia cases in children in the U.S. (3), as well as in 70Africa and Asia (4). Although HPIVs pose a significant health burden, there are no direct acting 71 antivirals or preventative vaccines. In order to identify targets for antiviral development, there is 72 a need to better understand fundamental aspects of paramyxovirus biology, including 73 mechanisms of virion assembly and particle production. 74Sendai virus (SeV), or murine parainfluenza virus 1, is closely related to human 75 parainfluenza viruses 1 and 3 with 69% homology at the RNA level between SeV and HPIV1. 76SeV has long served as a well-established model for understanding paramyxovirus replication. 77SeV genome replication occurs in the cytoplasm of infected cells (5) and virions bud from the 78 plasma membrane. The viral genomes are tightly coated in the viral nucleocapsid protein (NP) 79 with one NP molecule for every six nucleotides of genome (6). This viral RNA-protein complex 80 is referred to as the viral ribonucleoprotein (vRNP) (7, 8). Viral genomes a...