Dysfunction of Bovine Endogenous Retrovirus K2 Envelope Glycoprotein Is Related to Unsuccessful Intracellular Trafficking

Abstract

Endogenous retroviruses (ERVs) are the remnants of retroviral infection of ancestral germ cells. Mutations introduced intoERVs halt the production of infectious agents, but their effects on the function of retroviral proteins are not fully understood. Retroviral envelope glycoproteins (Envs) are utilized in membrane fusion during viral entry, and we recently identified intact coding sequences for bovine endogenous retrovirus K1 (BERV-K1) and BERV-K2 Envs. Amino acid sequences of BERV-K1 Env (also called Fematrin-1) and BERV-K2 Env are similar, and both viruses are classified in the genus Betaretrovirus. While Fematrin-1 plays an important role in cell-to-cell fusion in bovine placenta, the BERV-K2 envelope gene is marginally expressed in vivo, and its recombinant Env protein is defective in membrane fusion due to inefficient cleavage of surface (SU) and transmembrane subunits. Here, we conducted chimeric analyses of Fematrin-1 and BERV-K2 Envs and revealed that defective maturation of BERV-K2 Env contributed to failed intracellular trafficking. Fluorescence microscopy and flow cytometric analysis suggested that in contrast to Fematrin-1 Env, BERV-K2 Env could not be transported from the endoplasmic reticulum to the trans-Golgi network, where cellular proteases required for processing retroviral Envs are localized. We also identified that one of the responsive regions of this phenomenon resided within a 65-amino-acid region of BERV-K2 SU. This is the first report to identify that retroviral Env SU is involved in the regulation of intracellular trafficking, and it may help to elucidate the maturation process of Fematrin-1 and other related Envs. IMPORTANCERetroviruses utilize envelope glycoproteins (Envs) to enter host target cells. Mature retroviral Env is a heterodimer, which consists of surface (SU) and transmembrane (TM) subunits that are generated by the cleavage of an Env precursor protein in the trans-Golgi network. SU and TM mediate the recognition of the entry receptor and virus-host membrane fusion, respectively. However, unexplained issues remain for the maturation process of retroviral Env. We previously reported that bovine endogenous retrovirus K2 (BERV-K2) Env lost fusogenicity due to a defect in the cleavage of SU and TM. In this study, we identified that mutations residing in BERV-K2 SU disturbed intracellular trafficking of BERV-K2 Env and resulted its inefficient cleavage. Because SU is not known to play an important role in this process, our study may provide novel insights into the maturation mechanism of retroviral Envs.

Ancestral Mutations Acquired in Refrex-1, a Restriction Factor against Feline Retroviruses, during its Cooption and Domestication

“…Similarly, ERV-DC16 env also has a premature and a latent stop codon and the substitution Y431D. It is interesting that two similar molecules of Refrex-1 are present and that they occurred in parallel through the acquisition of similar mu- BERV-K2 Env and Fr-MLV Env mutants with mutations within glycosylation site 4 (gs4) are not cleaved because their mutation prevents intracellular trafficking from the rough endoplasmic reticulum (RER) to the trans-Golgi network (TGN), where Env cleavage occurs (11,63). Uncleaved Mo-MLV Env mutants with mutations within the cleavage motif (RXRR) are trapped in the TGN without transport to the cell surface (62).…”

“…Several uncleaved Env mutants have been previously reported, and some of these were defective in membrane trafficking toward the cell surface (11,62,63). To determine if ERV-DC7 fl and ERV-DC16 fl shared this defect, we used FACS analysis to test if ERV-DC7 fl and ERV-DC16 fl were transported normally to the cell surface.…”

“…They are gradually attenuated or inactivated by preinsertional mutations that occur during viral replications and by postinsertional mutations that occur during host genome replication. Nucleotide changes in the long terminal repeats (LTR) diminish the transcription activity of the virus, and amino acid changes in the viral genes disrupt or modify the functions of their coding proteins (7)(8)(9)(10)(11)(12).…”

Baton pass hypothesis: successive incorporation of unconserved endogenous retroviral genes for placentation during mammalian evolution

“…In contrast, gag , pol , and env ORFs are retained in BERV‐K2 (Baba et al . ; Nakaya & Miyazawa ). The expression of BERV‐K1, both mRNA and protein, is much greater than that of BERV‐K2 in bovine trophoblasts.…”

The Roles of Syncytin-Like Proteins in Ruminant Placentation

“…We also conducted fusion assays using BERV-K2 Env; however, BERV-K2 Env was not able to induce cell-to-cell fusion with endometrial cells [ 11 ]. This phenomenon was attributed to a failure of Env glycoprotein maturation [ 67 ].…”