Retrovirus glycoprotein functionality requires proper alignment of the ectodomain and the membrane-proximal cytoplasmic tail.

Nonnative viral glycoproteins, including Friend murine leukemia virus envelope (F-MLV Env) are actively recruited to HIV-1 assembly sites by an unknown mechanism. Because interactions with the lipid microenvironment at budding sites could contribute to recruitment, we examined the contribution of the hydrophobicity of the F-MLV Env membrane-spanning domain (MSD) to its incorporation into HIV-1 particles. A series of F-MLV Env mutants that added or deleted one, two, or three leucines in the MSD were constructed. All six mutants retained the ability to be incorporated into HIV-1 particles, but the -1L, -2L, -3L, +1L, and +2L mutants were not capable of producing infectious particles. Surprisingly, the +3L Env glycoprotein was able to produce infectious particles and was constitutively fusogenic. However, when the cytoplasmic tail domains (CTDs) in the Env constructs were deleted, all six of the MSD mutants were able to produce infectious particles. Further mutational analyses revealed that the first 10 amino acids of the CTD is a critical regulator of infectivity. A similar phenotype was observed in HIV-1 Env upon addition of leucines in the MSD, with +1 and +2 leucine mutations greatly reducing Env activity, but +3 leucine mutations behaving similar to the wild type. Unlike F-MLV Env (+1L and +2L), HIV-1 Env (+1L and +2L) infectivity was not restored by deletion of the CTD. We hypothesize that the CTD forms a coiled-coil that disrupts the protein's functionality if it is not in phase with the trimer interface of the ectodomain.

Kinetic studies of the effect of a 17-nucleotide difference in the 0.3-kb region containing the R-U5-5' leader sequence of Friend murine leukemia virus on viral gene expression.

In addition to the env gene, a 0.3-kb fragment containing the R-U5-5' leader sequence is essential for the induction of spongiform neurodegeneration by Friend murine leukemia virus (Fr-MLV) clone A8 and it also influences expression of the Env protein. Kinetic studies were carried out using two recombinant viruses, R7f, carrying the A8 0.3-kb fragment, and Rec5, carrying the 0.3-kb fragment of the non-neuropathogenic Fr-MLV clone 57. These analyses suggested that the 0.3-kb fragment influenced the expression level of the Env protein by regulating the amount of spliced env-mRNA rather than the amount of total viral mRNA or viral production.

Identification of the receptor binding domain of the mouse mammary tumor virus envelope protein.

Mouse mammary tumor virus (MMTV) is a betaretrovirus that infects rodent cells and uses mouse transferrin receptor 1 for cell entry. To characterize the interaction of MMTV with its receptor, we aligned the MMTV envelope surface (SU) protein with that of Friend murine leukemia virus (F-MLV) and identified a putative receptor-binding domain (RBD) that included a receptor binding sequence (RBS) of five amino acids and a heparin-binding domain (HBD). Mutation of the HBD reduced virus infectivity, and soluble heparan sulfate blocked infection of cells by wild-type pseudovirus. Interestingly, some but not all MMTV-like elements found in primary and cultured human breast cancer cell lines, termed h-MTVs, had sequence alterations in the putative RBS. Single substitution of one of the amino acids found in an h-MTV RBS variant in the RBD of MMTV, Phe(40) to Ser, did not alter species tropism but abolished both virus binding to cells and infectivity. Neutralizing anti-SU monoclonal antibodies also recognized a glutathione S-transferase fusion protein that contained the five-amino-acid RBS region from MMTV. The critical Phe(40) residue is located on a surface of the MMTV RBD model that is distant from and may be structurally more rigid than the region of F-MLV RBD that contains its critical binding site residues. This suggests that, in contrast to other murine retroviruses, binding to its receptor may result in few or no changes in MMTV envelope protein conformation.

In vivo distribution of receptor for ecotropic murine leukemia virus and binding of envelope protein of Friend Murine leukemia virus.

