In vitro assembly of feline immunodeficiency virus capsid protein: biological role of conserved cysteines.
Core assembly, a key step in the retroviral life cycle, is poorly understood. Previous studies have shown that the entire gag region is needed to form the assembled particles. In this report, we have shown that the assembly process is driven by recombinant capsid protein (p26) of feline immunodeficiency virus itself. Proteins are expressed in a bacterial system and soluble forms of wild-type and modified proteins are purified from bacterial extracts and are examined on gel-filtration chromatography fitted to an HPLC system. It has also been shown that changing residue Cys190 (one of the two conserved cysteines of feline immunodeficiency virus which are also conserved for all the immunodeficiency viruses including HIV) to serine by site-directed mutagenesis disrupts the assembly process. In addition, this modification causes considerable thermal instability of the protein while substitutions at nonconserved cysteines do not significantly affect the thermal stability and assembly of the protein. These findings indicate that conserved cysteine residues play a vital role in the capsid protein assembly and, therefore, are critical for virus infectivity.
Nath
M D
MD
National Cancer Institute, NIH, Frederick, Maryland 21702, USA. nathm@ncifcrf.gov
Peterson
D L
DL
D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D002213:Capsid / Q000737:chemistry*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry / Q000502:physiology*; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015151:Immunoblotting; D016089:Immunodeficiency Virus, Feline / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017510:Protein Folding; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013696:Temperature; D013997:Time Factors
D004220:Disulfides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D003545:Cysteine