Short Communication: Expression Profiles of Endogenous Retroviral Envelopes in Macaca mulatta (Rhesus Monkey)

Abstract

Endogenous retroviruses (ERVs), which are footprints of ancient germline infections, were inserted into the genome during the early stages of primate evolution. Human endogenous retroviruses (HERVs) occupy approximately 8% of the human genome. Although most ERV genes are defective, with large deletions, stop codons, and frameshifts in their open reading frames (ORFs), some full-length sequences containing long ORFs are expressed in several tissues and cancers. Several envelope glycoproteins that are encoded by env genes have retained some characteristics of their ancestral infectious viruses. These glycoproteins play essential physiological roles in the organs in which they are expressed. Previous studies have demonstrated the expression of ERV env at the mRNA level in cells and tissues rather than at the protein level, which is more difficult to detect. However, it is not known whether Env is functionally conserved in primates. To understand the possible role of Env in primates, we examined the expression of the env genes of four ERVs (ERV-R, -K, -W, and -FRD) at the protein as well as mRNA levels in various tissues of the rhesus monkey. The ERV env gene products were observed at moderate to high levels in each tissue that was examined and showed tissue-specific expression patterns. Our data suggest a biologically important role for retroviral proteins in healthy tissues of the rhesus monkey.

Introduction

Endogenous retroviruses (ERVs) are remnants of ancient germline infections integrated into the genome during the early stages of primate evolution.^1,2 Human endogenous retroviruses (HERVs) account for approximately 8% of the human genome; their numbers range from a single copy to 1,000 copies.^3,4 They are classified into 31 distinct types and are named according to their unique tRNA primer-binding sites (PBS), which are located between the 5′-long terminal repeat (LTR) region and the gag gene.^5,6 Although a majority of the ERV elements contain incomplete and deficient sequences with large deletions, multiple stop codons, and frameshifts in their open reading frames (ORFs), some of them are composed of LTRs and functional long ORFs that encode the gag, pol, and env proteins.^7
–9 ERVs are differentiated from other retrotransposons by the presence of the envelope (env) gene, which codes for viral membrane proteins. The Env proteins aid the entry of viral particles into target cells during the infectious cycle.^10,11

Growing evidence suggests that ERVs have significantly contributed to human evolution, development, and physiology; in addition, they play a role in the pathogenesis of human diseases.^12,13 Several endogenous retroviral envelope genes encode envelope glycoproteins, which have retained some characteristics of the cognate proteins produced by their ancestral infectious viruses, such as the ability to attach to a cell receptor, with crucial physiological consequences for the tissues in which they are expressed.¹⁴ For example, six retroviral env genes containing an ORF are transcribed in the placenta: HERV-K, HERV-R(b), and ERV-T at low levels and HERV-R, HERV-W, and HERV-FRD at high levels.¹⁵ Syncytin-1 and -2, which are glycoproteins encoded from HERV-W and HERV-FRD, respectively, have been detected in the cytotrophoblast and syncytiotrophoblast of the human placenta.^16

–21 Although its fusiogenic activity might correlate with cell fusion in terms of physiology and pathology in various cells and organs other than placenta,^22,23 the roles of the envelopes encoded from ERVs in a variety of organs remain largely unexplored.

To determine the potential functions of ERVs, it is necessary to analyze the expression of HERV mRNAs and proteins. In a previous study, we performed a quantitative analysis of the expression profiles of HERVs. Our results indicated high expression levels of the env gene in testis tumor tissues for HERV-K, in liver and lung tumors for HERV-R, in liver, lung, and testis tumors for HERV-H, and in colon and liver tumor tissues for HERV-P.²⁴ Based on the chromosomal localization of HERV-R (chromosome 7q11.21), determined by radiation hybrid mapping, we also demonstrated that HERV-R env mRNA is expressed in diverse human tissues and cancer cells.⁹ ERV3-1 Env expression at the protein level has been detected in various cancerous human tissues; this implies an association between ERV3-1 and tumor formation.²⁵ However, whether the env gene products are functionally conserved in various primate species is not well understood.

Most of the earlier studies have investigated the functional gene expression of ERVs at the mRNA level by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Northern blot in situ hybridization, rather than examine protein expression in cells and tissues, which is more difficult. In this study, to understand the possible functions of Env in primates, we screened the expression profile of env genes from four ERVs (ERV-R, -K, -W, and -FRD), not only at the mRNA level but also at the protein level, in various tissues of the rhesus monkey by qRT-PCRs and immunoassays. Our results revealed that the products of the ERV env genes are expressed at moderate to high levels in various tissues of the rhesus monkey.

Materials and Methods

Ethics statement

Samples of an adult rhesus macaque were acquired from animals culled for other purposes, as approved by the Animal Ethics Committee of the Kyoto University's Primate Research Institute (no. 2013-002).

Quantitative reverse transcriptase PCR amplification

Total RNA was isolated from 17 different whole-body tissues of the rhesus macaque using TRIzol reagent (Ambion, Austin, Texas), according to the manufacturer's instructions. The total RNA was quantified using a NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE). A PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Kyoto, Japan) was used for the reverse transcriptase reaction and gDNA removal from 1 μg of total RNA.

To design primers that were specific for rhesus macaque ERV env genes, the env sequences of rhesus macaque were obtained from the University of California, Santa Cruz (UCSC) genome browser (http://genome.ucsc.edu/) based on a basic local alignment search tool (BLAST) search against human sequences. Primer pairs were designed using the Primer3 program (http://frodo.wi.mit.edu/primer3/)²⁶ (Table 1). The amplification efficiencies and correlation coefficients (R ²) of the four ERV env genes were generated using the slopes of the standard curves obtained from serial dilutions. qRT-PCR with SYBR Green was performed using a Rotor-Gene Q real-time PCR cycler (Qiagen, Valencia, CA). The reaction mixture (19 μl) consisted of cDNA (1 μl), H₂O (5 μl), QuantiTect SYBR Green PCR mastermix (Qiagen, Valencia, CA; 10 μl), and the forward and reverse primers (1 μl each). qRT-PCR amplification of the four HERV env genes was carried out for 40 cycles of 94°C for 10 s, annealing temperature for 15 s, and 72°C for 15 s. Melting curve analyses were conducted for 30 s at 55–99°C. Each primer pair showed a single, sharp peak, indicating that the primers amplified a single specific PCR product. All samples were amplified in triplicate and analyzed in duplicate according to the ΔΔC _t method.

Table 1.

Primers for Endogenous Retrovirus env Genes of Rhesus Macaque Used for Quantitative Reverse Transcriptase Polymerase Chain Reaction

Gene	Primer sequences (5′-3′)	Annealing temperature (°C)	Amplicon size (bp)
ERV-R env	F: GCTCAACCCAACAGTTCCG	55	142
	R: TAACATGAAGCGACGTGCAG
ERV-K env	F: TGGCTTATGAGCTTGGAACA	56	111
	R: AACCATGTCCCAGTGATGCT
ERV-W env	F: TCTCATTTTTAGTGCCCCCA	56	158
	R: GAGGTTGTGATACCGCCAAT
ERV-FRD env	F: GACATGTTAACGGCAGCACA	56	155
	R: TTTAACCAACCCTGAGTGGC

Western blot analysis

Tissue extracts (20 μg) were prepared using the PRO-PREP Protein Extraction Solution (Intron Biotechnology, Kyunggi, Korea) and separated by electrophoresis on a Novex 4–12% Bis-Tris gel (Invitrogen, Carlsbad, CA). Protein concentrations were determined using a bicinchoninic acid protein assay system (Pierce, Rockford, IL), and equal amounts of each sample were separated by electrophoresis on Novex 4–12% Bis-Tris gels. Equal protein loading was confirmed by Coomassie blue staining of duplicate gels after electrophoresis. The gels were incubated in a blotting buffer containing 1× NuPAGE Bis-Tris transfer buffer (Invitrogen, Carlsbad, CA) and 20% methanol for 30 min at room temperature. The proteins were transferred to a nitrocellulose membrane (Invitrogen, Carlsbad, CA) by electrotransfer.

The membrane was preincubated for 2 h in Tris-buffered saline (TBS) containing 5% skimmed milk and 0.05% Tween 20 (TBS-T). The membrane was incubated overnight at 4°C in TBS-T containing anti-HERV-R Env (rabbit polyclonal anti-HERV-R, 1:500 dilution; Abcam, Cambridge, MA), anti-HERV-K Env (mouse polyclonal anti-HERV-K env, 1:2,000 dilution; Abcam, Cambridge, MA), anti-HERV-W env (rabbit polyclonal anti-HERV-W Env, 1:1,000 dilution; Abcam, Cambridge, MA), and anti-HERV-FRD Env (rabbit polyclonal anti-HERV-FRD Env, 1:2,000 dilution; Abcam, Cambridge, MA). The membranes were washed five times with PBS-T and incubated with a species-appropriate horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech, Piscataway, NJ) for 1 h at room temperature. The membranes were washed five times with TBS-T, and the bound antibody was detected with an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ). The specificity of antibodies in various human normal cell and cancer cells has been validated (Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/aid). In addition, the specificity of the ERV-R Env antibody has previously been published.²⁵

Results and Discussion

To investigate the expression profiles of the ERV env genes at the protein as well as the mRNA levels, qRT-PCR and western blot analyses were performed for various tissues of the rhesus macaque. The results revealed moderate to high levels of ERV env expression in the rhesus monkey (Figs. 1 and 2).

FIG. 1.

Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analyses of the four ERV env in various tissues of the rhesus monkey. The mean±SD (n=3) of (A) ERV-R, (B) ERV-K, (C) ERV-W, and (D) ERV-FRD env mRNA were determined in various tissues (1, testis; 2, rectum; 3, kidney; 4, cecum; 5, adrenal gland; 6, pancreas; 7, colon; 8, ileum; 9, heart; 10, stomach; 11, spleen; 12, esophagus; 13, liver; 14, jejunum; 15, duodenum; 16, lung; 17, gallbladder). The error bars indicate standard deviation. The experiments were repeated three times to ascertain the reproducibility of the results.

FIG. 2.

Expression of ERV (ERV-R, -K, -W, and -FRD) Env proteins in the rhesus monkey. The expression profiles of the Env proteins in various tissues of the rhesus monkey were analyzed by western blotting. The western blot was performed in more than three independent experiments. Each band was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

env mRNA levels of ERV-R, one of the first ERVs to be discovered, varied in each sample (Fig. 1A). The env gene was transcribed at a relatively high level in the testis, adrenal gland, and gallbladder, while low levels were detected in some gastrointestinal systems. Our previous study revealed that the HERV-R env gene is expressed only in the brain, prostate, testis, kidney, placenta, thymus, and uterus of humans.⁹ However, in the rhesus monkey, the env transcripts could be detected in the heart, spleen, and lung. As shown in Fig. 2, the Env expression levels of ERV-R varied in different tissues of the rhesus monkey. The Env protein was highly expressed in the rectum, kidney, adrenal gland, liver, jejunum, duodenum, and gallbladder, moderately expressed in the cecum, pancreas, and heart, and weakly expressed in the testis, colon, ileum, and spleen. This tissue-specific expression pattern suggests a physiologically important role for retroviral proteins in various tissues.

On the other hand, the ERV-K family, which is the youngest and most biologically active family among the ERVs, is known to be associated with the pathogenesis of several diseases, including cancer. Noninfectious ERV-K particles are expressed in various cells, especially in tumors and cancer cells derived from teratocarcinomas and melanomas.^27
–29 However, whether ERV-K functions in healthy tissues is not known. In this study, ERV-K env mRNA levels were found to be significantly high in the testis and gallbladder of the rhesus monkey (Fig. 1B). Conversely, the ERV-K envelope protein was detected at high levels in a number of tissues, including the testis, stomach, esophagus, lung, and gallbladder of the rhesus monkey (Fig. 2). This tissue-specific expression pattern is consistent with the fact that ERVs retain the ability to encode functional retroviral proteins and produce retrovirus-like particles.^30
–32 A comparative analysis of the expression patterns of ERV-K Env across other primates, including humans, is necessary to elucidate the biological roles of ERV-K.

Envelopes encoded from HERV-W and –FRD, which have similarities to exogenous gammaretroviruses, are known to play important roles during placental development, where they mediate cell fusion between placental cytotrophoblast cells.^33

–37 After the isolation of a multiple sclerosis-associated retrovirus (MSRV) from retroviral particles in cell cultures from MS patients, extensive quantitative analyses of HERV-W env gene expression in neuroinflammatory diseases have been carried out.^38
–40 Moreover, the ERV-W envelope has been reported to be involved in the pathogenesis of breast and testicular cancers.^22,41 In this study, we found that the transcription levels of ERV-W env were high in the liver and lungs (Fig. 1C); this did not correlate with the protein expression profile. The ERV-W Env protein, syncytin-1, was found to be expressed in diverse tissues of the rhesus monkey (Fig. 2). Env was more highly expressed than the other ERV families in almost all the tissues of the rhesus monkey (Fig. 2).

ERV-FRD is thought to have been inserted into the primate branch after the prosimian and simian divergence, 40 million years ago.³³ The ERV-FRD Env, syncytin-2, has been identified in all primates, from New World monkeys to humans; this implies that syncytin-2 plays an important role in primate physiology.^33,42 In this study, we detected high transcription levels of ERV-FRD env in the testis, ileum, and lungs (Fig. 1D). ERV-FRD-encoded proteins were highly expressed in the kidneys, moderately expressed in the small and large intestines, and weakly expressed in the stomach and liver (Fig. 2). A previous study that involved the examination of primate FRD envelope sequences revealed positive selection for a physiological function that has not yet been elucidated.⁴² Seen in this light, the expression profiles of syncytin-1 and -2 in the rhesus monkey suggest that ERV-W and -FRD Env might play physiological roles in various organs, including the placenta.

In some rhesus tissues, no evident correlation could be detected between the expression profiles of env mRNA and protein. For instance, the expression of ERV-W env in the lungs was high at the transcriptional level but could not be detected at the protein level (Figs. 1C and 2). In the case of ERV-K, lung tissue showed no transcripts but high levels of envelope proteins were observed (Figs. 1B and 2). Such a weak correlation between gene products might result from several causes, including diverse posttranscriptional processes or differences in the specificity profiling the two targets.

In summary, we examined the expression patterns of the env genes from the four ERVs found in the rhesus monkey at the mRNA and protein levels. As discussed above, in ERV-W and -FRD, the main function of the endogenized retroviral envelope is fusogenic activity to facilitate cell–cell fusion. In addition, envelope proteins function in retrovirus-induced immunosuppression.⁴³ These observations suggest that retroviral envelope proteins might play biologically crucial roles in different organs. A comparative analysis of the expression profiles of ERVs in humans and nonhuman primates might help elucidate the role of ERV elements in primate evolution and physiology.

Footnotes

Acknowledgments

This research was supported by the Cooperative Research Program of the Primate Research Institute, Kyoto University. Jungwoo Eo and Hee-Jae Cha contributed equally to this work.

Author Disclosure Statement

No competing financial interests exist.

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