[PMC free article] [PubMed] [CrossRef] [Google Scholar] 11. HAE-ALI. Thus, our study identified three novel CL2-SN-38 NS proteins, NS2, NS3, and NS4, and suggests an important function of the NS2 protein in HBoV1 replication in HAE-ALI. IMPORTANCE Human bocavirus 1 infection causes respiratory diseases, including acute wheezing in infants, of which life-threatening cases have been reported. of the family (1, 2). HBoV1 causes lower respiratory tract infections, especially in infants less than 2 years old (3,C7). Severe and deadly cases associated with high viral load, anti-HBoV1 IgM antibody detection, or increased IgG antibody production have been documented (7,C9). express one large NS protein (NS1) from the left viral genome, VP proteins from the right side of the viral genome, and at least one small NS protein (NP1) that is encoded by an open reading frame (ORF) located in the middle of the viral genome (11, 16, 17). During MVC infection, we have detected another small NS protein (NS166kd [NS1 protein with an approximate molecular mass of 66 kDa]), which contains the N terminus of CL2-SN-38 the NS1 (18). The NS1, like the NS1 or Rep78 and Rep68 (Rep78/68) of other parvoviruses, is a multifunctional protein that has a site-specific origin DNA binding domain (OBD) and endonuclease activity at its N terminus (20), ATPase and helicase activities in the middle (21, 22), and a transactivation domain at its C terminus (23, 24). NS1 is essential to viral DNA replication, while NP1 is required for efficient viral DNA replication (11, 17). MVC NP1 protein plays a role in facilitating VP-encoding mRNAs to read through the proximal polyadenylation site that lies in the middle of the viral genome (18). Nothing is known about the functions of the newly identified MVC NS166kd protein. The protein expression profile of HBoV1 has been characterized via transfection using an incomplete HBoV1 genome lacking both the left and right inverted termini (19). Expression of NS1, NP1 and VP from their respective encoding transcripts was identified; however, the identities of the NS proteins with approximate molecular masses of 66 kDa and 34 kDa that were detected by an anti-NS1 C terminus antibody are unknown (19). In this study, we explored the expression profile of HBoV1 NS proteins in either nonreplicating plasmids or replicating infectious plasmids transfected into HEK293 cells or during HBoV1 infection of HAE-ALI. We identified three novel HBoV1 NS proteins, NS2, NS3, and NS4, both in the HBoV1 genome-replicating HEK293 cell system and in HBoV1 infection of HAE-ALI system. The functions of these proteins were further explored in these two CL2-SN-38 systems. MATERIALS AND METHODS Cell culture. (i) Cell line. Human embryonic kidney 293 (HEK293) cells (CRL-1573) were obtained from American Type Culture Collection (ATCC, Manassas, VA) and were cultured in HyClone Dulbecco’s modified Eagle’s medium (DMEM; GE Healthcare Bio-Sciences, Piscataway, NJ) with 10% fetal calf serum (FCS) (product number VRP F0926; Sigma-Aldrich, St. Louis, MO). (ii) Primary human airway epithelium cultures. Polarized human airway epithelium cultures at an air-liquid interface (ALI), termed HAE-ALI, were generated by growing isolated human bronchial airway epithelial cells on collagen-coated, semipermeable membrane inserts (0.33 cm2, Transwell; Corning, Corning, NY). They were then allowed to differentiate at an air-liquid interface, either in an CL2-SN-38 Ultraser G-containing medium, as described previously (11), or in PneumaCult-ALI medium (StemCell, Vancouver, BC, Canada), in 5% CO2 at 37C. After 3 to 5 5 weeks, the polarity of the HAE-ALI cultures was determined based on the transepithelial electrical resistance (TEER); the cultures that had a TEER of over 1,000 cm2 were used for HBoV1 infection..