In addition to viral entry occurring by a CD4-independent mechanism, neutralization of SIV/17E-Fr with rhesus mAbs from 3 different neutralization groups blocked entry into xKLuSIV cells by both CD4-dependent and -independent mechanisms. into xKLuSIV cells by both CD4-dependent and -impartial mechanisms. Triggering the glycoprotein of SIV-17EFr with soluble CD4 had no significant effect in infectivity, but triggering of the same glycoprotein of SIV/17E-Cl allowed it to enter cells in a CD4-independent fashion. Using mutant molecular Wortmannin clones, we studied the determinants for CD4 independence, all of which are confined to the gene. We report here that truncation of the TM at amino acid 764 and changing a single amino acid (R751G) in the SIV envelope transmembrane protein (TM) conferred the observed CD4-impartial phenotype. Our data suggest that the envelope from the neurovirulent SIV/17E-Fr interacts with CCR5 in a CD4-independent manner, and changes in the TM protein of this virus are important components that contribute to neurovirulence in SIV. may play a role in neurotropism (Barber et al., 1998; Flaherty et al., 1997, 1998). We therefore investigated the role of mutations in TM and Nef in the infection of cells by the neurotropic virus SIV/17E-Fr and its possible implications in the CD4-independent mechanism previously observed. Results Creation of xKLuSIV cell lines To study SIV usage of chemokine receptors for cell entry by simian immunodeficiency viruses of different tropisms, we used the K562 human chronic myelogenous leukemia cell line that has been used extensively in chemokine receptor experiments because it expresses few, Mouse monoclonal to FOXA2 if any, chemokine receptors, and none that have been shown to be utilized for SIV or HIV entry (Baba et al., 1997; Gao and Murphy, 1995). We stably transformed a reporter construct, pLuc (Roos et al., 2000), into K562s to create a parental cell line, xKLuSIV, that could be used to readily identify cells that were infected by a straightforward, quantitative luciferase assay (Roos et al., 2000). Transfection of an infectious molecular clone into xKLuSIV cells resulted in activation of the luciferase gene (Fig. 3A), while inoculating the same cells with virus from either the macrophage-tropic non-neurovirulent SIV/17E-Cl or with the macrophage-tropic neurovirulent SIV/17E-Fr (see Fig. 1 for maps of these two strains) resulted in no detectable luciferase activity (Fig. 3B); neither virus replication was detected in basic xKLuSIV cells 4 days post-inoculation. These data showed that the basic xKLuSIV cells were devoid of any functional cellular receptors for SIV, but that they also had all the necessary intracellular components to support replication. xKLuSIV cells were then transformed with either or both RhCD4 and Wortmannin RhCCR5 cDNA. Each cell line bearing different receptors was drug selected and each molecules surface expression was confirmed by flow cytometry, (Table 1 and Fig. 2; see Materials and methods). Open in a separate window Fig. 1 Maps of the 3 regions of the genomes of SIV/17E-Cl and Wortmannin SIV/17E-Fr. The molecular clone SIV/17E-Cl was constructed by shuttling the SU region of the env gene from the previously described neurovirulent SIV/17E-Br (black bars) into the genetic background of the non-neurovirulent SIVmac239 (white bars; Flaherty et al., 1997). SIV/17E-Fr differs in that it also contains the entire env and nef genes of SIV/17-Br (black bars) in the genetic context of SIVmac239 (white bars; Flaherty et al., 1997), including TM and part of the 3 LTR. The TM of SIV/17E-Fr contains one mutation that codes for an R751G change (*) and a second mutation that results in a truncated envelope glycoprotein at amino acid 763 (open triangle). The dark diamond represents a stop codon, present in the nef gene of SIV/17E-Cl to create a truncated nef, but absent in the genome of SIV/17E-Fr, which expresses a full-length nef (Flaherty et al., 1997). Open in a separate window Fig. 2 Expression of receptors on the surface of xKLuSIV cells as quantitated by flow cytometry. Wortmannin (A, B, and E) Expression of CCR5; (C, D, and E) expression of CD4. Staining on xKLuSIV cells is depicted.