Effects Of The C-terminus On The Subcellular Localization Of Class Ⅰ Viral Membrane Proteins

Abstract:Coronavirus, influenza virus and human immunodeficiency virus (HIV) are three major pathogens of human infectious diseases. The spike (S) protein of coronavirus, hemagglutinin (HA) of influenza virus A and envelope (Env) protein of HIV-1are class I viral membrane fusion proteins which play multiple roles in the viral life cycle and are often cleaved into two subunits:the N-terminal subunit determines viral tropism through receptor binding and the ectodomain of C-terminal subunit take part in membrane fusion activity. The extreme C-terminus is composed of transmembrane domain and endodomain, which is associated with membrane integration, trafficking and assembly into virions of the protein.Previously, our group focused on C-terminal functions of murine hepatitis virus (MHV) S protein and results indicate that multiple cysteines are required for viral replication through S-palmitoylation and sequential incorporation into lipid rafts. And some negatively charged residues in the endodomain are critical for S assembly into virions specifically. In this thesis, we investigated the effects of the positively charged residues and cysteines on the subcellular location of S protein. We also explored the effects of three motifs MPER, Y7i2XXLand LL855/856on the subcellular location of HIV-1Env fusion proteins. Finally, we analysed the mechanisms of improvement of human DC-SIGN and L-SIGN on replication and protein expression of recombinant MHVs.The results showed that all EGFP fusion proteins contained the transmembrane domain plus endodomains originated from three control resources MS2, PDGFR, HA H3and MHV S respectively are effective expressed and properly located in murine Neuro-2a and human He La cells. Non-trans membrane fusion protein MS2is only located in cytoplasm. Fusion protein with truncated human cellular receptor PDGFR is located intracellular membrane and plasma membrane. Class I viral membrane fusion protein from influenza HA H3showed co-localization with ER marker (KDEL). Fusion protein with wild type of MHV S (Mwt) had similar distribution to the PDGFR and fusion protein with the truncated Cys-rich motif (M2C and M6C) showed co-localization with ER marker. M6C showed co-localization with Golgi marker (TGN46) as well. For HIV-1Env fusion proteins, the partial deletion of MPER did not effect the distribution of the fusion proteins. However, alanine substitution of motif Y712XXL and LLg55/856changed the subcellular distribution of the fusion proteins, which might be involved in the interaction between both motifs and the vesicle trafficking protein AP-1or AP-2in cells.In addition, we noted that the expression and distribution of most of fusion proteins by recombinant MHVs are similar to those by eukaryotic vector pIRES2-EGFP. And human DC-SIGN and L-SIGN improved the infection and protein expression of recombinant MHVs. These results provided valuable insights and research base for the investigation of trafficking mechanism of class I viral membrane protein in host cells.