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Previous Article | Next Article 
Journal of Virology, August 2006, p. 7295-7307, Vol. 80, No. 15
0022-538X/06/$08.00+0 doi:10.1128/JVI.00679-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Xiaoyu Wang, and
Michele M. Fluck*
Department of Microbiology and Molecular Genetics, Interdepartmental Program in Cell and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1101
Received 4 April 2006/ Accepted 12 May 2006
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ABSTRACT |
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1, could contribute in an additive way. Unexpectedly, a mutant lacking all these connections because the three major tyrosines had been converted to phenylalanine retained some activity. A mutant in which all six MT C-terminal tyrosines had been mutated was inactive. This suggests a novel signaling pathway for MT that uses the minor tyrosines. What is common to ST and the individual MT signaling pathways is the ability to signal to the polyomavirus enhancer, in particular to the crucial AP-1 and PEA3/ets binding sites. This connection explains the pleiotropy of MT and ST effects on transcription and DNA replication. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The enhancer exerts a cis-acting simultaneous control on early and late gene expression as well as DNA replication (15, 44, 59, 62). Fine mapping of the enhancer has attributed a key role for a 20-bp "
core" subdomain (nucleotides [nt] 5110 to 5130) and its bipartite PEA1/AP-1 and PEA3 binding site and minor roles to additional subdomains that contain, among others, PEA3 and/or c/EBP binding sites (43, 44, 46, 57, 62, 70).
Considerable support can be found for the linkage of MT and to a much lesser extent ST signaling to the activation of AP-1 and PEA3/ets (reviewed in the Discussion). Both AP-1 and PEA3/ets family members are bona fide downstream targets of the MT signaling pathways. Indeed, MT induces a high upregulation of a reporter gene under the control of the PYV bipartite PEA1 and PEA3 binding site, resulting from synergistic actions of AP-1 and PEA3 (65-67). ST has not been similarly tested so far. The convergence of the requirement for AP-1 and PEA3 transcription factors, their simultaneous roles in transcription and replication, and the effects of MT and likely ST downstream signaling on the activation of these factors provide a compelling rationale for a major multifunctional if indirect role for MT (and ST) in viral gene expression and genome amplification, in addition to the previously described role for MT in the maturation process (21-23). Both transcription factors PEA1 (the murine analog of AP-1) and PEA3 (a member of a large family of factors that includes ets proteins) also play key roles in cancer (14). Therefore, the same pathways that are used for lytic infection also serve in transformation by the viral oncogenes.
In the present studies, we sought to further define, separate, and contrast the effects of MT from those of ST, as well as to identify the MT tyrosines that mediate the MT role in lytic infection processes. The results of these experiments considerably expand our understanding of ST and MT functions and reveal additional roles for MT tyrosines than those derived from transformation studies.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T transitions in primers spanning the corresponding nt 985, 1009, 1099, 1126, 1180, and 1201, using the QuikChange mutagenesis kit (Stratagene). In all six cases, these mutations are neutral in the overlapping large T antigen frame. MT-Ter was constructed with a GA transversion at nt 825, causing a Glu termination at residue 197 in the MT frame. The 196-amino-acid truncated MT protein produced is identical to ST except for the five C-terminal residues (Lys-Arg-Arg-Ser-Glu replacing Ser-Leu-Ser-Pro). This mutation also causes a Gly89Val change in the LT frame. All mutations were confirmed by sequencing an approximately 600-bp region spanning the mutations. The 1387T mutant, a generous gift from Tom Benjamin, contains a termination codon at nt 1387, lacks a 37-amino-acid C-terminal membrane anchor, and is in a different genetic background (7).
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Cells and infections. A murine NIH 3T3 cell subline (originally from Tom Benjamin's laboratory) was used for the study as well as to grow virus stocks. This line has a functional p53, as it induces the expression of p21 in response to treatment with actinomycin D; however, it is negative for p16 expression. Fischer rat FR3T3 cells were also used. Cells were plated in 60-mm culture plates in Dulbecco's modified Eagle's medium (GIBCO BRL) supplemented with 10% newborn calf serum (GIBCO BRL) up to 25% confluence. Prior to infection, cells were brought into synchrony in the G0 state by lowering the serum supplement to 0.5% for 24 h. Infections were carried out in phosphate buffer (1x phosphate-buffered saline) supplemented with 0.5% serum. After 1 to 2 h, the unadsorbed virus was removed, and cells were washed once with 1x phosphate-buffered saline and refed with medium supplemented with either 10% serum or 0.5% as stated. A multiplicity of infection (MOI) of 10 PFU was used. Since the major focus of these studies concerns transcription and DNA replication, it was important to have the same levels of viral templates in comparisons of different mutants. Thus, the titers of stocks of wild-type and mutant viral strains were matched so that input genome levels were equal. This strategy bypasses problems such as inaccurate titers of the A185 MTnull/STnull small plaque mutant. Genome equivalency was verified in all experiments by assaying genome levels between 4 and 12 h postinfection (hpi). The times given throughout also correspond to times post-release from G0.
