Amino Acids

, Volume 40, Issue 3, pp 981–989 | Cite as

Biophysical characterization of recombinant HIV-1 subtype C virus infectivity factor

  • Daniela Gallerano
  • Siva Charan Devanaboyina
  • Ines Swoboda
  • Birgit Linhart
  • Irene Mittermann
  • Walter Keller
  • Rudolf Valenta
Original Article

Abstract

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.

Keywords

HIV Virus infectivity factor (Vif) E.coli expression Circular dichroism Thermal stability 

Abbreviations

HIV

Human immunodeficiency virus

Vif

Virus infectivity factor

rVif

Recombinant virus infectivity factor

Notes

Acknowledgments

This study was supported by a research grant from Biomay, Vienna, Austria (http://www.biomay.com/).

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Daniela Gallerano
    • 1
  • Siva Charan Devanaboyina
    • 2
  • Ines Swoboda
    • 1
    • 3
  • Birgit Linhart
    • 1
  • Irene Mittermann
    • 1
  • Walter Keller
    • 2
  • Rudolf Valenta
    • 1
    • 3
  1. 1.Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and ImmunologyMedical University of ViennaViennaAustria
  2. 2.Institute of Molecular Biosciences, Structural BiologyKarl-Franzens-University GrazGrazAustria
  3. 3.Christian Doppler Laboratory for Allergy Research, Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and ImmunologyMedical University of ViennaViennaAustria

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