Transactivation of elements in the human endogenous retrovirus W family by viral infection
© Nellåker et al; licensee BioMed Central Ltd. 2006
Received: 31 March 2006
Accepted: 06 July 2006
Published: 06 July 2006
Aberrant expression of human endogenous retrovirus (HERV) elements in the W family has previously been associated with schizophrenia, multiple sclerosis and preeclampsia. Little is know regarding the basal expression, transcriptional regulation and functional significance of individual HERV-elements. Since viral infections have previously been reported to transactivate retroviral long terminal repeat regions we examined the basal expression of HERV-W elements and following infections by influenza A/WSN/33 and Herpes simplex 1 viruses in human cell-lines.
Relative levels of transcripts encoding HERV-W elements and cellular genes were analyzed by qPCR methods. An analysis of amplicon melting temperatures was used to detect variations in the frequencies of amplicons in discrete ranges of such melting temperatures. These frequency-distributions were taken as proxy markers for the repertoires of transcribed HERV-W elements in the cells.
We report cell-specific expression patterns of HERV-W elements during base-line conditions. Expressed elements include those with intact regulatory long terminal repeat regions (LTRs) as well as elements flanked by truncated LTRs. Subsets of HERV-W elements were transactivated by viral infection in the different cell-lines. Transcriptional activation of these elements, including that encoding syncytin, was dependent on viral replication and was not induced by antiviral responses. Serum deprivation of cells induced similar changes in the expression of HERV-W elements suggesting that the observed phenomena are, in part, an effect of cellular stress.
We found that HERV-W elements, including elements lacking regulatory LTRs, are expressed in cell-specific patterns which can be modulated by environmental influences. This brings into light that mechanisms behind the regulation of expression of HERV-W elements are more complex than previously assumed and suggests biological functions of these transcripts.
Human endogenous retroviruses (HERV) are assumed to be remnants of ancient retroviral infections of our ancestors' germ-line cells. HERV sequences constitute approximately 3–8% of the human genome and can be classified into at least 31 families [1, 2]. Tissue-specific hybridization patterns toarrays of sequences representative of different HERV families was recently reported, indicating a discrete and diversified regulation of their transcriptional activities [3, 4].
The differential detection of pol transcripts related to one of these families, HERV-W , was previously observed in cerebrospinal fluids obtained from patients with multiple sclerosis  and patients experiencing their first manifestations of schizophrenia or schizoaffective disorder  as compared to control individuals. A recent study reported similar hybridization signals to a HERV-W pol sequence in prefrontal cortex samples from postmortem brains from patients with a long standing history of schizophrenia or bipolar disorder and control individuals .
According to Pavlicek et.al.  the human genome contains 654 HERV-W elements, the majority of which are comprised of long terminal repeat regions (LTR) lacking internal sequence. The remaining elements were classified into 2 major categories, a total of 77 retroelements with proviral structure containing intact LTRs and complete or partial internal sequences (gag, pol and env genes). In addition, 149 pseudoelements with internal sequences were found, lacking the regulatory U3 region of the 5'-LTR and the U5 region of the 3'-LTR. Structurally these copies resemble retroviral mRNAs and are thought to originate from LINE-mediated reverse transcription of such mRNAs. The remaining elements were grouped together in a third category based on lack of diagnostic regions due to truncations . Due to the absence of regulatory promoter regions, these latter groups have been suggested to be non-transcribed [9, 10]. However, except for a proviral element in ERVWE1 locus, which contains an intact env gene encoding syncytin , basal transcriptional activities of individual HERV-W elements remain poorly defined. Furthermore, potential regulation of individual HERV-W element expression is even less studied.
Herpes simplex viruses are known to transactivate retroviral regulatory LTR regions of both exogenous and endogenous human retroviruses, reviewed in . With regard to HERV-W elements, induction of protein expression by HSV-1 was recently reported . Although Influenza A virus has in one study been reported to transactivate the HIV-1 LTR , the influence RNA viruses on the transcriptional activities of HERV-W elements has, however, not been studied. Consequently we investigated the transcriptional activities of different HERV-W elements in human cell-lines during baseline conditions and how these are modulated by viral infections.
