A phylogenetic tree of both CLP-repressed and CLP-induced U3 clones was established based on the alignment data (Fig. 3). As expected, primarily based on their size differences with regard to the repressed group, the CLP-induced U3 clones were segregated on a unique branch (highlighted in a dotted box). Interestingly, the branching pattern of the CLP-repressed LU(r)06 and LU(r)07 U3 clones was closer to the CLP-induced U3 clones rather than the other CLP-repressed U3 clones. In addition, one distinct branch of five CLP-repressed U3 clones was established from the liver (highlighted in gray) but not the lung.
Tropism traits and transcription potentials of the CLP-repressed and CLP-induced U3 clones
A total of 38 (35 unique) U3 clones isolated from differentially amplified U3 regions were subjected to tropism trait analysis (Table 1). The sequence characteristics, primarily the direct repeat, unique sequence, and ∼190-bp insertion, of the individual U3 clones were surveyed to determine their putative tropism traits. The survey resulted in 29 polytropic and six xenotropic clones (Table 1). Interestingly, among the 31 unique CLP-repressed U3 clones, only two, [Lu(r)06 and Lu(r)07] clones, were xenotypic, whereas all four CLP-induced clones were xenotropic as well, which parallels the phylogenetic data described above. However, the significance of this in silico finding is yet to be determined by further in vitro and in vivo experiments.
To evaluate the transcription potential of individual U3 promoters/clones identified in this study, the profiles of transcription regulatory elements were determined by mapping the consensus sequences of each element. A total of 83 transcription regulatory elements, including the TATA box and CCAAT enhancer, were identified from the 38 (35 unique) U3 clones examined [Table 2. The transcription regulatory element profiles from the CLP-repressed U3 clones were different from those induced by CLP. Five transcription regulatory elements (column labeled as "A") were present only in U3 clones derived from the CLP repressed, and three (column labeled as "B") were from the CLP induced, whereas four elements (column labeled as "C") were shared by all U3 clones examined].
CLP-mediated changes in expression of MuERV variant transcripts
In this study, we examined how the CLP-elicited stress signals affect the profile of full-length as well as variant MuERV transcripts in the liver and lung. We also identified specific MuERV variant transcripts responding to the stress signals. Reverse transcriptase-polymerase chain reaction analyses using a set of primers that are capable of amplifying full-length MuERV as well as subgenomic transcripts revealed that several variant transcripts were regulated in the liver and lung in response to CLP (Fig. 4). However, we were not able to detect any full-length transcripts, presumably ∼7.5 to ∼8 kb in size, in any experimental groups examined. There were unique and substantial changes in variant transcripts in the liver at 12 h (LI-C1(12H), LI-C2(12H), LI-C3(12H), and LI-C4(12H)) compared with changes in the liver and lung at 48 h (LI-S1(48H), LI-S2(48H), LI-C1(48H), and LU-C1(48H)). Four different sizes of variant transcripts (∼5 kb [LI-C1(12H)], ∼2.3 kb [LI-C2(12H)], ∼1.2 kb [LI-C3(12H)], and ∼0.9 kb [LI-C4(12H)]) were induced in the liver at 12 h after CLP, whereas only the ∼0.9-kb transcript was induced in the lung. In contrast, at 48 h after CLP, a variant transcript of ∼2.8 kb (LI-C1(48H)/ LI-S1(48H)), which is about the size of the typical MuERV envelope transcript, was induced in two of three CLP mice, and one variant transcript of ∼1.5 kb (LU-C1(48H)) was induced in the lung (two of three mice). Another envelope size variant transcript (∼2.9 kb [LI-S2(48H)]) was present in the livers of two sham mice at 48 h, but it was not detected in any of the CLP mice.
Characterization of splicing patterns and coding potentials of CLP-associated MuERV variant transcripts
To determine the splicing patterns of the variant transcripts identified above, each transcript cloned from the amplified products (indicated in Fig. 4) was examined for splicing junctions. In addition, coding potentials of these variant transcripts were evaluated for three retroviral genes (group specific antigen [gag], polymerase [pol], and envelope [env]) essential for viral replication. Four near-identical (>99% sequence identity) transcripts (5,063 or 5,064 bp) were cloned from the LI-C1(12H) fragment. There were four ORFs (I ∼ IV) in these transcripts, and none were intact for any of the three main retroviral polypeptides (gag, pol, and env) (Fig. 5). Interestingly, a single-nucleotide deletion in one clone led to a premature termination of ORF III. Putative splicing junctions were surveyed in these transcripts using the reference sequences for splice donors (S/D) and splice acceptors (S/A) yielding a negative result, suggesting that the transcripts are nonsplicing variants (NSVs) transcribed from defective MuERV provirus(es) (19, 29). In fact, a BLAST search of the C57BL/6J genome using one of these transcripts revealed two genomic loci in chromosomes 4 and 8 with more than 99.9% sequence identity with one nucleotide gap and no gap, respectively.
It has been documented that the U3 promoters are highly polymorphic and are the primary determinants of MuERV transcriptional activity and tropism (27). Comparison analysis of the U3 promoter sequences of all variant transcripts identified in this study revealed that all transcripts induced at 12 h in the liver (LI-C1(12H), LI-C2(12H), LI-C3(12H), and LI-C4(12H)) share more than 99% sequence identity within their U3 sequences of 346-bp size (Fig. 6). In contrast, four different sizes (434, 556, 600, and 611 bp) of U3 promoters were found in the transcripts isolated from the liver and lung at 48 h (LI-S1(48H), LI-S2(48H), LI-C1(48H), and LU-C1(48H)). Alignment analyses of variant transcripts of LI-C1(12H), LI-C2(12H), LI-C3(12H), and LI-C4(12H) revealed more than 99% identity within the overlapping sequences among them. We then examined whether the variant transcripts are splicing products derived from the transcripts of LI-C1(12H). The variant transcripts were surveyed for potential splicing junctions using reference sequences (Fig. 5) (19, 29). Five different splicing variants (SVs) were identified from the transcripts isolated from the liver at 12 h, and they are presumed to be the splicing products of the 5,063- and/or 5,064-bp NSV transcripts using combinations of previously described as well as novel splicing signals. There was a single splicing event in four variants (SV-I [S/D(III)-S/A(env)], SV-II [S/D(env)-S/A(env)], SV-III [S/D(V)-S/A(IV)], and SV-IV [S/D(IV)-S/A(III)]) and two events in one variant (SV-V [S/D(env)-S/A(II) and S/D(III)-S/A(env)]). Interestingly, one of the variant transcripts identified in the CLP liver at 48 h, called SV-VI, had more than 99% identity within the overlapping sequences with the above variants (SV-I ∼ SV-V) and was generated by four splicing events [S/D(env)-S/A(I), S/D(I)-S/A(II), S/D(III)-S/A(III), S/D(V)-S/A(env)]. None of these SVs (SV-I ∼ SV-VI) were able to encode intact polypeptides of gag, pol, or env.
Sequence analysis and splicing survey of the variant transcripts of ∼2.8 to ∼2.9 kb from the liver at 48 h (LI-S1(48H), LI-S2(48H), and LI-C1(48H)) yielded five different full-length env transcripts (SV-VII [env/env]) (Fig. 6). In addition, the variant transcripts isolated from the CLP lung at 48 h (LU-C1(48H)) were determined to be two different defective env transcripts (SV-VIII and SV-IX) derived from at least two different MuERV loci on the genome that have major deletions in the env and/or other genes (Fig. 5). Two defective MuERVs presumed to be proviral templates for SV-VIII and SV-IX were mapped on chromosomes 11 and 13 of the C57BL/6J genome with 94% and 100% sequence identity within the relevant U3 sequences, respectively.
The splicing signals used to generate the variant transcripts identified in this study are summarized in Figure 5C. In addition to the env S/D and S/A, eight well-conserved MuERV splicing signals (four donors [S/D(I), S/D(III), S/D(IV), and S/D(V)] and four acceptors [S/A(I), S/A(II), S/A(III), and S/A(IV)]) were used in combinations to generate the variant transcripts. Four [S/D(IV), S/D(V), S/A(III), and S/A(IV)] of these splicing signals have not been reported previously, whereas the other four signals [S/D(I), S/D(III), S/A(I), and S/A(II)] were identified in one of our previous burn studies (19, 29).
