Biophysical characterization of recombinant HIV-1 subtype C virus infectivity factor.
HIV-1 virus infectivity factor (Vif) is one of the four accessory proteins that are characteristic of primate lentiviruses and critically required for the infection of host cells. Vif plays a key role in replication and transmission of the virus in non-permissive cells, such as primary T cells and macrophages. Using co-precipitation and co-fractionation techniques, evidence has been provided that Vif interacts with a variety of host proteins, such as the cytidine deaminases APOBEC3G and 3F, the Cullin5/EloBC ubiquitin-ligase complex, Fyn and Hck tyrosine kinases, as well as with viral components, such as the immature Gag precursor and viral RNA. We report on the expression, purification and molecular characterization of a folded recombinant subtype C Vif. Vif was expressed in E. coli with a C-terminal hexahistidine tag and purified by nickel affinity chromatography. We obtained approximately 5 mg protein per liter of bacterial culture, with a purity >95%. The expected molecular mass of 23.7 kDa was confirmed by mass spectrometry. Although dynamic light scattering and small angle X-ray scattering measurements revealed the presence of high molecular weight aggregates in the protein preparation, circular dichroism analysis showed that the protein contains mainly folded β-sheet elements and exhibits remarkable thermal stability (T (m) > 95°C). Recombinant Vif may be used as a tool to study its biological functions and tertiary structure, as well as for the development of diagnostic, therapeutic and preventive strategies for HIV-1 infections.
Gallerano
Daniela
D
Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
Devanaboyina
Siva Charan
SC
Swoboda
Ines
I
Linhart
Birgit
B
Mittermann
Irene
I
Keller
Walter
W
Valenta
Rudolf
R
D055592:Biophysical Phenomena; D016346:Gene Products, vif / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D016346:Gene Products, vif; D011993:Recombinant Fusion Proteins