Virus infection transactivates HERV-W elements
Relative increases in HERV-W element transcription after serum deprivation
Since influenza A viruses induce the expression of a variety of cytokine and pro-apoptotic genes in infected cells, we next investigated the levels of HERV-W transcripts in response to double-stranded RNA or replication-incompetent virus. CCF-STTG1 cultures were therefore exposed to poly(cytidylic-inosinic) acid (poly(I:C)) to activate protein kinase R (PKR) or heat-inactivated influenza A/WSN/33 virus to simulate effects of viral binding and fusion in the absence of viral replication. Poly(I:C)-treatment elicited a 33-fold increase in the levels of transcripts encoding IFN-β indicating adequate stimulation (data not shown). However, no significant alterations in the relative levels of HERV-W transcripts were observed in cells treated with poly(I:C) or heat-inactivated virus as compared to untreated controls (data not shown). In addition to mechanisms mediated by PKR, influenza A virus infections can induce apoptosis in infected cells by complex mechanisms not yet fully understood . We next serum deprived the different cell-lines to induce stresses including cellular events leading to apoptosis [16–18]. CCF-STTG1 cells showed a small but significant increase only in the levels of env transcripts after serum deprivation. Serum deprived U937 cells exhibited significantly elevated levels of both HERV-W gag and env transcripts whereas the expression of these transcripts remained at baseline levels in 293F cells (Figure 2B). Thus, induction of HERV-W expression by influenza A/WSN/33 virus appears to be, at least partially, an effect of events leading to apoptosis during infection. Whether the relatively larger responses observed in infected cells as compared with serum deprived cells stems from additional actions of viral proteins or cellular responses to viral load remains undetermined.
Qualitative analysis of HERV-W env and gagexpression
To identify the specific HERV-W elements expressed, all sequences obtained were mapped to the human genome using Blat searches (Table 2). For those sequences without assignment to a unique genomic position, the numbers of homologous matches found are given in Table 2. For unambiguously mapped elements, 5000 upstream bases were subsequently downloaded and subjected to RepeatMasker analyses for the presence of HERV-W related 5'-LTR regions. According to these analyses, one transcribed element on chromosome 1q42 contains an intact HERV-W 5'-LTR whereas three transcribed elements on chromosomes 3q26, 12p13 and 15q21 all have partial HERV-W 5'-LTRs lacking the U3 region. An additional three transcribed elements on chromosomes 5p13, 12p12 and Xq22, however, lack discernible HERV-W 5'-LTRs.
Transactivation of specific HERV-W elements by influenza A virus
EZ4U assaying for syncytin mediated cytotoxicity
The transcripts encoding syncytin, found to be transactivated in all cell types studied are normally only found in a cell population in the placenta. To resolve possible functional consequences of ectopic expression of this element CCF-STTG1 cells were transfected with an expression plasmid containing the full-length env gene from 7q21 encoding syncytin. These cells were subsequently assayed for mitochondrial function through the reduction of tetrazolium salts. 24 hours after transfection, 25% less tetrazolium salts were reduced in syncytin-transfected cells as compared to mock-transfected cells (Unpaired t test with Welch's correction P < 0.0001, data not shown). However, as compared to cells over-expressing enhanced GPF, syncytin expression caused a 12% decrease in cell proliferation/viability (Unpaired t test with Welch's correction P = 0.0150).
Through melting-temperature differences of amplicons generated using SYBR-Green chemistry followed by cloning and sequencing, we here report the constitutive expression of several different HERV-W elements in human cell-lines. Transcripts from genomic elements with complete, partial as well as absent 5'-LTRs were detected. Furthermore, we report that viral infections in vitro elevate the transcript levels from select HERV-W elements, including elements lacking HERV-W 5'-LTR regulatory regions.
Transcripts from elements in the HERV-W family have previously been detected by RT-PCR in most human organs as well as in different cell-lines of human origin . Transcripts from HERV-W pol genes were recently reported to be present at high levels in the placenta, whole brain, adrenal glands and testis . The relative contribution of the different HERV-W elements to the total levels of pol transcripts in different organs was not examined. The differences in the levels of transcripts from HERV-W gag, pol and env genes observed in different tissues and cell-lines might be attributed to the documented variations in promoter activities of U3-regions of HERV-W LTRs [22, 23]. In addition, enhancer elements outside of the LTR can influence the transcriptional activities of HERV-W LTR promoters. This has been documented for the ERVWE1 locus on chromosome 7q21 which is regulated by promoter activity in the U3 region of the 5'-LTR as well as an upstream regulatory region . Based on our present data there is constitutive, albeit low, expression of various HERV-W elements in human cell-lines. Moreover, the base-line relative transcript levels from different elements appeared to differ between these cell-lines. Sequencing of amplified products and mapping to genomic regions followed by RepeatMasker analysis indicated the presence of transcripts from HERV-W elements previously assumed to be transcriptionally silent due to truncations of the U3-region or complete lack of identifiable 5'-LTR [9, 10]. The presence of such transcripts was subsequently verified by element-specific assays. We suggest that unidentified promoters, direct the expression of such HERV-W elements as has been described for cellular genes  in other studies. An analysis of eight different HERV-W gag transcripts previously identified in plasma samples from recent onset schizophrenia patients  revealed transcripts from one proviral element, six pseudoelements and one element lacking identifiable HERV-W 5'-LTR. Thus, elements lacking LTR regulatory regions appear to be transcribed in vivo and not only in cell-lines. In the present study, many of the transcribed HERV-W elements which could be mapped to single genomic sites were located in intronic regions of host genes. This is noteworthy as this is the case for only a minority of HERV elements in general . These intronic HERV-W elements were all oriented opposite to the direction of the host gene transcription. Our findings illustrate the importance of detailed characterization of disease-associated transcripts in order to approach the mechanisms underlying their aberrant expression.