Ecotropic infection by Murine leukemia virus (MuLV) infection is initiated by the interaction between the receptor-binding domain of the viral surface glycoprotein (SU) and the cell-surface receptor, mCAT-1. To study the in vivo localization of viral binding site in mice, green fluorescence protein (GFP)-tagged Friend SU (F-SU/GFP) was incubated with tissue sections. Lymphohematopoietic organs and a part of the glandular tissues of C3H as well as C57BL/6 mice revealed positive signals for F-SU/GFP binding on the cell surface. In contrast, C4W mice, which is a partial congenic mouse strain carrying the Fv-4 (r) gene on a BALB/c genetic background, exhibited negative signals in most of the organs except for a very weak binding in the pancreas. The expression of mCAT-1 mRNA determined by reverse transcriptase (RT)-polymerase chain reaction (PCR) revealed a similar distribution in C3H, C57BL/6 and C4W mice. Most of the organs including lymphohematopoietic organs and glandular organs revealed significant expression of mRNA for mCAT-1 gene, while the liver, heart and muscle did not. The results from binding assay were consistent with the fact that Friend MuLV-induced pathogenesis was usually associated with lymphohematopoietic systems, although mRNA expression for mCAT-1 was rather ubiquitous. The discrepancy between F-SU/GFP binding and mRNA expression for mCAT-1 in lymphohematopoietic organs of C4W mice would support the receptor interference effect by the Fv-4 (r) gene causing the resistance of C4W mouse to Friend MuLV infection.

Definition of an amino-terminal domain of the human T-cell leukemia virus type 1 envelope surface unit that extends the fusogenic range of an ecotropic murine leukemia virus.

Murine leukemia viruses (MuLV) and human T-cell leukemia viruses (HTLV) are phylogenetically highly divergent retroviruses with distinct envelope fusion properties. The MuLV envelope glycoprotein surface unit (SU) comprises a receptor-binding domain followed by a proline-rich region which modulates envelope conformational changes and fusogenicity. In contrast, the receptor-binding domain and SU organization of HTLV are undefined. Here, we describe an HTLV/MuLV envelope chimera in which the receptor-binding domain and proline-rich region of the ecotropic MuLV were replaced with the potentially corresponding domains of the HTLV-1 SU. This chimeric HTLV/MuLV envelope was processed, specifically interfered with HTLV-1 envelope-mediated fusion, and similar to MuLV envelopes, required cleavage of its cytoplasmic tail to exert significant fusogenic properties. Furthermore, the HTLV domain defined here broadened ecotropic MuLV envelope-induced fusion to human and simian cell lines.

The hypervariable domain of the murine leukemia virus surface protein tolerates large insertions and deletions, enabling development of a retroviral particle display system.

The surface proteins (SU) of murine type-C retroviruses have a central hypervariable domain devoid of cysteine and rich in proline. This 41-amino-acid region of Friend ecotropic murine leukemia virus SU was shown to be highly tolerant of insertions and deletions. Viruses in which either the N-terminal 30 amino acids or the C-terminal 22 amino acids of this region were replaced by the 7-amino-acid sequence ASAVAGA were fully infectious. Insertions of this 7-amino-acid sequence at the N terminus, center, and the C terminus of the hypervariable domain had little effect on envelope protein (Env) function, while this insertion at a position 10 amino acids following the N terminus partially destabilized the association between the SU and transmembrane subunits of Env. Large, complex domains (either a 252-amino-acid single-chain antibody binding domain [scFv] or a 96-amino-acid V1/V2 domain of HIV-1 SU containing eight N-linked glycosylation sites and two disulfides) did not interfere with Env function when inserted in the center or C-terminal portions of the hypervariable domain. The scFv domain inserted into the C-terminal region of the hypervariable domain was shown to mediate binding of antigen to viral particles, demonstrating that it folded into the active conformation and was displayed on the surface of the virion. Both positive and negative enrichment of virions expressing the V1/V2 sequence were achieved by using a monoclonal antibody specific for a conformational epitope presented by the inserted sequence. These results indicated that the hypervariable domain of Friend ecotropic SU does not contain any specific sequence or structure that is essential for Env function and demonstrated that insertions into this domain can be used to extend particle display methodologies to complex protein domains that require expression in eukaryotic cells for glycosylation and proper folding.

In vitro binding of purified murine ecotropic retrovirus envelope surface protein to its receptor, MCAT-1.

An amino-terminal portion of the Friend murine leukemia virus (MLV) envelope surface protein [SU, residues 1 to 236 [SU:(1-236)]] and its receptor, MCAT-1, were each purified from insect cells after expression by using recombinant baculoviruses. Friend SU:(1-236) bound specifically to Xenopus oocytes that expressed MCAT-1 with an affinity (Kd, 55 nM) similar to that of viral SU binding to permissive cells. Direct binding of Friend SU:(1-236) to purified MCAT-1 was observed in detergent and after reconstitution into liposomes. Analysis of binding demonstrated that MCAT-1 and Friend SU:(1-236) interact with a stoichiometry of near 1:1. These findings demonstrate that the amino-terminal domain from the SU of ecotropic murine retroviruses contains an MCAT-1 binding domain.

Structure of a murine leukemia virus receptor-binding glycoprotein at 2.0 angstrom resolution.