Some experimental variability was expected due to the inherent difficulty in controlling for equal inputs in experiments involving a large number of mutants. As previously discussed (10), results for the severity of the defects of mutants in different categories also depended upon variations in host cell "permissivity," hence, the use of specific cell conditions for infections. All conclusions are based upon at least three independent experiments.
Protein analysis. Cells were lysed at various times with protein sample buffer (5% sodium dodecyl sulfate [SDS], 0.03% bromophenol blue, 20% glycerol, 5% ß-mercaptoethanol, 0.5 M Tris-HCl pH 6.8) and boiled for 5 min. One-third aliquots of the lysates were electrophoresed in 10% polyacrylamide and electroblotted onto polyvinylidene difluoride membranes (Amersham). A polyclonal rat antitumor serum, harvested as ascites fluid, was used as the primary antibody. This antibody recognizes all three early proteins, large, middle, and small T, as well as a few cellular proteins, which can serve as an internal loading control. Goat anti-rat-horseradish peroxidase (Pierce) was used as the secondary antibody. A rabbit anticapsid antibody, a gift from Robert Garcea, was used for the detection of the VP1 capsid protein, with goat anti-rabbit-horseradish peroxidase as the second antibody.
Analysis of viral transcripts.
Infected cells were lysed in TRIzol solution (GIBCO BRL) at various times. Total RNA was extracted with chloroform and precipitated with isopropanol. For Northern blot assays, RNA samples were electrophoresed in a 1% agarose gel containing 2.2 M formaldehyde and transferred to a nylon membrane (Amersham). Probes were labeled either with radioactive [-32P]dCTP or with digoxigenin as indicated in the figure legends.
To detect early transcripts, a pGEM-1-based plasmid, pG3PyH4, was used, which contains PYV MspI fragment 4 (nt 399 to 1101), cloned between the HindIII and EcoRI sites. The plasmid was cleaved with HindIII, and the T7 RNA polymerase was used to synthesize the anti-early probe. This probe spans the three overlapping early introns and can detect all three early mRNAs, i.e., the 19S MT and ST mRNAs and the 18S large T mRNA, as well as the unspliced precursor and multigenomic early RNAs. To score for late RNAs, sequences spanning nt 3918 to 2928 were inserted between the HindIII and BamHI sites of pSPT18 (Roche Molecular Biochemicals). The plasmid was cleaved with HindIII. This probe detects all major late RNAs, the major 16S VP1 mRNA, and the minor VP2/VP3 mRNAs as it spans the 3' terminus of the late RNAs, spliced and unspliced, as well as the multigenomic late RNAs.
The strand-specific RNA probes were labeled with digoxigenin using the kit from Roche Molecular Biochemicals, following the instructions from the manufacturer. The hybridizations with digoxigenin-substituted probes were carried out as follows. Blots were prehybridized for 2 h at 68°C in 50% formamide, 5x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.02% SDS, 0.1% N-laurylsarcosine, 2% blocking reagent (Roche Molecular Biochemicals), and then hybridized overnight at 68°C in a hybridization mixture containing digoxigenin-labeled RNA probe. The membranes were washed twice with 2x SSC and 0.1% SDS at room temperature for 15 min each and twice with 0.5x SSC and 0.1% SDS at 68°C for 15 min each time. The membranes were treated with blocking agent solution for 1 h and then with the anti-digoxigenin-alkaline phosphatase, diluted 10,000-fold in blocking buffer for 30 min. After extensive washing, the chemiluminescent phosphatase substrate detection reagent was applied for 1 min and the membranes were exposed to X-ray film.
Genome amplification analysis.