Comparison of MuERV U3 sequences of the variant transcripts differentially expressed after CLP
To determine the relationships among the MuERVs from which the variant transcripts were derived, their U3 sequences were compared, and a phylogenetic tree was established (Fig. 6). A 346-bp U3 sequence was shared by a total of the initial 18 variant transcripts cloned, which include all clones (SV-I ∼ SV-V) from the CLP liver at 12 h and one (SV-VI) from the CLP liver at 48 h with greater than 99% sequence identity. This 346-bp U3 sequence, which was mostly derived from the CLP-induced variant transcripts at 12 h, was almost identical (>99% sequence identity) to the CLP-induced U3 clones (Fig. 2). Two full-length env transcripts (LI-C1(48H)−2 and LI-S1(48H)−1 of SV-VII) had a 434-bp U3 sequence with one nucleotide difference between each other, whereas the other three members of SV-VII (LI-C1(48H)-1, LI-S2(48H)−1, and LI-S2(48H)−2) shared a 556-bp U3 sequence (two identical plus one with 11-nucleotide mismatch). In addition, there were two SV-VIII transcripts (LU-C1(48H)-1, LU-C1(48H)−2) sharing a 611-bp U3 sequence with greater than 99% sequence identity (four nucleotides mismatch), and SV-IX was represented by one transcript (LU-C1(48H)-3) with a 600-bp U3 sequence. Interestingly, phylogenetic analysis of the U3 sequences of all SVs (SV-I ∼ SV-IX) revealed that two members of SV-VII retaining a 434-bp U3 sequence were evolutionarily closer to NSV and SV-I ∼ SV-VI with a 346-bp U3 than they were to the other three SV-VII members, SV-VIII, and SV-IX (Fig. 6). The direct repeat 4/4* was present only in two U3 sequences of 346 and 434 bp, suggesting their unique tropism traits.
Putative env polypeptides encoded from CLP-associated, full-length env transcripts
Five different full-length env transcripts of SV-VII isolated in the liver of sham and/or CLP mice were translated. Their polypeptide sequences were subjected to multiple alignment and phylogenetic analyses (Fig. 7). Two env polypeptides encoded from env transcripts of LI-C1(48H)−2 and LI-S1(48H)−1 were placed on one main branch, whereas the other three (LI-C1(48H)−1, LI-S2(48H)−1, and LI-S2(48H)−2) segregated together into another branch. It is likely that the env polypeptides encoded from LI-C1(48H)−2 and LI-S1(48H)−1 transcripts (either or both) were induced in the CLP liver at 48 h (Fig. 4). In addition, the tropism traits of these env polypeptides were determined by comparison to the variable regions A and B (VRA and VRB) of reference sequences (Fig. 7) (30). Comparison analysis using VRA sequences revealed that three (LI-C1(48H)−1, LI-S2(48H)−1, and LI-S2(48H)−2) were presumed to be polytropic and two (LI-C1(48H)−2 and LI-S1(48H)−1) were close to being xenotropic. However, the results from the VRB were not clear enough to interpret except for the LI-C1(48H)−2 as xenotropic, probably because of a limited size of the sequences to be aligned. All the other variant transcripts (SV-II, SV-VIII, and SV-IX) with an env splicing junction retained only coding potentials for truncated env polypeptides.
The complex network of signaling events underlying the pathogenesis of sepsis is often unique for individual patients, and it has not been fully understood even after extensive investigations involving a range of molecules and pathways (5, 31, 32). In this study, we examined whether the expression of MuERVs is altered in response to sepsis-elicited stress signals. The key findings are as follows. First, there were substantial changes in the expression of certain MuERVs in the liver and lung of CLP-sepsis mice. Second, individual U3 promoter sequences of CLP-sepsis-regulated MuERVs had unique characteristics in regard to tropism traits and transcription regulatory elements. In addition, the findings from the phylogenetic analysis suggest that some MuERVs responding to the CLP-elicited stress signals share similar promoter sequences. Third, several MuERV SVs (SV-I ∼ SV-IX) were differentially regulated in the liver and lung after CLP. Fourth, among the five full-length env transcripts, two of them (LI-C1(48H)−2 and LI-S1(48H)−1 of SV-VII) were presumed to be induced in the liver at 48 h after CLP.