An essential step in retrovirus infection is the binding of the virus to its receptor on a target cell. The structure of the receptor-binding domain of the envelope glycoprotein from Friend murine leukemia virus was determined to 2.0 angstrom resolution by x-ray crystallography. The core of the domain is an antiparallel beta sandwich, with two interstrand loops forming a helical subdomain atop the sandwich. The residues in the helical region, but not in the beta sandwich, are highly variable among mammalian C-type retroviruses with distinct tropisms, indicating that the helical subdomain determines the receptor specificity of the virus.

Effects of subtle changes in the SU protein of ecotropic murine leukemia virus on its brain capillary endothelial cell tropism and interference properties.

PVC-211 murine leukemia virus (MuLV) is a neuropathogenic variant of Friend MuLV (F-MuLV) that causes a rapidly progressive neurodegenerative disease in susceptible rodents. PVC-211 MuLV, but not the parental F-MuLV, can infect rat brain capillary endothelial cells (BCEC) efficiently, and the major determinant for BCEC tropism of PVC-211 MuLV is localized within the XbaI-BamHI fragment of the viral genome containing the 5' half of the env gene. To further dissect the XbaI-BamHI region for its effects on BCEC tropism, we constructed recombinant viruses between PVC-211 MuLV and F-MuLV and tested their infectivity on a cell line established from rat BCEC. Our results indicated that Glu116-to-Gly and Glu129-to-Lys substitutions in the background of the F-MuLV envelope SU protein were sufficient for conferring BCEC tropism on the virus. Interference studies of these viruses on Rat-1 fibroblastic cells showed that the structure of the SU protein encoded by the XbaI-BamHI region also has significant effects on their affinity for the rat ecotropic MuLV receptor. These results support the possibility that structural elements I and II of the SU protein are important determinants for virus-receptor interaction.

Localization of the palmitoylation site in the transmembrane protein p12E of Friend murine leukaemia virus.

Friend murine leukaemia virus complex was propagated on murine cells in the presence of [9,10-3H]palmitic acid. Virus particles were harvested from the culture supernatant and lysed with detergents. The viral transmembrane protein, p12E, was isolated from the lysates by size-exclusion chromatography and purified by narrowbore reverse-phase HPLC. Analysis of the purified product by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) revealed that the protein is palmitoylated carrying one fatty acid residue. The radiolabelled fatty acid was released by hydroxylamine treatment at pH 7, indicating that acylation occurred via a thioester linkage. For allocation of the acylation site, p12E was digested with trypsin. The resulting peptides were either directly subjected to MALDI-TOF-MS or fractionated by microbore reverse-phase HPLC prior to mass spectrometry. The results revealed that p12E of Friend murine leukaemia virus is acylated at a cysteine residue situated at the C-terminal side of the putative transmembrane anchor of the polypeptide. Fatty acid analysis of the purified acylpeptide demonstrated that p12E carries almost exclusively palmitic acid.

Structural elements in glycoprotein 70 from polytropic Friend mink cell focus-inducing virus and glycoprotein 71 from ecotropic Friend murine leukemia virus, as defined by disulfide-bonding pattern and limited proteolysis.

The disulfide-bonding pattern of glycoprotein 70 (gp70), the surface glycoprotein (SU) encoded by the envelope gene of polytropic Friend milk cell focus-inducing virus, was elucidated and compared with that of glycoprotein 71 (gp71), the corresponding glycoprotein of the ecotropic Friend murine leukemia virus, which had previously been determined (M. Linder, D. Linder, J. Hahnen, H.-H. Schott, and Stirm, Eur. J. Biochem. 203:65-73, 1992). In the carboxy-terminal constant domain, in which these glycoproteins have about 97% sequence homology, the location of the four disulfide bonds was found to be analogous. In the amino-terminal differential domain, with about 37% sequence homology, 8 of the 12 cysteine residues of the ecotropic SU are conserved in the polytropic SU. In this domain, a similar clustering of disulfide bonds was detected, which led to the identification of three distinct disulfide-bonded regions in both glycoproteins. However, because of deletions and sequence deviations, the glycoproteins must have significantly different three-dimensional structures in these regions. Since the receptor-binding functions of both glycoproteins have been attributed to their amino-terminal domains and since each binds to a different receptor, these disulfide-bonded structures are likely candidates for receptor-binding functions. Limited proteolysis of both glycoproteins with various endoproteinases led to the identification of preferential proteolytic sites between disulfide-bonded regions, at the beginning of the hypervariable proline-rich region, and between differential and constant domains, further confirming the structural organization of the folded glycoproteins.