Infected cells were lysed in 10 mM Tris-HCl, 10 mM EDTA, 0.2% SDS (pH 7.6) supplemented with 0.1 µg/ml of proteinase K (Sigma). DNA was extracted with phenol-chloroform. DNA was digested with the restriction endonuclease EcoRI (GIBCO BRL), which has a single recognition site in the PYV genome and linearizes it. Digested DNA was electrophoresed in 1% agarose, stained with ethidium bromide, and blotted onto nylon membranes (Amersham Pharmacia Biotech). Hybridization was carried out at 65°C in 1x Denhardt's solution, 2x SSPE (1x SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA [pH 7.7]) by standard procedures with a 32P-radiolabeled probe containing the whole PYV genomic DNA, labeled with [-32P]dCTP (3,000 mCi/mmol; New England Nuclear) with a multiprime DNA labeling kit (Amersham). The hybridized blots were washed under stringent conditions. The blots were exposed to X-ray film (Kodak) for a few days at –70°C with an intensifying screen. To quantitate the level of viral genomes, membranes were scanned with a Storm PhosphorImager.
Plaque assays. NIH 3T3 cells were grown to 80% confluence and infected with virus dilutions for 1 h at 37°C. Infected cells were refed with medium containing 5% serum and 0.9% agar and incubated for 7 days at 37°C. The plates were stained with neutral red. The plaques were counted after a >4-h incubation at 37°C.
Complementation with MT-expressing retroviral vectors. MT wild-type cDNA introduced in the pLZRS-based Pinco vector (Pinco-MT) was obtained from Luca Primo (24, 28). DrdI-EcoR1 fragments (nt 961 to 1562) from various MT mutants were used to shuttle between PYV or pMex-MT and Pinco-MT. Retroviral stocks were produced by cotransfections of the Pinco vectors and a Moloney virus-based helper plasmid in 293T cells by using Lipofectamine. Stocks were titrated by determining the frequency of green fluorescent protein-positive cells following infection and by quantitating MT protein expression by Western blotting. NIH 3T3 cells were infected with MT retroviruses in the presence of Polybrene and refed with serum containing medium. Twenty hours later, cells were superinfected with the A185 PYV mutant and cells were refed with medium supplemented with 0.5% serum. Samples were taken at 6 and 48 h post-A185 infection for protein, genome, and mature virus quantitation, similarly to what was described for PYV-only infections. In the case of infection with A185 only, equal loading of protein samples is reflected by equal intensities of the cross-reacting cellular bands.
Several other attempts to develop a complementation strategy failed. Stable MT-expressing cell lines were found to be quite resistant to PYV infection (at least strain A2) as previously noted (2, 31, 63), as were inducible cell lines based on constitutive Tet-VP16 expression. We also did not succeed in coinfecting with PYV and adenovirus vectors.
Transformation assays. Fischer rat FR3T3 cells were set at 2 x 105 cells per 60-mm dish and infected 24 h later at an MOI of 10 PFU/cell. Equivalency of genome input levels was confirmed at or before 24 hpi, as described for infection of NIH 3T3 cells. To assay for focal overgrowth of the monolayers, cultures were maintained in medium containing 5% calf serum that was refreshed every 3 or 4 days. At 14 days postinfection (dpi), monolayers were rinsed, fixed, stained with methylene blue, and photographed. To assay for anchorage-independent growth, infected monolayers were trypsinized 24 hpi and passed into semisolid agar (0.36%) containing Dulbecco's modified Eagle's medium supplemented with 15% calf serum.
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RESULTS |
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Gene expression of these four strains was tested by Western blotting in infections of NIH 3T3 cells carried out in medium supplemented with 0.5% or 10% serum. A representative set of results obtained in early (24 hpi) (Fig. 2A) and late (48 hpi) (Fig. 2B) phases of infection is shown. The pattern of early proteins produced by wild-type A2 was as described previously, with higher LT expression (and earlier [data not shown]) than MT or ST (9). The absence of both MT and ST (A185) resulted in a large decrease in the LT level compared to wild type, as expected from our previous results (10). The presence of ST in MT-Ter and 1387T partially restored this defect, demonstrating ST participation in polyomavirus gene expression. The higher level of gene expression in 1387T relative to MT-Ter was seen in all experiments. We believe that this likely reflects differences in the genetic background of these two strains rather than differences in the residual activities of the two truncated proteins. 1387T was generated in the same genetic background as the hr-t mutants, in which the loss of MT and ST functions does not seem to lead to deleterious effects on gene expression (10), presumably because of the selection of compensatory mutations in large T (39).
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Because MT downstream signaling overlaps with that triggered by growth factors (16), we examined the capacity of serum factors to compensate for the gene expression defect(s) caused by the absence of MT and ST. High (10%) serum treatment was most beneficial in complementing the defect of the most deficient mutants, e.g., A185, while not fully compensating for their defect. The magnitude of the serum effect was close to that induced by ST: the LT level in A185 infection in the presence of 10% serum was comparable to that seen in infection of MT-Ter in low serum (compare lanes 3[+] and 2[–] in Fig. 2A and B).
