Analysis of HIV-2 Vpx by modeling and insertional mutagenesis.
Vpx facilitates HIV-2 nuclear localization by a poorly understood mechanism. We have compared Vpx to an NMR structure HIV-1 Vpr in a central helical domain and probed regions of Vpx by insertional mutagenesis. A predicted loop between helices two and three appears to be unique, overlapping with a known novel nuclear localization signal. Overall, Vpx was found to be surprisingly flexible, tolerating a series of large insertions. We found that insertion within the polyproline-containing C-terminus destabilizes nuclear localization, whereas mutating a second helix in the central domain disrupts viral packaging. Other insertional mutants in the predicted loop and in a linker region between the central domain and the C-terminus may be useful as sites of intramolecular tags as they could be packaged adequately and retained preintegration complex associated integration activity in a serum starvation assay. An unexpected result was found within a previously defined nuclear localization motif near aa 71. This mutant retained robust nuclear localization in a GFP fusion assay and was competent for preintegration complex associated nuclear import. In summary, we have modeled helical content in Vpx and assessed potential sites of intramolecular tags which may prove useful for protein-protein interactions studies.
Mahnke
Lisa A
LA
Department of Medicine, Divisions of Oncology and Infections Diseases, Washington University School of Medicine, PO Box 8069, 660 South Euclid Avenue, Saint Louis, MO 63110, USA. lmahnke@im.wustl.edu
Belshan
Michael
M
Ratner
Lee
L
D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000737:chemistry; D016336:Gene Products, vpr / Q000737:chemistry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis; D015498:HIV-2 / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D021381:Protein Transport; D016415:Sequence Alignment; D054334:Viral Regulatory and Accessory Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D019065:Virus Assembly / Q000235:genetics; D054325:vpr Gene Products, Human Immunodeficiency Virus
D016336:Gene Products, vpr; C074286:VPX protein, Human immunodeficiency virus 2; D054334:Viral Regulatory and Accessory Proteins; D054325:vpr Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins