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 into ERVs 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.

Importance: Retroviruses 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.

FIG 1

Sequence comparison of Fematrin-1 and BERV-K2 Env. Amino acid sequences of Fematrin-1 and BERV-K2 Env are aligned. Identical amino acids and gap sequences are shown as dots and hyphens, respectively. Each Env subunit is indicated as SP, SU, and TM, and putative cleavage motifs are boxed.

FIG 2

Processing of Fematrin-1 and BERV-K2 chimeric mutants. (A to C) Schematic representation of expression constructs which were transfected into Cos-7 cells. All constructs contain FLAG epitope tags and 3′ long germinal repeats (LTR) downstream of the TM C termini. (D to F) Immunoblotting analyses of lysates of Cos-7 cells transfected with chimeric mutants. Mutants are indicated above the gels. The names of antibodies and detected proteins are at right. Molecular masses are displayed at left. (G to I) Fusion assay of chimeric mutants. Cos-7 cells cotransfected with indicated expression vectors for chimeric Envs and the pT7EMCVluc and pRL-TK vectors were cocultured with the other Cos-7 cells transfected with pCMVT7pol at 37°C for 24 h. Then, cocultured cells were subjected to a dual-luciferase reporter assay. Assays were performed in triplicate and repeated as three independent experiments (n = 9). Values are exhibited as means ± SE of relative luciferase activities. Significantly higher (P < 0.05) activities are marked with asterisks. Fematrin-1 is abbreviated as Fema-1. ShPep indicate the short peptides, under 20 kDa in mass.

FIG 3

Processing of Fematrin-1 SU and BERV-K2 SU chimeric mutants. (A) Schematic representation of expression constructs which were transfected into Cos-7 cells. All constructs contain FLAG epitope tags and 3′ LTR downstream of the TM C termini. (B) Immunoblotting analysis of lysates of Cos-7 cells transfected with chimeric mutants. Mutants' names are shown at the top. The names of used antibodies and detected proteins are at right. Molecular masses are displayed at left. “ShPep” indicates the short peptides under 20 kDa. (C) Fusion assay of chimeric mutants. Cos-7 cells cotransfected with indicated expression vectors for chimeric Envs and the pT7EMCVluc and pRL-TK vectors were cocultured with the other Cos-7 cells transfected with pCMVT7pol at 37°C for 24 h. Then, cocultured cells were subjected to a dual-luciferase reporter assay. Assays were performed in triplicate and repeated as three independent experiments (n = 9). Values are shown as means ± SE of relative luciferase activities. Significantly higher (P < 0.05) activities are marked with asterisks (*). Fematrin-1 is abbreviated as Fema-1.

FIG 4

Expression levels of FLAG-tagged Fematrin-1 SU and BERV-K2 SU in culture supernatants and those of fusion proteins of Fematrin-1 SU or BERV-K2 SU on the cellular surface. (A) Cell lysates and culture supernatants (Sup) of Cos-7 and HEK293T cells, which were transfected with each SU expression plasmid, were subjected to immunoblotting analysis using indicated antibodies (α-FLAG or α-Tubulin). (B) Schematic representation of pDisplaySU plasmids. Each BERV-K SU was inserted between HA and Myc tags. pDisplaySUs encode fusion proteins of MIKLS, HA, SU, Myc, and PDGFR TM. (C) Cell lysates of Cos-7 cells transfected with each pDisplaySU plasmid were subjected to immunoblotting analysis using anti-HA antibody (6E2). Molecular masses are indicated at left. (D) Flow cytometric analysis was conducted to measure the expression levels of the fusion proteins on surface of Cos-7 cells, which were transfected with each pDisplaySU plasmid. Anti-HA (6E2) and anti-mouse IgG Alexa Fluor 488 were used as the primary and the secondary antibodies, respectively. Values are represented as means ± SE for three independent experiments (n = 3). Significant differences were considered to be P values < 0.05 and are indicated with asterisks (*). Fematrin-1 is abbreviated as Fema-1.

FIG 5

Intracellular sublocalization of FLAG-tagged Fematrin-1 and BERV-K2 Env in Cos-7 cells. (A and C) Cos-7 cells were cotransfected with pER-mAG1 (A) or pEYFP-Golgi (C) and each Env expression plasmid, which was C-terminally tagged with FLAG, and were subjected to IFA 48 h posttransfection. Anti-FLAG M2 and anti-mouse IgG Alexa Fluor 555 were used as the primary and secondary antibodies, respectively. Enlarged images are shown in small windows. Signals of Envs and ER-mAG1/EYFP-Golgi are highlighted with red and green colors, respectively, and merged signals are shown in yellow. Nuclei were stained with DAPI (blue). (B and D) The bar graphs indicate the relative numbers of merged spots of ER-mAG1 and Envs (B) or EYFP-Golgi and Envs (D). Fluorescent spots were quantified using ImageJ software and colocalization finder. Values are represented as means ± SE for each 10 microscopic fields. Significant differences were considered to be P values < 0.05 and are indicated with asterisks (*).

FIG 6

Determination of the region responsible for functional defects in BERV-K2 Env. (A and D) Schematic representation of expression constructs which were transfected into Cos-7 cells. All constructs contain FLAG epitope tags and 3′ LTR downstream of the TM C termini. Plasmid names indicate ranges of amino acid sequences which originated with BERV-K2 Env. For example, “1-491” means that amino acids from 1 to 491 of BERV-K2 Env were recombined into the corresponding region of Fematrin-1. (B and E) Immunoblotting analysis of lysates of Cos-7 cells transfected with chimeric mutants. Mutants' names are shown at the top. The names of used antibodies and detected proteins are indicated at right. Molecular masses are displayed at left. (C and F) Fusion assay of chimeric mutants. Cos-7 cells cotransfected with indicated expression vectors for chimeric Envs and pT7EMCVluc and pRL-TK vectors were cocultured with other Cos-7 cells transfected with pCMVT7pol at 37°C for 24 h. Then, cocultured cells were subjected to dual-luciferase reporter assay. Assays were performed in triplicate and repeated as three independent experiments (n = 9). Values are shown as means ± SE of relative luciferase activities. Significantly higher (P < 0.05) activities are marked with asterisks (*). Fematrin-1 is abbreviated as Fema-1. “ShPep” indicates the short peptides, under 20 kDa in mass.