       Document 0391
 DOCN  M9620391
 TI    Mutagenic analysis of human immunodeficiency virus type 1 Vpr: role of a
       predicted N-terminal alpha-helical structure in Vpr nuclear localization
       and virion incorporation.
 DT    9602
 AU    Yao XJ; Subbramanian RA; Rougeau N; Boisvert F; Bergeron D; Cohen EA;
       Departement de Microbiologie et Immunologie, Faculte de; Medecine,
       Universite de Montreal, Quebec, Canada.
 SO    J Virol. 1995 Nov;69(11):7032-44. Unique Identifier : AIDSLINE
       MED/96013806
 AB    The Vpr gene product of human immunodeficiency virus type 1 is a
       virion-associated protein that is important for efficient viral
       replication in nondividing cells such as macrophages. At the cellular
       level, Vpr is primarily localized in the nucleus when expressed in the
       absence of other viral proteins. Incorporation of Vpr into viral
       particles requires a determinant within the p6 domain of the Gag
       precursor polyprotein Pr55gag. In the present study, we have used
       site-directed mutagenesis to identify a domain(s) of Vpr involved in
       virion incorporation and nuclear localization. Truncations of the
       carboxyl (C)-terminal domain, rich in basic residues, resulted in a less
       stable Vpr protein and in the impairment of both virion incorporation
       and nuclear localization. However, introduction of individual
       substitution mutations in this region did not impair Vpr nuclear
       localization and virion incorporation, suggesting that this region is
       necessary for the stability and/or optimal protein conformation relevant
       to these Vpr functions. In contrast, the substitution mutations within
       the amino (N)-terminal region of Vpr that is predicted to adopt an
       alpha-helical structure (extending from amino acids 16 to 34) impaired
       both virion incorporation and nuclear localization, suggesting that this
       structure may play a pivotal role in modulating both of these biological
       properties. These results are in agreement with a recent study showing
       that the introduction of proline residues in this predicted
       alpha-helical region abolished Vpr virion incorporation, presumably by
       disrupting this secondary structure (S. Mahalingam, S. A. Khan, R.
       Murali, M. A. Jabbar, C. E. Monken, R. G. Collman, and A. Srinivasan,
       Proc. Natl. Acad. Sci. USA 92:3794-3798, 1995). Interestingly, our
       results show that two Vpr mutants harboring single amino acid
       substitutions (L to F at position 23 [L23F] and A30F) on the hydrophobic
       face of the predicted helix coded for relatively stable proteins that
       retained their ability to translocate to the nucleus but exhibited
       dramatic reduction in Vpr incorporation, suggesting that this
       hydrophobic face might mediate protein-protein interactions required for
       Vpr virion incorporation but not nuclear localization. Furthermore, a
       single mutation (E25K) located on the hydrophilic face of this predicted
       alpha-helical structure affected not only virion incorporation but also
       nuclear localization of Vpr. The differential impairment of Vpr nuclear
       localization and virion incorporation by mutations in the predicted
       N-terminal alpha-helical region suggests that this region of Vpr plays a
       role in both of these biological functions of Vpr.
 DE    Amino Acid Sequence  Base Sequence  Cell Line  Cell Nucleus/*METABOLISM
       Cloning, Molecular  Comparative Study  DNA Primers  Gene Products,
       vpr/BIOSYNTHESIS/*CHEMISTRY/*METABOLISM  *Genes, vpr  Human  HIV Long
       Terminal Repeat  HIV-1/*GENETICS/*PHYSIOLOGY  Kinetics
       Methionine/METABOLISM  Molecular Sequence Data  Mutagenesis,
       Site-Directed  Polymerase Chain Reaction  Protein Conformation  *Protein
       Structure, Secondary  Recombinant
       Proteins/BIOSYNTHESIS/CHEMISTRY/METABOLISM  Support, Non-U.S. Gov't
       Virion/GENETICS/PHYSIOLOGY  Virus Replication  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

