title: Requirement of an additional Sam68 domain for inhibition of human immunodeficiency virus type 1 replication by Sam68 dominant negative mutants lacking the nuclear localization signal.
abstract: Human immunodeficiency virus type 1 (HIV-1) replication requires active nuclear export of unspliced and incompletely spliced HIV-1 RNA transcripts. This process is evolutionally made possible by expression of HIV-1 Rev, one of the three HIV-1 proteins encoded by completely spliced HIV-1 RNAs. Evidence has accumulated to suggest that Sam68 plays an important role in HIV-1 replication through HIV-1 Rev protein. In the present study, we further examined the structure-function relationship of Sam68 protein in relation to HIV-1 replication. We identified a Sam68 domain located between aa269 and aa321 to be involved in the HIV-inhibitory effects of Sam68 dominant negative mutants lacking the nuclear localization signal (NLS). Deletion of this domain abrogated inhibition of HIV-1 replication by these mutants. HIV-1 Rev protein appeared to mediate the HIV-inhibitory effects of these mutants and by this domain, as assessed by Rev-dependent chloramphenicol acetyltransferase reporter gene assay, in trans rev-defective HIV-1 complementation assay, and RNase protection assay. The HIV-inhibitory mutants containing this domain were further found to have diminished binding affinity to the wild-type Sam68 and to be associated with cytoplasmic retention of exclusively nuclear localized wild type Sam68. Taken together, these results further ascertain the important role of Sam68 in HIV-1 Rev function and viral replication, and suggest that the HIV-inhibitory effects of Sam68 dominant negative mutants directly result from their binding to endogenous Sam68 and their interference with nuclear localization of endogenous Sam68.
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, R2 302, 950 W. Walnut St., Indianapolis, IN 46202, United States.
authors.forename: Jizhong
authors.initials: J
authors.lastname: Zhang
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Jorge
authors.initials: J
authors.lastname: Henao
authors.forename: Maria T
authors.initials: MT
authors.lastname: Rugeles
authors.forename: Jinliang
authors.initials: J
authors.lastname: Li
authors.forename: Tie
authors.initials: T
authors.lastname: Chen
authors.forename: Johnny J
authors.initials: JJ
authors.lastname: He
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D015694:Gene Products, rev / Q000502:physiology; D005799:Genes, Dominant; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D047468:Immunoprecipitation; D009154:Mutation; D019913:Nuclear Localization Signals; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D013329:Structure-Activity Relationship; D020798:Two-Hybrid System Techniques; D014779:Virus Replication / Q000235:genetics* / Q000502:physiology; D054321:rev Gene Products, Human Immunodeficiency Virus
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D017931:DNA Primers; D004268:DNA-Binding Proteins; D015694:Gene Products, rev; C489559:KHDRBS1 protein, human; D019913:Nuclear Localization Signals; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus