The hr1 and Fusion Peptide Regions of the Subgroup B Avian Sarcoma and Leukosis Virus Envelope Glycoprotein Influence Low pH-Dependent Membrane Fusion

The avian sarcoma and leukosis virus (ASLV) envelope glycoprotein (Env) is activated to trigger fusion by a two-step mechanism involving receptor-priming and low pH fusion activation. In order to identify regions of ASLV Env that can regulate this process, a genetic selection method was used to identify subgroup B (ASLV-B) virus-infected cells resistant to low pH-triggered fusion when incubated with cells expressing the cognate TVB receptor. The subgroup B viral Env (envB) genes were then isolated from these cells and characterized by DNA sequencing. This led to identification of two frequent EnvB alterations which allowed TVB receptor-binding but altered the pH-threshold of membrane fusion activation: a 13 amino acid deletion in the host range 1 (hr1) region of the surface (SU) EnvB subunit, and the A32V amino acid change within the fusion peptide of the transmembrane (TM) EnvB subunit. These data indicate that these two regions of EnvB can influence the pH threshold of fusion activation

Common <i>envB</i> mutations in the R6 (pH 5.6) cell population.

<p>(A) Schematic of the EnvB protein showing host range regions hr1and hr2 of SU and the fusion peptide (FP) and membrane spanning domain (MSD) of TM. The amino acid sequences of wild-type (WT) Env B and of three common mutations found in the R6 (pH 5.6) cell population are shown below. The frequencies of each mutation in the R6 (pH 5.6) population are indicated in parentheses, measured as described in the text (n = number of cloned copies of the <i>envB</i> genes that were characterized). (B) Representative screen for the A32V mutation. Individual plasmid DNAs containing <i>envB</i> genes were screened for the A32V mutation by digestion with PvuII as described in the text. Plasmids containing wild-type <i>envB</i> gave rise to a linear 6 kb DNA fragment (lanes 1, 3, 5, and 6) whereas those containing A32V <i>envB</i> remain undigested (lanes 2 and 4). Lanes 1 and 2: wild-type and A32V <i>envB</i> controls. Lanes 4–6: Individual <i>envB</i> genes cloned from the R6 (pH 5.6) population. (C) Representative PCR-amplification based screen for the <i>Δ</i>152–164 mutation as described in the text. Plasmid DNA containing wild-type <i>envB</i> give rise to a 209 bp DNA fragment (lanes 1 and 4–8), while those containing the <i>Δ</i>152–164 mutant give rise to a 170 bp DNA fragment (lanes 2 and 3). Lane 1; wild-type <i>envB</i> control, Lanes 2–7: Individual <i>envB</i> genes cloned from the R6 (pH 5.6) population.</p

Selection of ASLV-B infected DF-1 cells that do not undergo low pH-mediated cell-cell fusion.

<p>(A) Selection scheme used to identify subgroup B ASLV-infected cells that are resistant to low pH-mediated syncytia formation. (B) The numbers of hygromycin B-resistant virus-infected colonies that resulted from cell-cell fusion experiments preformed with 293:TVB^S3<i>Δ</i>DD cells and either starting population of RCASH-B infected cells or R6 (pH 5.6) cells, are shown. This experiment was performed in triplicate and the average mean values obtained are shown along with the standard deviation of the data. (C) Flow cytometric analysis of EnvB expression. Uninfected cells (green histogram), the starting population of RCASH-B infected DF-1 cells (red histogram, upper panel) and the R6 (pH 5.6) cells (blue histogram, lower panel) were incubated with TVB^S3-rIgG and a FITC-conjugated anti-rabbit antibody and analyzed by flow cytometry as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000171#pone.0000171-Adkins2" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000171#pone.0000171-Melikyan1" target="_blank">[27]</a>.</p

The mutant EnvB proteins exhibit altered low pH thresholds for fusion activation.

<p>(A) DF-1 cells (blue histogram) and DF-1 cells expressing wild-type (WT), and either of the mutant forms of EnvB (red histograms) were analyzed by flow cytometry as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000171#pone-0000171-g001" target="_blank">Fig. 1</a> legend. (B)The virus-infected DF-1 cells (GFP-positive green) were mixed with 293:TVB^S3<i>Δ</i>DD cells labeled by expression of eGFP. The 293:TVB^S3<i>Δ</i>DD cells were labeled with R18 red. Images were taken using an Axiovert25 microscope at 50× magnification. Shown are representative panels from the whole cell cultures.</p