We here report that influenza A/WSN/33 can induce an elevation in the levels, to various degrees depending on host cell type, of transcripts related to HERV-W gag and env. The transactivating capacity of infectious agents on retroviral LTRs has previously been documented, e.g. HSV-1 has been reported to transactivate HIV-1 . In vitro, the HIV-1 LTR has been reported to be stimulated also by influenza A virus . Members of the Herpesviridae family can also activate LTRs of endogenous retroviruses including those related to HERV-W [28–30], which is also supported by our present study. Increased expression of HERV-W related envelope protein was also reported in response to HSV-1 but not rabies virus infection in neuroblastoma cells . HSV-1 was recently reported to induce the expression of HERV-W gag protein expression .
We find that the mechanisms conferring transcriptional activation of HERV-W elements upon influenza A/WSN/33 virus infection were not related to the antiviral response of cells to either double-stranded RNA or to viral capsid binding and fusion. Induction of cellular stress responses through serum deprivation did however, to some extent, mimic the effects of virus infection in terms of transcription of HERV-W elements. The reported relative sensitivities to serum deprivation of the cell-lines is; U937, CCF-STTG1 and 293F in falling order [16–18]. This sensitivity appears to correlate with the relative increases in HERV-W element transcript levels. Interestingly, in serum deprived 293F cells, despite no discernible influence on the relative amount of HERV-W transcripts, alterations in the relative levels of the transcribed elements were detected. The differences observed in the expression patterns of gag or env transcripts between influenza A/WSN/33 infection and serum deprivation suggest that the virus has specific effects beyond those related to cellular stresses.
Specific analysis of transcripts from 7q21 showed that the proviral element was transactivated by influenza A/WSN/33 virus in all cell-lines tested. Surprisingly, in U937, virus infection elevated the levels of transcripts from elements lacking 5'-U3 regulatory regions. In the other cell types the degree of transactivation was less pronounced. Transcripts from the element lacking identifiable LTR were not even detectable in CCF-STTG1 or 293F cells at either baseline or following infection. Thus, when examined at the individual level, the transcriptional regulation of HERV-W elements is considerably more complex than can be revealed by studying only the promoter activities of HERV-W LTRs.
The proviral element on 7q21, found to be transactivated in all cell-lines studied, contains the only HERV-W gene known to have been "domesticated" into the human genome . The product of this conserved gene is called syncytin in light of its fusogenic activity [11, 33]. Expression of syncytin is normally largely restricted to the placenta, where it is proposed to contribute to the biogenesis of the syncytiotrophoblast layer. In the present study, ectopic expression of syncytin in an astrocytoma cell-line was associated with a lower activity of mitochondrial dehydrogenases as a measure of cytotoxicity. Although the mechanisms mediating this effect remain to be identified, our findings support possible negative influences of ectopically expressed syncytin in multiple sclerosis [34, 35]
That viral insults induce expression of endogenous retroviral sequences raises questions as to the evolutionary origins of this effect. The observed HERV expression could constitute a cellular defense reaction, with syncytin and/or other envelope proteins acting as potential receptor blockers, preventing further spread of the virus in analogy to the protection it offers to Spleen Necrosis Virus infections . This scenario could also be considered a case of a viral hijacking of envelope expression in order to utilize the immunosuppressive properties of the transmembrane region of syncytin (reviewed in ). This is further supported by the presence of syncytin at the fetomaternal interface, a region of immunological conflict between mother and foetus [33, 38]. Thus, aberrant syncytin expression could promote immune-system evasion and viral spread, implied indirectly by the fact that syncytin expression is greatly enhanced by virus replication. However, the vast majority of HERV-W elements have no identifiable long ORFs. We speculate that functional consequences of the expression of such sequences should be sought at the level of non-coding RNA (for a review ).
The present study gives no evidence of a link between exogenous virus infection and aberrant expression of HERV-W elements in human disease. It does, however, raise some points of relevance for future studies regarding the expression of HERV-W elements;
i) Environmental stressors can modulate the transcriptional activities of certain HERV-W elements which could thereby be markers for such insults. ii) Disease specific frequency distribution patterns of different transcripts need not be reflected in levels of HERV-W transcripts and can be missed by generic methods. iii) Different cell-types exhibit specific quantitative and qualitative differences in the detectable patterns of transcribed HERV-W elements. Thus, transcripts detected in one tissue can differ from those detected in other tissues in response to a common insult. iv) Non-coding HERV-W elements are apparently transcribed which should merit studies into their transcriptional regulation and biological relevance.