All of the variant transcripts whose expression was induced at 12 h after CLP shared a 346-bp U3 promoter (with >99% sequence identity), and they were presumed to be splicing products processed from a variant transcribed from a defective MuERV. In contrast, at 48 h after CLP, four different sizes of U3 promoters (434, 556, 600, and 611 bp) were involved in changes in the expression profile of variant transcripts. The differential usage of MuERV U3 promoters at 12 and 48 h after CLP might be closely linked to the formulation of a unique transcriptional environment at each time point. In particular, it would be important to identify the transcription factors and components of the splicing machinery responsible for the changes in MuERV expression after CLP.
Coincidentally, the two genomic MuERV loci presumed to be proviral templates for CLP-sepsis-induced transcripts (5,063- and 5,064-bp NSVs from ICR stock mice) were shared by a transcript, which was induced in the liver of C57BL/6J mice after burn injury (20). Interestingly, these two different types of stressors (sepsis and burn) influenced the transcription, including splicing, of one specific MuERV (two genomic loci) out of the numerous MuERV copies on the genome. Furthermore, CLP-sepsis and burn experiments were performed in mice with two different backgrounds, ICR and C57BL/6J, respectively (19, 20). The sepsis and burn-associated profiles of the transcriptional environment and epigenetic modification in the liver were presumed to be specific enough to control the MuERV's U3 promoter. In addition, it might be interesting to examine whether MuERV regulation after CLP-sepsis and/or burn at these genomic loci is linked to the expression of neighboring host genes.
Murine endogenous retroviruses have been regarded as simple retroviruses primarily because of limited diversity in their ORFs associated with splicing potentials compared with the complex retroviruses with several splicing events and resulting ORFs. Only two splicing signals (env S/D and S/A) were previously known for MuERVs until five additional signals (three donors and two acceptors) were reported from our laboratory, and four novel splicing signals (two donors and two acceptors) are identified in this study (19, 29). These findings suggest that the genomic organization of MuERVs, in regard to splicing and coding potentials, is more complex than previously thought. It might be interesting to investigate the underlying mechanisms of how the stress signals from CLP-sepsis influence the differential splicing events of MuERVs.
There are three potential mechanisms of how certain MuERVs contribute to the pathogenic processes of CLP-sepsis. First, MuERVs encode viral proteins such as env and gag polypeptides that could participate in CLP-sepsis-associated signaling events. Second, MuERVs may retain coding potentials required for the assembly of pathogenic virions, resulting in infection into susceptible host cells, such as hepatocytes and alveolar epithelial cells, accompanied by pathological effects. Third, changes in the transcriptional activities of MuERV loci on the genome may influence the expression of neighboring genes, leading to phenotypic alterations in affected cells and tissues.
The results from our preliminary study demonstrated that overexpression of certain MuERV env proteins in a macrophage cell line induced differential expression of a few inflammatory mediators (e.g., COX-2, IL-6) (data not shown). These findings may suggest that the CLP-sepsis-activated MuERVs in Kupffer cells and/or alveolar macrophages may participate in a cascade of signaling events associated with early inflammatory response in the liver and lung, respectively. It is likely that the MuERV expression levels will return to normal within 1 week; however, it will be interesting to investigate the long-term pathological effects of infection and genomic random reintegration of the CLP-activated MuERVs.
The CLP-activated MuERVs may exert deleterious and/or beneficial effects during the course of sepsis. One potential positive outcome from the treatment of CLP-sepsis mice with antiretroviral agents (e.g., reverse transcriptase inhibitor, siRNA against env gene) will be alleviation of the inflammatory response by reducing the production of inflammatory cytokines.
The findings from this study suggest that the stress signals from CLP-sepsis affect the expression and splicing of specific MuERVs. It may lead to a justification for further investigation into the roles of MuERVs and their gene products in the complex network of pathogenic processes of sepsis.
The authors thank the staff of Minnesota Molecular Inc (Minneapolis, Minn) for the generous gift of molecular weight markers.
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Endogenous retrovirus; transcription; splicing variant; polymicrobial sepsis; cecal ligation and puncture
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