Effects of MT and MT tyrosine mutations in the absence of ST expression.
To examine the specific role of MT and the effect of MT YF mutations in the absence of ST signaling, we adopted a complementation strategy in which wild-type or mutant MT was introduced in cells infected with the MTnull/STnull mutant A185. This strategy has a number of advantages. Use of a single viral strain and hence of a single virus stock bypasses unavoidable experimental differences in input levels in infections with many different strains. Furthermore, since MT and/or ST expression was shown to be responsible for the acetylation of virion chromatin (50), the use of a single mutant (A185) eliminates differences in input chromatin structure that may impact gene expression by the parental genomes. Coinfections with MT retroviral vectors and PYV (which to our knowledge had not been tried previously) were very successful. NIH 3T3 cells were first infected with wild-type or mutant MT retroviruses in the presence of serum, followed by infection with PYV MTnull/STnull A185 and withdrawal of serum (to 0.5%) as described in Materials and Methods. The percentage of green fluorescent protein-positive cells was high, suggesting expression of MT in most cells (80 to 100%). Effects on early (LT) and late (VP1) gene expression were determined by Western blotting (Fig. 3). Comparison of LT expression between coinfection with the empty vector and with wild-type MT at 48 hpi demonstrates robust complementation of the A185 defect with wild-type MT, with an 
5-fold increase in LT level (compare lanes 1 and 2). The level of MT was elevated, since the LT/MT abundance ratio was reversed compared to what is seen in natural infection with wild-type virus (e.g., Fig. 2A and B, lanes 1).
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(PLC), respectively. Combinations of tyrosine-to-phenylalanine (YF) mutations encompassing the three major Y250, Y315, and Y322 tyrosines were generated (see Materials and Methods and Table 1 for mutant construction). Fairly even MT expression was achieved for the various MT mutants, as tested prior to LT synthesis (Fig. 3A) and at 48 hpi (Fig. 3B). These data also demonstrate that no tyrosine MT mutant is particularly unstable, as also tested in a pulse-chase experiment (data not shown). Surprisingly, MT complementation activity was not totally eliminated by mutation of all three major tyrosines, Y3FM (compare lanes 1 and 3 for VP1 or LT). (This is further documented in experiments with genomic Y3FM mutations.) In view of the result with Y3FM, we considered the possibility that signaling could be mediated by one or more of the remaining C-terminal tyrosines, Y258, Y288, and Y297 (referred to as the minor [m] tyrosines). Although no biochemical functions have been associated so far with them, previous results have shown phosphorylation for at least Y297 (33). In line with the situation for the three major tyrosines, pY297 might provide a phosphotyrosine binding site for a signaling intermediate. Hence, a sextuple mutant (Y6F) was constructed to eliminate contributions from all three minor tyrosines (Table 1). Y6F MT was unable to induce any complementation (compare lanes 1 and 5). The difference in degree of complementation by Y3FM and Y6F MT (lanes 3 and 5) suggests a role for at least one among the minor tyrosines. In contrast, mutating all three minor tyrosines (Y3Fm) did not result in a detectable change compared to wild-type MT (compare lanes 2 and 4). This may reflect the limitation of the assay. A 25% decrease in genome amplification was observed (see "MT and ST effects in genome amplification," below). In most assays, the effects of mutating Y258, Y287, and Y297 were more pronounced when Y250, Y315, and Y322 were also mutated (comparing Y6F to Y3Fm) than when the latter were wild type (comparing Y3Fm wild type).
To examine the role of each major tyrosine, each was tested one at a time in "add back" quintuple mutants. Each major tyrosine caused an increase in LT expression over that produced by Y6F MT (compare lanes 6, 7, and 8 to lane 5 or 1); similarly, each of the three combinations of two functional major tyrosines showed an increase over single tyrosine-induced complementation (compare lanes 8, 9, and 10 with lanes 6, 7, and 8). The data suggest that each major tyrosine was able to contribute in an additive manner.
The examination of the effect of MT tyrosine mutations on late capsid gene expression was hampered by the high background in VP1 major capsid in A185 infections, presumably due to an excess of empty capsids in the input (i.e., detected prior to initiation of gene expression; see below). However, the lower levels in the empty vector control (Fig. 3C, lane 1) and Y6F MT (lane 5) compared to wild type (lane 2) are indicative of effects on late expression, further demonstrated below. Some difference between Y3FM and Y6F expression on VP1 late gene expression is also visible.