Human neuroepithelioma cells, SK-N-MC (HTB-10), were grown in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (D-MEM/F-12) supplemented with 4 mM L-glutamine, 15 mM HEPES and 12.5% FBS. Human astrocytoma cells, CCF-STTG1 (CRL-1718), and human histiocytic lymphoma cells, U-937 (CRL-1593.2), were grown in RPMI 1640 adjusted to contain 1,5 g/l NaHCO3, 4,5 g/l glucose, 10 mM HEPES and 1.0 mM Na-pyruvate. 293F cells, derived from human kidney, purchased from Invitrogen (Carlsbad, CA), were grown in D-MEM/F-12. All other cell-lines were obtained from the American Type Culture Collection, Manassas, VA. All cell culture media were supplemented with 10% FBS and penicillin/streptomycin (Invitrogen) unless otherwise specified.
Virus infection of cells in culture
SK-N-MC cells, plated 18 hours prior to infection in 6-well tissue culture trays, were rinsed twice with Hank's Balanced Salt Solution w/o Ca2+ or Mg2+ and then duplicate wells were inoculated with MEM 4% FBS without (negative control) or with known amounts (as determined by standard plaque assays) of HSV-1 (102 - 106 plaque forming units (pfu) per well) or influenza A/WSN/33 virus (103 -107 pfu/well). The infections were allowed to proceed for 24 (HSV-1) or 48 hours (influenza A) at 37°C/5% CO2 before RNA isolation, see below.
CCF-STTG1, 293F and U937 cells were washed with one plating volume of MEM and inoculated with MEM containing influenza A/WSN/33 (0.5 MOI) . After 1 hr at 37°C in 5% CO2, cells were washed thrice in one plating volume of MEM. Complete media was added and infections were allowed to proceed for 24 hrs in a humidified 5% CO2 incubator at 37°C before RNA isolation, see below.
Heat-inactivated virus and poly(I:C) treatment
Influenza A/WSN/33 virus was inactivated by heating at 56°C for 90 minutes . CCF-STTG1 cells were washed once in a plating volume of MEM before inoculation as described above with the exception that heat-inactivated virus was also added to the culture medium. Poly(I:C) (Sigma-Aldrich, St. Louis, MO) was dissolved in nuclease-free water (Ambion Inc., Austin, TX) at 10 mg/ml and added to CCF-STTG1 cells at a final concentration of 100 μg/ml .
CCF-STTG1, 293F and U937 cells were cultured in 35 mm plates with normal growth medium. Cells were washed with one plating volume of their corresponding growth medium without FBS. Cells were subsequently allowed to incubate in another plating volume of serum deprived culture media for 24 hrs in a humidified 5% CO2 incubator at 37°C before RNA isolation, see below.
RNA preparation and reverse transcription
RNA was isolated using the RNeasy Mini kit in accordance with instructions supplied by the manufacturer (Qiagen). RNA was quantified by spectrophotometric analysis. Oligo(dT)-primed cDNA was subsequently generated from 150–500 ng of DNaseI-treated RNA (as previously described ) using Superscript II reagents (Invitrogen) according to instructions from the manufacturer. Control reactions without the addition of reverse transcriptase were included.
Real-time PCR and data analysis
Targets, primer and probe sequences and GenBank accession numbers of sequences used for assay design.
HSV-1 glycoprotein D
Seg. 8 A/WSN/33
HERV-W 3q26.32 gag ORF, MGB-probe
Sequences and genomic positions of mapped HERV-W gag (A) and env (B) elements. Dashes indicate nucleotides that are identical with the prototypical HERV-W sequences. Open circles indicate gaps in sequence. Sequences that could not be unambiguously mapped to one genomic loci are indicated by the number of indistinguishable genomic loci found.
5' HERV-W gag3'
Genomic location (strand)
5' HERV-W env 3'
Genomic location (strand)
Mitochondrial viability in response to syncytin expression
CCF-STTG1 cells were transfected with the plasmid PH74  containing the full length ORF encoding syncytin using Lipofectamine 2000 reagents in accordance with the manufacturer's instructions (Invitrogen). Transfection with the pEBFP expression plasmid (Clontech, Mountain View, CA) encoding a variant of green fluorescent protein was used as a control for the effects of forced protein expression. CCF-STTG1 cells were transfected with expression plasmids at 70% confluence in 96-well plates. After 24 hours of incubation at 37°C and 5% CO2, toxicity was determined using the EZ4U kit  according to the manufacturer's instructions (Biomedica Medizinprodukte GmbH & Co KG, Wien).
The present study was generously supported by the Stanley Medical Research Institute, Bethesda, MD and the Swedish Research Council (21X-20047).
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