The murine AIDS virus Gag precursor protein binds to the SH3 domain of c-Abl

Abstract

The Pr60 gag protein of the murine AIDS (MAIDS) defective virus promotes the proliferation of the infected target B cells and is responsible for inducing a severe immunodeficiency disease. Using the yeast two-hybrid system, we identified the SH3 domain of c-Abl as interacting with the proline-rich p12 domain of Pr60 gag. The two proteins were shown to associate in vitro and in vivo in MAIDS virus-infected B cells. Overexpression of Pr60 gag in these cells led to a detectable increase of the levels of c-Abl protein and to its translocation at the membrane. These results suggest that this viral protein serves as a docking site for signaling molecules and that c-Abl may be involved in the proliferation of infected B cells. Murine AIDS (MAIDS) is a severe immunodeficiency disease characterized by lymphadenopathy, splenomegaly, hypergammaglobulinemia, T-and B-cell dysfunctions, and late appearance of B-cell lymphomas and opportunistic infections (19, 23). This severe immune disease is caused by a defective strain of murine leukemia virus (MuLV) (2, 4). The main target cells of this defective virus appear to be peripheral B lymphocytes which are induced to proliferate after infection (17). The defective viral genome encodes a single Gag precursor (Pr60 gag) protein (15) whose p12 region is highly divergent from those of the p12 proteins of other helper MuLVs. Mutational analyses have confirmed that the Pr60 gag is necessary and sufficient for disease induction (14, 20, 25). In addition, myristylation-negative mutant MAIDS viruses were found to be nonpathogenic, indicating that myristylation and tight membrane association are required for Pr60 gag to be pathogenic (16). These results suggested that intact Pr60 gag may interact with some cellular effectors and possibly serve as a docking site at the membrane to initiate target cell expansion and pathogenesis. We searched for some proteins interacting with Pr60 gag and found that the c-Abl protein is one of these interacting proteins. MATERIALS AND METHODS Construction of plasmids for yeast two-hybrid screening. The construction of the clone encoding the complete Pr60 gag fused to the LexA DNA-binding domain (DB) (pBTM/DuGAG) was made by PCR site-directed mutagenesis using oligonucleotides 5Ј-CCGGAATTCATGGGACAGACCGTAACCACTC-3Ј (sense) and 5Ј-AGTACCATCTAGTGGCCACC-3Ј (antisense). An EcoRI site was introduced at nucleotide (nt) 970 from Du5H (2). The amplified fragment was digested with EcoRI and AatII (nt 1025) and subcloned into pBS-SK together with an AatII (nt 1025)-HindIII (nt 3264) fragment from plasmid pDu5Hneo (2) to generate Du5H R1-970 , which was sequenced. Digestion with EcoRI and SalI (of pBS-SK) from Du5H R1-970 generated a fragment which was cloned into pBTM116 to generate pBTM/DuGAG. Plasmids pBTM/DuGAG/⌬CA, pBTM/DuGAG/⌬12, and pBTM/DuGAG/⌬MA were constructed by swapping the AatII-HindIII fragment of plasmid Du5H R1-970 with those of plasmids pDu5H-A, pDu5H-B, and pDu5H-C, encoding Pr60 gag deletion mutants described previously (14), before subcloning into pB...

Radioactive Scandium in the Youngest Galactic Supernova Remnant G1.9+0.3

“…It appears that G1.9+0.3 shows the first definite detection of this transition. Our line strength implies a flux in the 1.157 MeV line of about 1.2 × 10 −5 ph cm −2 s −1 , well below the COMP-TEL limit of about 2 × 10 −5 ph cm −2 s −1 (Dupraz et al 1997).…”

Three supernova remnants observed by BeppoSAX

“…During its decay to 44 Sc 44 Ti emits two photons at 67.9 keV and 78.4 keV. The flux in theses lines should correspond to the flux at 1.157 MeV of (3.4 ± 0.9)10 −5 cm −2 s −1 reported for Comptel [15]. Attempts to measure these lines with RossiXTE [23] and CGRO/OSSE [18] are not yet successful, although the results are not inconsistent with the Comptel results.…”

“…So can we conclude that Cas A contains a black hole? Possibly, but this seems at odds with the recent discovery of emission from radio-active 44 Ti by the CGRO/Comptel experiment [14,15], as we will explain below. A puzzling fact is also the rather small amount of neon and magnesium [13], which is not expected for an oxygen rich remnant.…”

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Increased cAMP levels and protein kinase (PKA) type I activation in CD4+ T cells and B cells contribute to the retrovirus‐induced immunodeficiency of mice (MAIDS). A useful in vivo model for drug testing in PKA type I‐ induced immunodeficiency

“…The mechanism by which the variant Pr60 gag protein induces T-cell abnormalities is not known. Pr60 gag could interact with the c-abl oncogene and induce aberrant activation of signaling pathways in infected B cells (13). Soluble factors secreted by infected cells have been claimed to influence the function of T cells at a distance (14), but the nature of such mediators has never been elucidated.…”