Gene expression defects of MTYF/ST+ mutants.
Having established a role for the three major tyrosines in complementing the defects of an MTnull/STnull virus, we examined the same issues in the context of viral mutant strains when MT was expressed in cis from the same promoter as LT and ST by alternate splicing of a precursor mRNA. To this end, we introduced combinations of tyrosine-to-phenylalanine (YF) mutations encompassing the three major tyrosines, Y250, Y315, and Y322, and three minor tyrosines, Y258, Y288, and Y297, in the A2 genetic background (see Materials and Methods and Table 1). As discussed above, all such mutants express a wild-type ST protein and are thus designated MTYF/ST+. Comparisons were focused on MTYF/ST+ mutants relative to wild type (MT+/ST+) on the one hand and to MTnull/ST+ mutants on the other. A representative set of results obtained by Western blotting in early (24 hpi) (Fig. 4A) and late (48 hpi) (Fig. 4B) phases of infection are shown. In the presence of ST, the loss of any single major tyrosine did not lead to a consistent detectable decrease in early gene expression in multiple experiments in NIH 3T3 cells, while the loss of two major tyrosines in combinations involving Y250, especially the Y250/322F combination, resulted in lower gene expression levels (Fig. 4A and B, lanes 9). Surprisingly, the triple mutant Y3FM (Y250/315/322F) showed either no or a smaller defect than Y250/Y322F in most of the NIH 3T3 gene expression tests. This result is in general agreement with findings in early studies that examined the growth of deletion mutants encompassing MT tyrosines (27). The loss of all six C-terminal tyrosines did result in the loss of MT function in gene expression, as the Y6F mutant was found to be close to or equally as defective as the MTnull/ST+ MT-Ter and 1387T mutants in most tests (compare lanes 12 with lanes 2 and 4). As also seen in the complementation experiments described above, Y3Fm did not show a defect in gene expression (data not shown). Not withstanding experimental variations, the relative defects of the various mutants were similar at 24 and 48 hpi, suggesting that most of the effect of MT loss in these mutants was established by 24 hpi. Defects in late gene expression (VP1) showed a good correlation with those seen for early gene expression (Fig. 4A and B), demonstrating effects of MT and ST on both early and late gene expression.
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As stated above, serum treatment was most beneficial in complementing the defect of the most deficient mutants, A185, MT-Ter, Y6F, Y250/322F, and Y250/Y315F, while not fully compensating for their defects. In addition, the presence of serum factors resulted in differential MT mobility as demonstrated in Fig. 4A and B, with a faster mobility in the presence of serum factors. The reason behind the intriguing persistent difference in MT migration is unknown. It suggests differences in calf serum factors and MT downstream signaling pathways that affect MT modifications.
In summary, comparisons between these mutant viral strains support the following order for defects in gene expression (from no defect to pronounced defects): WT 
Y3Fm, Y1F > Y3FM > Y6F 
MT-Ter >> A185. These results also determined that the gene expression defect pattern of these mutants is established by 24 hpi, which is close to the "early-to-late" transition.
Timing of appearance of the gene expression defect. The development of the gene expression defect in the absence of MT and/or ST was further examined. One possible expectation is that the problems of MT- and/or ST-defective mutants become manifest only past the time when these proteins will have been expressed and have exerted their effects in upregulating gene expression in a wild-type infection. To determine this point, a set of wild type and mutants comprised of Y6F, MT-Ter, and A185 were examined from very early times (8 hpi) representing an "immediate-early" phase through the very beginning of the late phase (16 hpi). As can be seen in Fig. 5, LT protein could be detected by 10 hpi in infections with the wild type, while ST and then MT appeared with the previously reported delay, by 12 and 16 hpi, respectively (9). Kinetics studies of the induction of the MT-associated kinase activity place its detection from approximately 14 hpi (data not shown). No delay, or a small 2-h delay, in LT expression was seen with Y6F, A185, and MT-Ter. Furthermore, in contrast to the results at 24 hpi shown in Fig. 2 and 4, no or a minimal defect in LT and ST expression relative to wild type was observed up to 16 hpi for MT-Ter. At that time, Y6F showed only a relatively very small reduction in expression of all three early proteins compared to findings at 24 and 48 hpi. In the case of A185, a defect in LT expression became apparent from about 12 hpi; however, even at 16 hpi, the ratio of wild-type to A185 LT levels was considerably less than that seen at 24 hpi and 48 hpi. This places the onset of MT- and ST-dependent activation of viral gene expression between 16 and 24 hpi. By 16 hpi, the late phase had started, as deduced from the presence new VP1 in wild-type, MT-Ter, and Y6F infections. At a higher serum concentration, the A185 defect in early expression was partially relieved (not shown).
