The five highly related envelope subgroups of the avian sarcoma and leukosis viruses (ASLVs), subgroup A [ASLV(A)] to ASLV(E), are thought to have evolved from an ancestral envelope glycoprotein yet utilize different cellular proteins as receptors. Alleles encoding the subgroup A ASLV receptors (Tva), members of the low-density lipoprotein receptor family, and the subgroup B, D, and E ASLV receptors (Tvb), members of the tumor necrosis factor receptor family, have been identified and cloned. However, alleles encoding the subgroup C ASLV receptors (Tvc) have not been cloned. Previously, we established a genetic linkage between tvc and several other nearby genetic markers on chicken chromosome 28, including tva. In this study, we used this information to clone the tvc gene and identify the Tvc receptor. A bacterial artificial chromosome containing a portion of chicken chromosome 28 that conferred susceptibility to ASLV(C) infection was identified. The tvc gene was identified on this genomic DNA fragment and encodes a 488-amino-acid protein most closely related to mammalian butyrophilins, members of the immunoglobulin protein family. We subsequently cloned cDNAs encoding Tvc that confer susceptibility to infection by subgroup C viruses in chicken cells resistant to ASLV(C) infection and in mammalian cells that do not normally express functional ASLV receptors. In addition, normally susceptible chicken DT40 cells were resistant to ASLV(C) infection after both tvc alleles were disrupted by homologous recombination. Tvc binds the ASLV(C) envelope glycoproteins with low-nanomolar affinity, an affinity similar to that of binding of Tva and Tvb with their respective envelope glycoproteins. We have also identified a mutation in the tvc gene in line L15 chickens that explains why this line is resistant to ASLV(C) infection.Retroviruses require an interaction between the viral glycoproteins and a specific cell surface protein (receptor) to initiate entry into a cell (reviewed in references 32 and 56). The envelope glycoproteins of retroviruses are composed of trimers of two glycoproteins: the surface glycoprotein (SU), which contains the domains responsible for interaction with the host receptor, and the transmembrane glycoprotein (TM), which anchors SU to the membrane and mediates fusion of the viral and host membranes. The interaction of the SU glycoprotein with the host receptor usually involves multiple, noncontiguous determinants in both proteins that specify receptor choice and binding affinity and trigger a conformational change in the envelope glycoproteins that initiates the fusion process. Despite the complexity and specificity of the interaction between the viral glycoproteins and host receptors, closely related retroviruses carry envelope glycoproteins with mutations that alter receptor usage. The natural selection of retroviral subgroups with altered receptor usage may help the virus overcome host resistance and promote coinfection and may lead to heterotransmission.The five highly related envelope subgroups of...

