Novel Monoclonal Antibody Directed at the Receptor Binding Site on the Avian Sarcoma and Leukosis Virus Env Complex

Abstract

We report here on the generation of a mouse monoclonal antibody directed against Rous sarcoma virus (RSV) subgroup A Env that will be useful in functional and structural analysis of RSV Env, as well as in approaches employing the RCAS/Tva system for gene targeting. BALB/c mice were primed and given boosters twice with EnvA-expressing NIH 3T3 cells. Resulting hybridomas were tested by enzyme-linked immunosorbent assay against RCANBP virions and SU-A-immunoglobulin G immunoadhesin. One highly reactive hybridoma clone, mc8C5, was subcloned and tested in immunofluorescence, immunoprecipitation (IP), and Western blotting assays. In all three assays, mc8C5-4 subgroup-specifically recognizes SR-A Env, through the SU domain, expressed from different vectors in both avian and mammalian cells. This multifunctionality is notable for a mouse monoclonal. We furthermore observed a preference for binding to terminally glycosylated Env over core-glycosylated Env precursor in IPs, suggesting that the epitope is at least partially conformational and dependent on glycosylation. Most importantly, we found mc8C5-4 inhibited Env function: in vitro, the monoclonal not only interferes with binding of the EnvA receptor, Tva, but it also blocks the Tva-induced conformational change required for activation of the fusion peptide, without inducing that change itself. Infection of Tva-expressing avian or mammalian cells by avian sarcoma and leukosis virus (ASLV) or EnvA-pseudotyped murine leukemia virus, respectively, is efficiently inhibited by mc8C5-4. The apparent interference of the monoclonal with the EnvA-Tva complex formation suggests that the epitope seen by mc8C5 overlaps with the receptor binding site. This is supported by the observation that mutations of basic residues in hr2 or of the downstream glycosylation site, which both impair Tva-binding to EnvA, have similar effects on the binding of mc8C5. Thus, anti-ASLV-SU-A mc8C5-4 proves to be a unique new immunoreagent that targets the receptorbinding site on a prototypical retroviral envelope.Not only are the avian retroviruses (alpharetroviruses, or avian sarcoma and leukosis viruses [ASLV]) relevant pathogens in poultry with economic impact, but they also have long served as an important model system for studying the biological functions of retroviruses and other enveloped viruses. Attachment to and infection of target cells is mediated by the ASLV envelope (Env) glycoproteins. Env is synthesized as a precursor molecule (Pr95) that is proteolytically processed in the Golgi into two disulfide-linked subunits, SU/gp85 (surface) and TM/gp37 (transmembrane) (18). The TM region is divided into an ectodomain involved in membrane fusion, a membrane-spanning domain anchoring the protein, and a cytoplasmic C terminus containing endocytosis and potentially other trafficking signals (39). The ectodomains of both SU and TM are extensively glycosylated. SU contains two hypervariable regions, hr1 and hr2, that are determinants of host range and thus of the interaction of SU with its respectiv...

Replication competent molecular clones of HIV-1 expressing Renilla luciferase facilitate the analysis of antibody inhibition in PBMC

“…The T2A/ nef Forward primer was designed to be partially complementary to the LucR/T2AReverse primer allowing the two purified PCR products to anneal in a third PCR reaction using primers LucForward and nef Reverse to generate a “fusion” PCR product. The fusion PCR product was then cloned into the shuttle vector pCB6_NL_env/nef, a vector derived from plasmid pCB6-EnvA (Ochsenbauer-Jambor et al, 2002) and modified to contain a fragment of the HIV-1 NL4-3 genome from BamHI (NL4-3 nt 8465) in env to KpnI (NL4-3 nt 9005) in nef , utilizing the restriction sites NheI (a specifically engineered restriction site between the env stop codon and the nef start codon) and XhoI. The env /LucR/T2A/ nef DNA fragment was then subcloned into the parental pNL4-3 genome using native viral sites BamHI (NL4-3 nt 8465) and XhoI (NL4-3 nt 8887).…”

Foreign Glycoproteins Can Be Actively Recruited to Virus Assembly Sites during Pseudotyping

“…The RSV Schmidt-Ruppin A Env expression vector pCB6.SR-A was generously provided by Eric Hunter and Christina Ochsenbauer-Jambor (18). The RSV Env gene was subcloned into the vector pIRES2-DsRedExpress (Clontech) to provide fluorescent expression in transfected cells.…”

Receptor-Induced Thiolate Couples Env Activation to Retrovirus Fusion and Infection

“…Goat antisera against Tween/ether disrupted Rous sarcoma virus Prague C (74S-389) was originally made at the NCI and distributed through Viromed. The anti-SU mAB mc8C5 was obtained from the hybridoma facility at the University of Alabama at Birmingham [ 37 ].…”