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Effects of MT and/or ST mutations on viral transcription patterns.
MTnull/STnull, MTnull/ST+, and the most-defective MTYF/ST+ mutant (Y6F) were chosen for a comparative analysis of their transcription patterns. As described previously (9) the abundance of transcripts is too low for detection by Northern blotting until immediately prior to or simultaneously with the onset of late gene expression. Thus, a kinetic analysis of early and late transcript accumulation was followed between 16 and 24 hpi. This analysis was carried out in the same experiment shown in Fig. 5, in which early proteins were analyzed at earlier times. Based upon our previous studies carried out under similar conditions (9), the 16- to 24-hpi period spans the "early to late transition," with the onset of detectability of the early (16 hpi) and late (18 hpi) transcripts by Northern blot analysis, a very large increase in the abundance of both classes of mRNAs, and the onset of viral DNA replication. We chose Northern blot analysis of total RNA, which reveals the presence of giant transcripts. The data are shown in Fig. 6. In the case of early transcripts (Fig. 6A), wild-type transcripts were barely detectable at 16 hpi and their abundance increased substantially between 16 and 24 hpi. As described previously (9), most of the RNA is spliced and bands corresponding to the LT (18S) and MT plus ST (19S) transcripts (which differ by 14 nt) were detected. By 24 hpi, giant early transcripts were observed (the top of the gel was cut). These results recapitulate previously described patterns, as do those for the A185 mutant, demonstrating a severe defect in early transcription (10). From 18 hpi on, Y6F early transcripts were produced in substantially lower abundance than wild type and only slightly exceeded the level produced by A185. Additionally, no giant transcripts were seen by 24 hpi. As was shown in Fig. 2 and 4, by 24 hpi the Y6F defect in early viral gene expression was evident at the protein level. Perhaps due to the twofold dosage of ST function, MT-Ter early transcripts levels were higher than those seen for Y6F. In the case of the late transcripts (Fig. 6B), these were detected from 18 hpi in wild-type infection, with the inclusion of giant species by 21 hpi. Their very large increase in abundance, as well as their size heterogeneity, has been described previously (1, 9). The extensive deficit of late transcription of the A185 mutant recapitulates previous findings (10). The abundance of the late transcripts in mutant-infected cells followed that seen for the early transcripts. In this case, by 24 hpi the Y6F late transcript levels were clearly higher than A185 transcripts. For both early and late transcripts, the gradation in relative transcript levels was as follows: WT > MT-Ter > Y6F > A185.
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That there is a small delay (as previously documented [10]) in ST- and MT-mediated upregulation of late compared to early gene expression is likely due to the genome amplification requirement for late gene expression. Thus, the MT- plus ST-dependent upregulation of early gene expression leads to an increase in LT concentration, leading in turn to initiation of genome amplification in cells that are in S phase and, with a delay, initiation of late gene expression by dilution of a presumed late repressor (5, 40).
MT and ST effects in genome amplification. The genome amplification potential of the same set of mutants whose gene expression patterns were defined in Fig. 4 was examined by Southern blotting in the same experiment. Samples were taken simultaneously of the protein samples, namely, at 24 and 48 hpi. The data for 48 hpi are shown in Fig. 7 along with the input samples, and the quantitation of mutant and wild-type amplification for both 24 and 48 hpi is given in Table 2. Genome amplification defects were detected, and overall mutant-specific loss in gene amplification paralleled their loss in gene expression. The presence of serum factors led to a small relief of the replication defect. The previously described profound defect of A185 is apparent here as well (57-fold) (10). The much smaller defects of MTnull and trunc/ST+ mutants (MT-Ter and 1387T) (range, 3.9- to 5-fold decrease) demonstrate the impact of ST expression. In the case of MT tyrosine mutants, very small decreases in genome amplification accompanied the loss of any single major tyrosine (range, 1.5 to 2.6). Larger defects were observed with double mutants containing the Y250F mutation. Yet, as in the case of gene expression, the defect of the Y3FM triple mutant was similar to that of single mutants. The genome level in Y6F infections was in the same range as that observed for MTnull and trunc/ST+ mutants (MT-Ter and 1387T); its defect was clearly greater than Y3FM.
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Maturation analysis of MTYF/ST+ mutants.
