Melder, Deborah C.

The subgroup A through E avian sarcoma and leukosis viruses ASLV(A) through ASLV(E) are a group of highly related alpharetroviruses that have evolved to use very different host protein families as receptors. We have exploited genetic selection strategies to force the replication-competent ASLVs to naturally evolve and acquire mutations to escape the pressure on virus entry and yield a functional replicating virus. In this study, evolutionary pressure was exerted on ASLV(B) virus entry and replication using a secreted for of its Tvb receptor. As expected, mutations in the ASLV(B) surface glycoprotein hypervariable regions were selected that knocked out the ability for the mutant glycoprotein to bind the sTvbS3-IgG inhibitor. However, the subgroup B Rous associated virus 2 (RAV-2) also required additional mutations in the C-terminal end of the SU glycoprotein and multiple regions of TM highlighting the importance of the entire viral envelope glycoprotein trimer structure to mediate the entry process efficiently. These mutations altered the normal two-step ASLV membrane fusion process to enable infection.

ABSTRACTThe study of the interactions of subgroup A avian sarcoma and leucosis viruses [ASLV(A)] with the TVA receptor required to infect cells offers a powerful experimental model of retroviral entry. Several regions and specific residues in the TVA receptor have previously been identified to be critical determinants of the binding affinity with ASLV(A) envelope glycoproteins and to mediate efficient infection. Two homologs of the TVA receptor have been cloned: the original quail TVA receptor, which has been the basis for most of the initial characterization of the ASLV(A) TVA, and the chicken TVA receptor, which is 65% identical to the quail receptor overall but identical in the region thought to be critical for infection. Our previous work characterized three mutant ASLV(A) isolates that could efficiently bind and infect cells using the chicken TVA receptor homolog but not using the quail TVA receptor homolog, with the infectivity of one mutant virus being >500-fold less with the quail TVA receptor. The mutant viruses contained mutations in the hr1 region of the surface glycoprotein. Using chimeras of the quail and chicken TVA receptors, we have identified new residues of TVA critical for the binding affinity and entry of ASLV(A) using the mutant glycoproteins and viruses to probe the function of those residues. The quail TVA receptor required changes at residues 10, 14, and 31 of the corresponding chicken TVA residues to bind wild-type and mutant ASLV(A) glycoproteins with a high affinity and recover the ability to mediate efficient infection of cells. A model of the TVA determinants critical for interacting with ASLV(A) glycoproteins is proposed.IMPORTANCEA detailed understanding of how retroviruses enter cells, evolve to use new receptors, and maintain efficient entry is crucial for identifying new targets for combating retrovirus infection and pathogenesis, as well as for developing new approaches for targeted gene delivery. Since all retroviruses share an envelope glycoprotein organization, they likely share a mechanism of receptor triggering to begin the entry process. Multiple, noncontiguous interaction determinants located in the receptor and the surface (SU) glycoprotein hypervariable domains are required for binding affinity and to restrict or broaden receptor usage. In this study, further mechanistic details of the entry process were elucidated by characterizing the ASLV(A) glycoprotein interactions with the TVA receptor required for entry. The ASLV(A) envelope glycoproteins are organized into functional domains that allow changes in receptor choice to occur by mutation and/or recombination while maintaining a critical level of receptor binding affinity and an ability to trigger glycoprotein conformational changes.

ABSTRACT The entry process of the avian sarcoma and leukosis virus (ASLV) family of retroviruses requires first a specific interaction between the viral surface (SU) glycoproteins and a receptor on the cell surface at a neutral pH, triggering conformational changes in the viral SU and transmembrane (TM) glycoproteins, followed by exposure to low pH to complete fusion. The ASLV TM glycoprotein has been proposed to adopt a structure similar to that of the Ebola virus GP2 protein: each contains an internal fusion peptide flanked by cysteine residues predicted to be in a disulfide bond. In a previous study, we concluded that the cysteines flanking the internal fusion peptide in ASLV TM are critical for efficient function of the ASLV viral glycoproteins in mediating entry. In this study, replication-competent ASLV mutant subgroup A [ASLV(A)] variants with these cysteine residues mutated were constructed and genetically selected for improved replication capacity in chicken fibroblasts. Viruses with single cysteine-to-serine mutations reverted to the wild-type sequence. However, viruses with both C9S and C45S (C9,45S) mutations retained both mutations and acquired a second-site mutation that significantly improved the infectivity of the genetically selected virus population. A charged-amino-acid second-site substitution in the TM internal fusion peptide at position 30 is preferred to rescue the C9,45S mutant ASLV(A). ASLV(A) envelope glycoproteins that contain the C9,45S and G30R mutations bind the Tva receptor at wild-type levels and have improved abilities to trigger conformational changes and to form stable TM oligomers compared to those of the C9,45S mutant glycoprotein.

ABSTRACT 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.

ABSTRACT 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.

ABSTRACTThe subgroup A to E avian sarcoma and leukosis viruses (ASLVs) are highly related and are thought to have evolved from a common ancestor. These viruses use distinct cell surface proteins as receptors to gain entry into avian cells. Chickens have evolved resistance to infection by the ASLVs. We have identified the mutations responsible for the block to virus entry in chicken lines resistant to infection by subgroup A ASLVs [ASLV(A)]. Thetvagenetic locus determines the susceptibility of chicken cells to ASLV(A) viruses. In quail, the ASLV(A) susceptibility alleletvasencodes two forms of the Tva receptor; these proteins are translated from alternatively spliced mRNAs. The normal cellular function of the Tva receptor is unknown; however, the extracellular domain contains a 40-amino-acid, cysteine-rich region that is homologous to the ligand binding region of the low-density lipoprotein receptor (LDLR) proteins. The chickentvascDNAs had not yet been fully characterized; we cloned the chickentvacDNAs from two lines of subgroup A-susceptible chickens, line H6 and line 0. Two types of chickentvascDNAs were obtained. These cDNAs encode a longer and shorter form of the Tva receptor homologous to the Tva forms in quail. Two different defects were identified in cDNAs cloned from two different ASLV(A)-resistant inbred chickens, line C and line 72. Line Ctvarcontains a single base pair substitution, resulting in a cysteine-to-tryptophan change in the LDLR-like region of Tva. This mutation drastically reduces the binding affinity of TvaRfor the ASLV(A) envelope glycoproteins. Line 72tvar2contains a 4-bp insertion in exon 1 that causes a change in the reading frame, which blocks expression of the Tva receptor.

ABSTRACT 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 study, we demonstrate that evolutionary pressure on subgroup A ALV [ALV(A)] entry exerted by the presence of a competitive inhibitor, a soluble form of the ALV(A) Tva receptor linked to a mouse immunoglobulin G tag (quail sTva-mIgG), can select different populations of escape variants. This escape population contained three abundant ALV(A) variant viruses, all with mutations in the surface glycoprotein hypervariable regions: a previously identified variant containing the Y142N mutation in the hr1 region; a new variant with two mutations, W141G in hr1 and K261E in vr3; and another new variant with two mutations, W145R in hr1 and K261E. The W141G K261E and W145R K261E viruses escape primarily by lowering their binding affinities for the quail Tva receptor competitive inhibitor while retaining wild-type levels of binding affinity for the chicken Tva receptor. A secondary phenotype of the new variants was an alteration in receptor interference patterns from that of wild-type ALV(A), indicating that the mutant glycoproteins are possibly interacting with other cellular proteins. One result of these altered interactions was that the variants caused a transient period of cytotoxicity. We could also directly demonstrate that the W141G K261E variant glycoproteins bound significant levels of a soluble form of the Tvb S3 ALV receptor in a binding assay. Alterations in the normally extreme specificity of the ALV(A) glycoproteins for Tva may represent an evolutionary first step toward expanding viral receptor usage in response to inefficient viral entry.

ABSTRACT To better understand retroviral entry, we have characterized the interactions between subgroup A avian leukosis virus [ALV(A)] envelope glycoproteins and Tva, the receptor for ALV(A), that result in receptor interference. We have recently shown that soluble forms of the chicken and quail Tva receptor (sTva), expressed from genes delivered by retroviral vectors, block ALV(A) infection of cultured chicken cells (∼200-fold antiviral effect) and chickens (>98% of the birds were not infected). We hypothesized that inhibition of viral replication by sTva would select virus variants with mutations in the surface glycoprotein (SU) that altered the binding affinity of the subgroup A SU for the sTva protein and/or altered the normal receptor usage of the virus. Virus propagation in the presence of quail sTva-mIgG, the quail Tva extracellular region fused to the constant region of the mouse immunoglobulin G (IgG) protein, identified viruses with three mutations in the subgroup A hr1 region of SU, E149K, Y142N, and Y142N/E149K. These mutations reduced the binding affinity of the subgroup A envelope glycoproteins for quail sTva-mIgG (32-, 324-, and 4,739-fold, respectively) but did not alter their binding affinity for chicken sTva-mIgG. The ALV(A) mutants efficiently infected cells expressing the chicken Tva receptor but were 2-fold (E149K), 10-fold (Y142N), and 600-fold (Y142N/E149K) less efficient at infecting cells expressing the quail Tva receptor. These mutations identify key determinants of the interaction between the ALV(A) glycoproteins and the Tva receptor. We also conclude from these results that, at least for the wild-type and variant ALV(A)s tested, the receptor binding affinity was directly related to infection efficiency.