Retroviruses share a common strategy for entry into cells (reviewed in references 27 and 51). The entry process is initiated by an interaction between the viral envelope glycoprotein and a specific cell surface protein that acts as a receptor. This interaction triggers a conformational change in the structure of the viral glycoprotein that leads to the fusion of the viral and cellular membranes. Despite the complexity of the interaction between the viral glycoprotein and the receptor, closely related retroviruses carry envelope glycoproteins that use different cellular proteins as receptors. In the avian sarcoma and leukosis viruses (ASLVs), there are five highly related envelope subgroups, subgroups A to E, that are thought to have evolved from a common ancestor (reviewed in references 7 and 50). The presence of viral subgroups that utilize distinct receptors helps the virus overcome host resistance and promotes coinfection. We and others have developed strategies that mimic resistance to ASLV entry in cell culture to study the evolution of the envelope glycoprotein. These studies demonstrated that blocking virus entry could select viral variants with mutations in the viral glycoproteins that altered receptor usage (24,25,31,34,43). The goal of the present study was to identify and characterize the mutations in the subgroup A receptor in lines of chickens that cause resistance to infection by ASLVs carrying the subgroup A envelope glycoproteins.Three genetic loci in chicken cells determine the susceptibility and resistance to subgroup A to E ASLVs: tva (susceptibility to subgroup A viruses), tvb (susceptibility to subgroup B, D, and E viruses), and tvc (susceptibility to subgroup C viruses) (49, 50). Alleles that confer susceptibility to ASLV infection are dominant: two recessive resistance alleles are required at these loci to confer resistance. Because the tva r , tvb r , and tvc r resistance alleles are recessive, it is unlikely that these alleles encode dominant-negative forms of the receptor protein. The resistance alleles are likely to contain defects that either block receptor expression or prevent its use as an efficient ASLV receptor (29).Several alleles of the tvb genetic locus and three related Tvb receptors have been identified. Two different susceptibility alleles have been defined at the chicken tvb locus. The tvb s1 allele confers susceptibility to subgroups B, D, and E; the tvb s3 allele confers susceptibility to only subgroups B and D (1, 3). These alleles encode the chicken Tvb S1 (3) and Tvb S3 (12) receptors, respectively. Tvb S3 differs from Tvb S1 by a single amino acid change, cysteine to serine at position 62, which presumably alters the structure of the Tvb S1 protein so that it no longer functions as an ASLV(E) receptor. A third tvb receptor, the turkey Tvb T receptor (2), which confers susceptibility to only subgroup E ASLV, has also been cloned. The Tvb proteins are members of the tumor necrosis factor receptor (TNFR) family. The recessive tvb r resistance allele does not support the...

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A complex interaction between the retroviral envelope glycoproteins and a specific cell surface protein initiates viral entry into cells. The avian leukosis-sarcoma virus (ALV) group of retroviruses provides a useful experimental system for studying the retroviral entry process and the evolution of receptor usage. In this Retroviruses share a common overall strategy for entry into cells (for recent reviews, see references 26 and 40). The retroviral envelope glycoproteins are initially synthesized as a polyprotein precursor that is subsequently processed into two glycoproteins: the surface glycoprotein (SU), which contains the major domains that interact with the host receptor, and the transmembrane glycoprotein (TM), which anchors SU to the membrane and is directly involved in the fusion of viral and host membranes. The entry process is initiated by a complex interaction between SU and a specific cell surface protein that acts as a receptor, involving multiple, noncontiguous determinants in both proteins that specify receptor choice and binding affinity. Only a proper interaction triggers a conformational change in the structure of the viral glycoproteins which unlocks the fusion peptide located in TM. The exposed fusion peptide interacts with the target cell membrane, initiating a multistep process leading to fusion of the viral and cellular membranes and delivery of a subviral particle into the cell. Despite the complexity of the initial viral SU-cellular receptor interaction, retroviruses have the ability to evolve the structure of their envelope glycoproteins so that they can use a different cellular protein as a receptor (at times a protein that has no obvious homology to the original receptor) and retain efficient entry functions.The avian leukosis-sarcoma virus (ALV) group of retroviruses provides a useful experimental system for studying the initial interactions of retroviral entry and the evolution of receptor usage. ALV envelope subgroups A through E [ALV(A) through ALV(E)] are highly related, suggesting that these viruses have evolved from a common viral ancestor to use distinct cellular proteins as receptors in order to gain entry into chicken cells, presumably in response to the development of host resistance to viral entry. ALV(A) to ALV(E) SU glycoproteins are almost identical except for five hypervariable regions designated vr1, vr2, hr1, hr2, and vr3 ( Fig. 1) (6,7,13). Past analyses have suggested that the principal receptor interaction determinants are contained in the hr1 and hr2 domains of ALV SU, with vr3 playing a role in the specificity of receptor recognition but not in receptor binding affinity (14,36,37). The vr1 and vr2 hypervariable regions did not appear to be essential for receptor specificity or binding affinity.Five cell surface proteins have been identified as ALV receptors. The two subgroup A receptors, quail Tva and the chicken Tva homologue, are related to the low-density lipoprotein receptor family (4, 5, 41). The three receptors Tvb S1 (subgroups B, D, and E) (2), Tvb S3 (...

