Variable Sensitivity to Substitutions in the N-Terminal Heptad Repeat of Mason-Pfizer Monkey Virus Transmembrane Protein

Abstract

The transmembrane protein of Mason-Pfizer monkey virus contains two heptad repeats that are predicted to form amphipathic ␣-helices that mediate the conformational change necessary for membrane fusion. To analyze the relative sensitivity of the predicted hydrophobic face of the N-terminal heptad repeat to the insertion of uncharged, polar, and charged substitutions, mutations that introduced alanine, serine, or glutamic acid into positions 436, 443, 450, and 457 of the envelope protein were examined. Novel systems using Tat protein and the GHOST cell line were developed to test and quantitate the effects of the mutations on Env-mediated fusion and infectivity of the virus. While no single amino acid change at any of the positions interfered significantly with the synthesis, processing, or transport to the plasma membrane of glycoprotein complexes, 9 of the 12 nonconservative mutations in these residues completely abolished fusion activity and virus infectivity. Mutations in the central positions (443 and 450) of the heptad repeat region were the most detrimental to Env function, and even single alanine substitutions in these positions dramatically altered the fusogenicity of the protein. These results demonstrate that this N-terminal heptad repeat plays a critical role in Env-mediated membrane fusion and highlight the key function of central hydrophobic residues in this process and the sensitivity of all positions to charge substitutions. Mason-Pfizer monkey virus (M-PMV) was the firstBetaretrovirus to be isolated from a nonhuman primate and was recovered from a spontaneous breast carcinoma of a rhesus monkey (13,29). Subsequent studies revealed that this virus, although isolated from a mammary adenocarcinoma, was not oncogenic (19); instead, infected macaques suffered a severe immunodeficiency syndrome with pathology distinct from those of lentiviruses, such as simian immunodeficiency virus and human immunodeficiency virus (HIV) (14). Betaretroviruses are characterized by the assembly of intracytoplasmic capsids, which make their way to the plasma membrane and are released by budding. Most retroviruses, in contrast, assemble their capsids and bud simultaneously from the plasma membrane.Retrovirus glycoproteins are translated as a polyprotein precursor from a spliced env gene-specific mRNA. The glycosylated precursor is assembled into oligomers in the endoplasmic reticulum and then proteolytically cleaved by a host protease into two subunits, the surface (SU) and transmembrane (TM) proteins, in the late Golgi complex (28). These glycoprotein complexes are then incorporated into budding virions at the plasma membrane. The SU glycoprotein is responsible for cellular tropism for the virus, whereas the TM glycoprotein is responsible for anchoring the SU protein in the viral membrane and for mediating virus-cell membrane fusion during viral entry. The glycoprotein precursor of M-PMV, Pr86, is cleaved to yield the mature gp70 (SU) and gp22 (TM) proteins, and then, following virus release, a viral protease-mediated mat...

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