Previous work demonstrated that MT plays a key role in viral maturation (21, 22). Here we examined the capacity of MT mutants Y3FM, Y6F, and MT-Ter as well as MTnull/STnull A185 to produce mature virions. Data on yields of viral genomes and of mature virions (PFU) are summarized in Table 5. A maturation factor, defined as the ratio of plaque-forming virus per viral genome, was computed, and an arbitrary number of 1.0 was chosen for the wild type. As can be seen, for Y3F, Y6F, and MT-Ter this factor was between 20 and 50% of the wild-type maturation capacity. In contrast, in the case of A185, this maturation potency was only 10% that of the wild type. The examination of capsid protein levels (data not shown) does not account for the more pronounced maturation defect of A185. Rather, this likely reflects the previously reported maturation defect, which has been associated with the underphosphorylation of VP1 (22). This defect may result in an increased production of empty capsids (or pentamers), which are entering into the cells, and can be detected long before late proteins are expressed. Interestingly, the present data showing that MTnull/ST+ mutants had a three- to fivefold less severe maturation defect than MTnull/STnull suggest that ST may also carry out this maturation function. This observation fits with previous results obtained with mutant 1387T (MTtrunc/ST+) (23).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ST effects were examined by comparisons of mutant viral strains that express ST only (MTnull/ST+ MT-Ter and 1387T) with the MTnull/STnull A185 strain. ST expression led to an increase in the level of early and late transcripts and gene products as well as in genome levels. Isogenic strains that express both MT and ST (MT+/ST+ wild type) showed a further increase in all three parameters over MTnull/ST+ strains. This indicated a contribution from MT. To study the effect of MT in the absence of ST, a complementation strategy was required. Retroviral MT expression in coinfection with the MTnull/STnull A185 strain resulted in the upregulation of early and late viral gene expression and genome amplification, thus confirming the role of MT. Overall, the results show that the presence of either MT or ST, singly or together, complements the pleiotropic defect of MTnull/STnull mutants.
As summarized in the introduction, cis-acting elements involving at least the AP-1 and PEA3 transcription factor binding sites coordinately regulate viral early and late gene expression and DNA replication. This suggests that the effects of MT and ST are exerted, at least in part, at the level of AP-1 and PEA3/ets activation and that the absence of MT and ST results in a defect in primary transcription. Knowledge of MT and ST signaling pathways support this hypothesis. MT- and ST-dependent activation of ERK1 (and other mitogen-activated protein [MAP] kinases) provides a linkage to the activation of AP-1 and PEA3/ets factors, since the activation of both AP-1 partners c-fos and c-jun family and PEA3/ets members is dependent upon MAP kinases (13, 36, 52, 64). This linkage is better understood in the case of MT signaling. Most MT-dependent functions characterized to date have been linked with its capacity to bind to c-src family tyrosine kinases (11) (Fig. 1). The association leads to constitutive c-src activation and to the phosphorylation of MT tyrosines located in the MT unique C terminus. Biochemical downstream signaling pathways for Y250, Y315, and Y322 have also been characterized, as sketched in Fig. 1 (reviewed in references 16, 26, 34, and 35). pY250 serves as a binding site for the adapter Shc protein, connecting to the activation of the Ras Raf MEK plus ERK pathway (6, 17). The phosphotyrosine motif at Y315 provides a binding site for the 85-kDa subunit of PI3'K, resulting in PI3'K activation (12, 69). ERK1 phosphorylation, sustained activation, and nuclear translocation implicate a role for both Y315 and Y250 (60). Cross talk between the Shc and PI3'K pathways may be responsible for this double requirement (48). As for pY322, it binds PLC1 (56), presumably leading to protein kinase C activation and hence protein kinase C Raf-1 MEK activation (37).
The MT carboxy-terminal tyrosines were examined for their role in the regulation of PYV lytic functions. The tyrosine contributions were difficult to observe in the presence of ST. This may explain why early studies with deletion mutants involving Y315 and Y322 did not detect gene expression defects (27, 53). In contrast, in the complementation assay, each of Y250, Y315, and Y322 could contribute alone to early and late gene viral gene expression and genome amplification, and the signals from each tyrosine were additive. Effects of MT and each MT tyrosine as well as ST always coordinately affected early and late viral gene expression and genome amplification. These results are the first to demonstrate a role for MT tyrosines in viral gene expression and suggest that each tyrosine signals to the PYV enhancer. However, the requirement for Y250 and Y315 in the MT-dependent upregulation of a cellular gene controlled by AP-1 and PEA3, such as urokinase plasminogen activator, has been previously demonstrated (4, 60). These gene induction functions of Y250, Y315, and Y322 are congruent with their known signaling pathways. The functional role of MT tyrosines has been extensively studied previously in relation to neoplastic transformation. These studies revealed a major role for Y250 and lesser or minor roles for Y315 and Y322, respectively (8, 18, 41, 49). While it is likely that the activation of cellular genes controlled by AP-1 and PEA3/ets plays a role in transformation (14), it is interesting that the relative requirement for Y250, Y315, and Y322 in the lytic cycle is very different from that seen in transformation.
