Change of Tropism of SL3-2 Murine Leukemia Virus, Using Random Mutational Libraries

Abstract

SL3-2 is a polytropic murine leukemia virus with a limited species tropism. We cloned the envelope gene of this virus, inserted it into a bicistronic vector, and found that the envelope protein differs from other, similar envelope proteins that also utilize the polytropic receptor (Xpr1) in that it is severely impaired in mediating infection of human and mink cells. We found that two adjacent amino acid mutations (G212R and I213T), located in a previously functionally uncharacterized segment of the surface subunit, are responsible for the restricted tropism of the SL3-2 wild-type envelope. By selection from a two-codon library, several hydrophobic amino acids at these positions were found to enable the SL3-2 envelope to infect human TE 671 cells. In particular, an M212/V213 mutant had a titer at least 6 orders of magnitude higher than that of the wild-type envelope for human TE 671 cells and infected human, mink, and murine cells with equal efficiencies. Notably, these two amino acids are not found at homologous positions in known murine leukemia virus isolates. Functional analysis and library selection were done on the basis of sequence and tropism analyses of the SL3-2 envelope gene. Similar approaches may be valuable in the design and optimization of retroviral envelopes with altered tropisms for biotechnological purposes.The murine leukemia virus (MLV) group of gammaretroviruses has been divided into the ecotropic, amphotropic, and polytropic-xenotropic subfamilies according to their host range and interference properties, as determined primarily on the basis of the structures of their respective envelope glycoproteins.The envelope precursor protein is cleaved into two subunits in the endoplasmic reticulum by a cellular protease. The Nterminal surface subunit (SU) is mainly responsible for receptor recognition and binding, while the C-terminal transmembrane subunit is involved in the fusion of the viral and cellular membranes. Accordingly, the SU subunits of different MLV subfamilies show a considerable degree of residue disparity, especially in the N-terminal 200 to 250 residues, while the transmembrane subunits are very much identical. Interestingly, the N terminus of the SU subunit constitutes a receptor-binding domain (RBD), which can bind to the appropriate receptor independently of the rest of the envelope protein. Thus, the ecotropic RBD, which consists of the first 230 (4) or 245 (22) residues, contains almost all of the unique ecotropic envelope sequences. This domain is followed by a proline-rich region that can tolerate large insertions or deletions (15,36).Three segments of the RBD that show large variations in MLV subfamilies are conveniently known as hypervariable regions A, B, and C (VRA, VRB, and VRC, respectively). Several studies have suggested that the determinants of the tropisms of ecotropic and amphotropic envelopes are found in the N terminus of RBD, particularly VRA, while segments further downstream are partially responsible for polytropic envelope tropism (4,7,8,25,29).Ecotropic and...

Construction of a Gammaretrovirus with a Novel Tropism and Wild-Type Replication Kinetics Capable of Using Human APJ as Entry Receptor

“…Since this insertion does not remove the ability of the AP@165 envelope protein to use its natural receptor, while insertion at position 86 completely inactivates the envelope function, the binding site for Xpr1 might be expected to be found on the other end of this surface, mostly in VRA and VR3. This is supported by our previous finding that two amino acid alterations at VR3 can enable SL3-2 to use Xpr1 in other species (4) (Fig. 7).…”

“…SL3-2 envelope protein, because of its limited and ecotropiclike tropism, offers another candidate for a retargeting scaffold. This envelope belongs to the xenotropic-polytropic group but can infect only murine cells (4). Using it as a scaffold, we have produced a retargeted envelope protein that can infect through a heterologous receptor as well as its natural surface receptors.…”

“…4A, D17 APJ cells showed high levels of envelope protein, suggesting that the virus has replicated in these cells, while D17 cells without APJ were not infected. Control viruses containing the SL3-2 GI envelope protein (which can use Xpr1 for entry in both human, mink, and murine cells) (4,6) were unable to infect the D17 APJ cell line (Fig. 4B).…”

“…As seen in Fig. 1, alignment of the receptor binding domains (RBDs) from these two envelope proteins shows that the main differences are found in two clusters, which correspond to variable regions A and B (VRA and VRB, respectively) identified previously (4). Based on this information, three constructs were made by insertion of the apelin-13 sequence flanked by two linker sequences at positions 86, 155, and 165 of the SL3-2 envelope protein.…”

Retroviral insertional mutagenesis: past, present and future

“…This specificity is frequently attributable the LTR regions (DesGroseillers and Jolicoeur, 1984a, b) although in some cases subtle mutations can also alter the tropism of the virus envelope proteins (Bahrami et al, 2004). A series of variants of the SL3-3 murine leukaemia virus that normally induces T-cell lymphomas have been shown to have altered oncogenic capabilities.…”