Mutations in Both the Surface and Transmembrane Envelope Glycoproteins of the RAV-2 Subgroup B Avian Sarcoma and Leukosis Virus Are Required to Escape the Antiviral Effect of a Secreted Form of the TvbS3 Receptor †

Abstract

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.

Reverse Engineering Provides Insights on the Evolution of Subgroups A to E Avian Sarcoma and Leukosis Virus Receptor Specificity

“…The RCAS vectors can be used to chronically infect cultures of avian cells, and the same subgroup vector containing a reporter gene (e.g., AP and GFP) can be used to challenge the cells and quantitate the infectious titer. Two examples of this type of assay are shown in Figure 6A,B [63,64]. The receptor interference assay demonstrates the remarkable receptor specificity for using just one receptor attained by the wild type ASLV envelope glycoproteins with little or no cross-interference.…”

“…A DF-1 cell line was generated that expresses high levels of the sTvb S3 -mIgG immunoadhesin that binds to the subgroup B ASLV glycoproteins blocking access to cellular Tvb receptors and infection. The DF-1/sTvb S3 -mIgG cell line was challenged with RCASBP(B) or RCASBP(RAV2) that contained the entire wild type RAV-2 env gene [64]. A variant resistant to the antiviral effects of the sTvb S3 -mIgG immunoadhesin was only selected from the RCASBP(B) challenged culture; RCASBP(RAV2) failed to evolve a resistant variant even after multiple attempts.…”

“…The RCASBP(RAV2) envelope glycoprotein consisting of the entire RAV-2 Subgroup B envelope glycoprotein could not evade the antiviral effects of the Tvb S3 immunoadhesin. We constructed the same Del136-142 deletion into the RCASBP(RAV2) env gene to test whether this mutation would rescue the ability of RAV-2 to replicate in the presence of sTvb S3 -mIgG [64]. Unexpectantly, not only did the Del136-142 deletion not rescue RCASBP(RAV2)/Del136-142 replication on the sTvb S3 -mIgG selective cells until additional mutations were acquired, but the production of detectable RCASBP(RAV2)/Del136-142 virus on DF-1 cells was significantly delayed and also needed the acquisition of addition mutations.…”

“…Recombination between RCASBP(A) and RAV-2 envelope glycoproteins provided an advantage for replication in the presence of the Tvb S3 immunoadhesin. RCASBP(A) was used to deliver the sTvb S3 -mIgG immunoadhesin gene in chicken embryos to express the immunoadhesin in virtually all cells and tissues of the hatched chicks [64]. The chicks expressing sTvb S3 -mIgG were significantly and specifically resistant to a challenge with subgroup B wild type RAV-2 virus but not subgroup C RAV-49 virus: 15 of 17 birds (88%) challenged with RAV-2 did not produce detectable subgroup B ASLV; 100% of birds challenged with RAV-49 produced subgroup C ASLV.…”

“…Indeed, there are only eight amino acid differences between the RCASBP(B) and RCASBP(RAV2) viruses to account for the inability of RCASBP(RAV2) to escape sTvb S3 -mIgG inhibition: five differences in the C-terminal region of SU (2 nonconserved changes) and three differences in TM (2 nonconserved changes). The analysis of the biochemical properties of the RCASBP(B) and RCASBP(RAV2) envelope glycoprotein trimers demonstrate fundamental differences between the two glycoproteins in their ability to execute virus entry with the two-step mechanism of ASLV fusion process [64]. The mature RCASBP(B) glycoproteins appear to be more stable compared to the RCASBP(RAV2) glycoproteins upon conformation change triggered by heat with significant levels of triggered RCASBP(RAV2) glycoproteins occurring at low temperatures.…”