title: Transactivation and signaling functions of Tat are not correlated: biological and immunological characterization of HIV-1 subtype-C Tat protein.
abstract: BACKGROUND
  Of the diverse subtypes of Human Immunodeficiency Virus Type-1 (HIV-1), subtype-C strains cause a large majority of infections worldwide. The reasons for the global dominance of HIV-1 subtype-C infections are not completely understood. Tat, being critical for viral infectivity and pathogenesis, may differentially modulate pathogenic properties of the viral subtypes. Biochemical studies on Tat are hampered by the limitations of the current purification protocols. Tat purified using standard protocols often is competent for transactivation activity but defective for a variety of other biological functions. Keeping this limitation in view, we developed an efficient protein purification strategy for Tat.
  
  
  RESULTS
  Tat proteins obtained using the novel strategy described here were free of contaminants and retained biological functions as evaluated in a range of assays including the induction of cytokines, upregulation of chemokine coreceptor, transactivation of the viral promoter and rescue of a Tat-defective virus. Given the highly unstable nature of Tat, we evaluated the effect of the storage conditions on the biological function of Tat following purification. Tat stored in a lyophilized form retained complete biological activity regardless of the storage temperature. To understand if variations in the primary structure of Tat could influence the secondary structure of the protein and consequently its biological functions, we determined the CD spectra of subtype-C and -B Tat proteins. We demonstrate that subtype-C Tat may have a relatively higher ordered structure and be less flexible than subtype-B Tat. We show that subtype-C Tat as a protein, but not as a DNA expression vector, was consistently inferior to subtype-B Tat in a variety of biological assays. Furthermore, using ELISA, we evaluated the anti-Tat antibody titers in a large number of primary clinical samples (n = 200) collected from all four southern Indian states. Our analysis of the Indian populations demonstrated that Tat is non-immunodominant and that a large variation exists in the antigen-specific antibody titers.
  
  
  CONCLUSIONS
  Our report not only describes a simple protein purification strategy for Tat but also demonstrates important structural and functional differences between subtype-B and -C Tat proteins. Furthermore, this is the first report of protein purification and characterization of subtype-C Tat.
authors.affiliation: Molecular Virology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India. sidhu@jncasr.ac.in
authors.forename: Nagadenahalli Byrareddy
authors.initials: NB
authors.lastname: Siddappa
authors.forename: Mohanram
authors.initials: M
authors.lastname: Venkatramanan
authors.forename: Prasanna
authors.initials: P
authors.lastname: Venkatesh
authors.forename: Mohanbabu Vijayamma
authors.initials: MV
authors.lastname: Janki
authors.forename: Narayana
authors.initials: N
authors.lastname: Jayasuryan
authors.forename: Anita
authors.initials: A
authors.lastname: Desai
authors.forename: Vasanthapuram
authors.initials: V
authors.lastname: Ravi
authors.forename: Udaykumar
authors.initials: U
authors.lastname: Ranga
mesh_terms: D002460:Cell Line; D016207:Cytokines / Q000096:biosynthesis; D015696:Gene Products, tat / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology; D009000:Monocytes / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D015533:Transcriptional Activation; D014162:Transfection; D054322:tat Gene Products, Human Immunodeficiency Virus
chemical_list: D016207:Cytokines; D015696:Gene Products, tat; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus