Production and characterization of a soluble, active form of Tva, the subgroup A avian sarcoma and leukosis virus receptor. (1/332)

The receptor for the subgroup A avian sarcoma and leukosis viruses [ASLV(A)] is the cellular glycoprotein Tva. A soluble form of Tva, sTva, was produced and purified with a baculovirus expression system. Using this system, 7 to 10 mg of purified sTva per liter of cultured Sf9 cells was obtained. Characterization of the carbohydrate modification of sTva revealed that the three N glycosylation sites in sTva were differentially utilized; however, the O glycosylation common to Tva produced in mammalian and avian cells was not observed. Purified sTva demonstrates significant biological activity, specifically blocking infection of avian cells by ASLV(A) with a 90% inhibitory concentration of approximately 25 pM. A quantitative enzyme-linked immunosorbent assay, developed to assess the binding of sTva to ASLV envelope glycoprotein, demonstrates that sTva has a high affinity for EnvA, with an apparent dissociation constant of approximately 0.3 nM. Once they are bound, a very stable complex is formed between EnvA and sTva, with an estimated complex half-life of 6 h. The soluble receptor protein described here represents a valuable tool for analysis of the receptor-envelope glycoprotein interaction and for structural analysis of Tva.  (+info)

Substitutions in the receptor-binding domain of the avian sarcoma and leukosis virus envelope uncouple receptor-triggered structural rearrangements in the surface and transmembrane subunits. (2/332)

The retrovirus avian sarcoma and leukosis virus (ASLV) enters cells via pH-independent membrane fusion. This reaction is catalyzed by the viral glycoprotein Env, composed of a membrane-distal subunit, SU, and a membrane-anchored subunit, TM. Previous mutational analysis of a variable region, central within the SU subunit, indicates that this region constitutes part of the receptor-binding domain for subgroup A envelope (EnvA) and furthermore that basic residues (R210, R213, R223, R224, and K227) within this region are critical determinants of efficient ASLV infection. Substitutions of these basic residues exert effects on both receptor binding and postbinding events in EnvA-mediated entry. In this study, we performed biochemical analysis of the EnvA protein from three of the receptor-binding domain mutants (R213A/K227A, R213A/R223A/R224A, and R213S) to define the role of this domain in early molecular events in the entry pathway. Protease sensitivity assays demonstrated that receptor binding was sufficient to trigger conformational changes in the SU subunit of mutants R213A/K227A and R213S similar to those in the wild-type EnvA, while R213A/R223A/R224A was constitutively sensitive to protease. In contrast, all three receptor-binding domain mutants disrupted receptor-triggered conversion of EnvA to an active, membrane-binding conformation as assessed by liposome flotation assays. Our results demonstrate that mutations in the receptor-binding site can dissociate receptor-triggered conformational changes in the SU subunit from membrane binding. Furthermore, they suggest that communication between the receptor-binding subunit, SU, and the fusogenic subunit, TM, is crucial for efficient activation of the fusogenic state of EnvA. Analysis of these mutants continues earlier observations that binding to the cellular receptor provides the trigger for efficient activation of this pH-independent viral envelope protein.  (+info)

Development of a flexible and specific gene delivery system for production of murine tumor models. (3/332)

To develop models of human cancer we have expressed the avian retroviral receptor, TVA, under a variety of mammalian promoters in transgenic mice, thus rendering mice susceptible to infection with avian leukosis virus-derived gene vectors. TVA-based retroviral gene transfer offers advantages over current murine models of human cancer. A single transgenic mouse line can be used to evaluate multiple genetic lesions, individually and in combination. Furthermore, mutant genes are introduced somatically into animals, as occurs in the majority of naturally occurring tumors. Because the avian viral vectors replicate only in avian cells, the viral receptor in infected transgenic mouse cells remains available for multiple rounds of infection with different ASLV vectors. We discuss the theoretical and practical aspects of using recombinant avian retroviruses with TVA transgenic mice to generate cancer models.  (+info)

An Mpsi-containing heterologous RNA, but not env mRNA, is efficiently packaged into avian retroviral particles. (4/332)

Retroviruses preferentially package full-length genomic RNA over spliced viral messages. For most retroviruses, this preference is likely due to the absence of all or part of the packaging signal on subgenomic RNAs. In avian leukosis-sarcoma virus, however, we have shown that the minimal packaging signal, MPsi, is located upstream of the 5' splice site and therefore is present on both genomic and spliced RNAs. We now show that an MPsi-containing heterologous RNA is packaged only 2.6-fold less efficiently than genomic Rous sarcoma virus RNA. Thus, few additional packaging sequences and/or structures exist outside of MPsi. In contrast, we found that env mRNA is not efficiently packaged. These results indicate that either MPsi is not functional on this RNA or the RNA is somehow segregated from the packaging machinery. Finally, deletion of sequences from the 3' end of MPsi was found to reduce the packaging efficiency of heterologous RNAs.  (+info)

Secondary structure analysis of a minimal avian leukosis-sarcoma virus packaging signal. (5/332)

We previously identified a 160-nucleotide packaging signal, MPsi, from the 5' end of the Rous sarcoma virus genome. In this study, we determine the secondary structure of MPsi by using phylogenetic analysis with computer modeling and heterologous packaging assays of point mutants. The results of the in vivo studies are in good agreement with the computer model. Additionally, the packaging studies indicate several structures which are important for efficient packaging, including a single-stranded bulge containing the initiation codon for the short open reading frame, uORF3, as well as adjacent stem structures. Finally, we show that the L3 stem-loop at the 3' end of MPsi is dispensable for packaging, thus identifying an 82-nucleotide minimal packaging signal, microPsi, composed of the O3 stem-loop.  (+info)

The central proline of an internal viral fusion peptide serves two important roles. (6/332)

The fusion peptide of the avian sarcoma/leukosis virus (ASLV) envelope protein (Env) is internal, near the N terminus of its transmembrane (TM) subunit. As for most internal viral fusion peptides, there is a proline near the center of this sequence. Robson-Garnier structure predictions of the ASLV fusion peptide and immediate surrounding sequences indicate a region of order (beta-sheet), a tight reverse turn containing the proline, and a second region of order (alpha-helix). Similar motifs (order, turn or loop, order) are predicted for other internal fusion peptides. In this study, we made and analyzed 12 Env proteins with substitutions for the central proline of the fusion peptide. Env proteins were expressed in 293T cells and in murine leukemia virus pseudotyped virions. We found the following. (i) All mutant Envs form trimers, but when the bulky hydrophobic residues phenylalanine or leucine are substituted for proline, trimerization is weakened. (ii) Surprisingly, the proline is required for maximal processing of the Env precursor into its surface and TM subunits; the amount of processing correlates linearly with the propensity of the substituted residue to be found in a reverse turn. (iii) Nonetheless, proteolytically processed forms of all Envs are preferentially incorporated into pseudotyped virions. (iv) All Envs bind receptor with affinity greater than or equal to wild-type affinity. (v) Residues that support high infectivity cluster with proline at intermediate hydrophobicity. Infectivity is not supported by mutant Envs in which charged residues are substituted for proline, nor is it supported by the trimerization-defective phenylalanine and leucine mutants. Our findings suggest that the central proline in the ASLV fusion peptide is important for the formation of the native (metastable) Env structure as well as for membrane interactions that lead to fusion.  (+info)

Soluble receptor-induced retroviral infection of receptor-deficient cells. (7/332)

Current models of retroviral entry hypothesize that interactions between the host cell receptor(s) and viral envelope protein induce structural changes in the envelope protein that convert it to an active conformation, allowing it to mediate fusion with the membrane. Recent evidence supporting this hypothesis is the demonstration that Tva, the receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A), induces conformational changes in the viral envelope protein. These changes include conversion of the envelope protein to an active, membrane-binding state likely representing a fusogenic conformation. To determine whether binding of the soluble Tva (sTva) receptor was sufficient to activate fully the fusogenic potential of the ASLV-A envelope protein, we have evaluated the ability of ASLV-A to infect receptor-deficient cell lines in the presence of sTva. Soluble receptor efficiently mediated infection of cells devoid of endogenous Tva in a dose-dependent manner, and this infection was dependent absolutely on the addition of sTva. The infectivity of the virus was enhanced dramatically in the presence of the polycationic polymer Polybrene or when centrifugal forces were applied during inoculation, resulting in viral titers comparable to those achieved on cells expressing endogenous receptor. sTva functioned to mediate infection at low concentrations, approaching the estimated binding constant of the receptor and viral envelope protein. These results demonstrate that receptor binding can activate the ASLV-A envelope protein and convert it to a fusogenic conformation competent to mediate the fusion of the viral and cellular membranes.  (+info)

Selection of a subgroup A avian leukosis virus [ALV(A)] envelope resistant to soluble ALV(A) surface glycoprotein. (8/332)

The host developing resistance to retroviral infection is believed to be a major force in the evolution of multiple receptor usage by retroviruses. The avian leukosis-sarcoma virus (ALV) group of retroviruses provides a powerful system for studying the envelope-receptor interactions involved in retrovirus entry; different members of this group of closely related viruses use distinct cellular receptors. Analysis of the ALV envelope subgroups suggests that the different ALVs evolved from a common ancestor by mutations in the env gene. Cells and animals that express subgroup A ALV envelope glycoproteins are highly resistant to ALV(A) infection due to receptor interference. In this study, we tested whether expression of a soluble form of subgroup A surface glycoprotein (SU) would result in receptor interference and whether this interference would select for resistant viruses with altered receptor usage. Chicken cells expressing the secreted ALV(A) SU immunoadhesin SU(A)-rIgG, which contains the subgroup A SU domain fused to the constant region of a rabbit immunoglobulin (IgG) heavy chain, showed significant receptor interference. A variant virus resistant to SU(A)-rIgG receptor interference was obtained. This virus had a six-amino-acid deletion in the subgroup A hr1 that altered receptor usage. This approach may identify regions of SU that play a critical role in receptor specificity.  (+info)

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