Similar to the situation with growth factors, the effect of MT signaling on AP-1 and PEA3 sites is likely to be very complex, involving phosphorylation of c-fos, c-jun, and PEA3/ets/elk proteins by MAP and JNK kinases and transcriptional activation of the c-fos and c-jun (and perhaps PEA3/ets) promoters and, depending upon the intensity of signaling, the Fra-1 promoter as well. Previous work has shown that MT expression leads to the upregulation of the c-jun and c-fos promoters (51, 61), the activation of the elk member of the PEA3 family required for upregulation of the c-fos promoter (61), and the phosphorylation of the c-jun transactivation domain (on Ser63 and Ser73), which depends upon both Y250 and Y315 (55).
Interestingly, the function of MT in upregulating early and late transcription and DNA replication was not eliminated by the mutation of the three major tyrosines. By two independent methods, we found that the annulment of MT activity requires the additional mutation at least one among the three remaining minor tyrosines. Even in the presence of ST, biologically relevant differences between MTY3FM/ST+ and MTY6F/ST+ were observed in virus growth in two cell lines, as described above, as well as in acute and especially in persistent mouse infections (X. Wang and M. M. Fluck, unpublished data). Differences between Y6F and Y3FM MT were also detected in the MT-associated kinase assay (Wang and Fluck, unpublished). Thus, it is likely that at least one among the three minor tyrosines can provide some function in PYV transcription and DNA replication. At least one of them (Y297) is known to be phosphorylated in the MT-associated kinase assay (30). The mechanism of action of this tyrosine(s) remains to be determined. Interestingly, Y258, Y288, and Y297 are embedded in a conserved context (YPXT/S). These sites may serve as binding sites for a novel signaling intermediate, opening the possibility that each of them can carry out a similar function.
The effect of ST on PYV lytic functions suggests that, similarly to MT, ST also signals to the enhancer. Although ST signaling has been less well studied, the link to AP-1 has been established. In the case of PYV, ST-dependent upregulation of c-fos and c-jun transcripts has been reported (25, 45, 72); in the case of the simian virus 40 ST, stimulation of MAP kinases (including MEK, ERK2, and SAPK) was demonstrated along with increased AP-1 activity (20, 32, 54, 68). For ST it is the interaction with PP2A that is important for these functions. Thus, it appears that both ST and MT can signal to AP-1 and PEA3 factors which are crucial for PYV transcription and replication and that these functions are mediated by different pathways, at least partially PP2A dependent in the case of ST and src dependent in the case of MT. Whether the readouts of these pathways on AP-1 and PEA3/ets genes and proteins are partially overlapping and of similar intensity remains to be determined. MT and ST contributions have a graded effect in viral gene expression and DNA replication, with MT plus ST > ST > neither. Preliminary results suggest that the increased effect of MT plus ST compared to ST alone is not simply the result of dosage effect but perhaps a consequence of differences in signaling by these two proteins.
Our kinetic studies demonstrated that the expression of the defective phenotype generated by the absence of MT or of MT and ST is delayed almost to the early-late switch. We assume that this delay reflects the extent of the full establishment of MT and ST signaling pathways. Kinetic studies of the MT-associated kinase activity support this notion. The effects of MT/ST on early gene expression define them as autogenous regulators—regulating their own synthesis—as well as that of other gene products. Furthermore, the lag between the effect on early and late gene expression is likely a consequence of the dependence of late gene expression on the onset of DNA replication.
The present studies highlight the pleiotropic aspects of both MT and ST lytic roles in upregulating both early and late viral gene expression and DNA replication. All three functions were affected by both MT and ST expression and similarly by each MT mutant or mutant combination. This impact of MT/ST is a reflection of the central role of AP-1 and PEA3/ets in the life cycle of PYV, set by the viral bidirectional transcription strategy and the converging position of the two promoters, and the origin of DNA replication.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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L.C. and X.W. contributed equally to this work.
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