We previously showed that the cysteines flanking the internal fusion peptide of the avian sarcoma/leukosis virus subtype A (ASLV-A) Env (EnvA) are important for infectivity and cell-cell fusion. Here we define the stage of fusion at which the cysteines are required. The flanking cysteines are dispensable for receptor-triggered membrane association but are required for the lipid mixing step of fusion, which, interestingly, displays a high pH onset and a biphasic profile. Second-site mutations that partially restore infection partially restore lipid mixing. These findings indicate that the cysteines flanking the internal fusion peptide of EnvA (and perhaps by analogy Ebola virus glycoprotein) are important for the foldback stage of the conformational changes that lead to membrane merger.The avian sarcoma/leukosis virus (ASLV) Env protein is unusual among class I fusion proteins in that it contains an internal fusion peptide, a characteristic it shares with filovirus (Ebola virus and Marburg virus) glycoproteins. These fusion peptides are flanked by two Cys residues, which, based on structural and mutagenesis evidence, likely form a disulfide bond (9, 21). The ASLV fusion peptide contains one proline, and the filovirus fusion peptides contain two prolines, at their centers. Previous work has shown that these Pro residues are important for fusion (4,8). The fusion peptide of EnvA is operationally defined as residues 22 to 37 of the transmembrane (TM) subunit. We previously provided evidence that the Pro in the middle of this peptide (P29) requires a ␤-turn structure (4) at some stage of fusion. We subsequently showed that the two Cys residues (C9 and C45) that define the likely disulfide-bonded loop encompassing the fusion peptide are required for infectivity and cell-cell fusion (5). In this study, we identified the specific stage of fusion that requires the Cys residues that flank the EnvA fusion peptide.To begin our studies, we asked if the defect was in the first step of fusion: target membrane binding. Accordingly, we determined the ability of murine leukemia virus pseudotyped virions (10) bearing either wild-type EnvA or an EnvA harboring double Cys-to-Ser mutations at both positions 9 and 45 of the TM subunit (referred to below as EnvAC9,45S) to bind to target membranes in a receptor-dependent manner (6, 7, 15). As seen in Fig. 1, wild-type murine leukemia virus pseudovirions bound liposomes and floated to the top of the gradient when either the quail (lane 1) or the chicken (lane 5) form of the soluble Tva receptor (sTva) was used (7). In the absence of sTva, all of the pseudovirions remained at the bottom of the gradient (Fig. 1, lane 12). Similar receptor-dependent membrane association was observed for EnvAC9,45S (Fig. 1). These data show that the Cys residues (C9 and C45) that define the loop encompassing the fusion peptide are not required for receptor-triggered binding of EnvA-bearing virus particles to target membranes.We then asked whether the cysteines are required for EnvA to reach the lipid mixing ...

A number of unnatural D-3-deoxy-3-substituted myo-inositols were synthesized and their effects on the growth of wild-type NIH 3T3 cells and oncogene-transformed NIH 3T3 cells were studied. The compounds were found to exhibit a diversity of growth-inhibitory activities and showed selectivity in inhibiting the growth of some transformed cells as compared with wild-type cells. Remarkably, D-3-deoxy-3-azido-myo-inositol exhibited potent growth-inhibitory effects toward v-sis-transformed NIH 3T3 cells but had little effect on the growth of wild-type cells. The growth-inhibitory effects of the myo-inositol analogues were antagonized by myo-inositol. Since [3H]-3-deoxy-3-fluoro-myo-inositol was shown to be taken up by cells and incorporated into cellular phospholipids, we suggest that these unnatural myo-inositol analogues may act as antimetabolites in the phosphatidylinositol intracellular signalling pathways. Because cells transformed by oncogenes often exhibit elevated phosphatidylinositol turnover, the inhibition of signalling pathways that mediate oncogene action could offer new opportunities for controlling the growth of cancer cells.

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