Department of Medical Microbiology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands1
Author for correspondence: Frank van Kuppeveld. Fax +31 24 3540216. e-mail f.vankuppeveld{at}ncmls.kun.nl
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Abstract |
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Introduction |
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The 2B protein is a small hydrophobic membrane-active protein that is involved in an early step of vRNA replication (Johnson & Sarnow, 1991 ; van Kuppeveld et al., 1995
, 1996
), but its exact function is as yet unknown. So far, all genetic defects in the 2B protein have been found to be noncomplementable in trans. Poliovirus mutants carrying linker insertions at amino acid positions 28 and 34 of the 2B protein caused primary defects in vRNA replication that could not be trans-complemented by wild-type virus (Bernstein et al., 1986
; Li & Baltimore, 1988
; Johnson & Sarnow, 1991
). Instead, the mutants exhibited a dosage-dependent trans-dominance over wild-type poliovirus (Johnson & Sarnow, 1991
). Noncomplementability of a lesion in the 2B protein was also observed by Collis et al. (1992)
, who studied trans-complementation of subgenomic RNA transcripts carrying in-frame deletions by cotransfecting helper RNA.
Here, we report the first evidence of trans-complementation, albeit with low efficiency, of a replication-defective mutation in the enterovirus 2B protein. The genetic defect was the E[40]K mutation in the putative cationic amphipathic -helix in the 2B protein of coxsackie B3 virus (CVB3) (van Kuppeveld et al., 1996
). The 2B-E[40]K mutation caused a quasi-infectious (qi) phenotype, a definition introduced by V. I. Agol and his colleagues (Gmyl et al., 1993
) to indicate that the mutation disrupts vRNA replication to such an extent that (pseudo)reversion mutations can arise but that no virus progeny can be observed harbouring the original mutation. Remarkably, on one occasion, we observed that RNA transfection gave rise to a virus stock in which the introduced 2B-E[40]K mutation was retained. Here, we show that this is not due to a compensating second-site mutation elsewhere in the RNA genome. Instead, we present evidence that the growth of viruses carrying the 2B-E[40]K mutation was due to trans-complementation of the defective function by viable (pseudo)revertant viruses that had emerged in the transfection-derived virus population.
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Methods |
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Viruses and virus titrations.
All viruses used in this study are recombinant CB3 viruses obtained by transfection of RNA run-off transcripts from plasmid pCB3/T7 (Klump et al., 1990 ), which contains a full-length cDNA of CBV3 strain Nancy behind a T7 RNA polymerase promoter. The recovery of the virus stock containing mutation 2B-E[40]K has been described previously (van Kuppeveld et al., 1996
). Virus yields were determined by endpoint titration as described previously (van Kuppeveld et al., 1995
). Virus titres were calculated and expressed in 50% tissue culture infective dose (TCID50) values (Reed & Muench, 1938
).
Sequence analysis of viral RNA.
RNA was isolated from virus suspensions using guanidium thiocyanatephenolchloroform (Chomczynski & Sacchi, 1987 ). RNA was reverse transcribed into cDNA using Superscript reverse transcriptase (RT) according to the recommendation of the manufacturer (Gibco). Amplification by PCR was performed with SuperTaq DNA polymerase (HT Biotechnology). Dideoxy chain termination sequence analysis was performed according to standard procedures.
For the sequence analysis of the RNA genome of the 2B-E[40]K mutant virus, the following RTPCR products were generated. Nucleotides 1780 were amplified by RTPCR using forward primer 5'TTAAAACAGCCTGTGGGT 3' and reverse primer 5' ATGTGCCCCAGTCTTTTG 3'. Nucleotides 32805295 were amplified by RTPCR using forward primer 5' ACAATGACAAATACGGGCGCA 3' and reverse primer 5' TTGAAAACCCGCAAAGAGCTT 3'. Nucleotides 49337420 were amplified by RTPCR using forward primer 5' CAGGTAAGATACTCTCTAGACATG 3' and reverse primer 5' GGGGGGGTCGACTTTTTTTTTTTTTTTTTTTTC 3'.
For the verification of the 2A-N[14]S mutation, RTPCR was performed with forward primer 5' AACGTGAACTTCCAACCCAGCGGA 3' (nt 3235258) and reverse primer 5' CTGTTCCATTGCATCATCTTC 3' (nt 37243744). Sequence analysis was performed with reverse primer 5' TGTGGTCGTGCTCAATAAGAG 3' (nt 34123432).
For the sequence analysis of the 2B coding region of the 2B-E[40]K and 2B-E[40]T mutant viruses, RTPCR was performed with forward primer 5' TGGTGTCATTGGCATTGTGACCATGGGGGG 3' (nt 36483677) and reverse primer 5' TTGGGATGGCGCGCTCTGCTC 3' (nt 4231 4251). Sequence analysis was performed with reverse primer 5' CCATTCAATGAATTTCTG 3' (nt 41174134).
Site-directed mutagenesis.
In vitro mutagenesis was performed with single-stranded DNA generated from a subgenomic pALTER phagemid construct that contained the HindIII (nt 2080)XbaI (nt 4947) fragment of CBV3, using the Altered Sites in vitro Mutagenesis System according to the recommendations of the manufacturer (Promega). Synthetic oligonucleotides were used to introduce site-specific mutations. The nucleotide sequence of the oligonucleotides were: 5' ATTTACCACCCTGTAGCTCCCTACGTACACTGCCCCTGATTG 3' (mutation 2A-N[14]S); 5' TTTTAGAGATTTCTTTAAAATGGAGTCTTGACC 3' (mutation 2B-E[40]K); 5' GGCTTTTAGAGATTTCGTTAAGATGGAGTCTTG 3' (mutation 2B-E[40]T). The nucleotide sequence of the mutant pALTER clones was verified by sequence analysis. The 2A mutation was introduced into the pCB3/T7 plasmid using the unique PshAI (nt 2803) and SpeI (nt 3837) sites. The 2B mutations were introduced into the pCB3/T7 plasmid using the unique SpeI (nt 3837) and BssHII (nt 4238) sites.
Transfection of cells with RNA transcripts.
Plasmids were linearized with SalI, purified, and transcribed in vitro by T7 RNA polymerase as described previously (van Kuppeveld et al., 1995 ). BGM monolayers cells grown in 25 cm2 flasks to 75% confluency were transfected with 2·5 µg of RNA transcripts using the DEAE-dextran method as described previously (van Kuppeveld et al., 1995
). After transfection, cells were grown at 36 °C. When virus growth was observed, the cultures were incubated until cytopathic effect (CPE) was complete. The cultures were then subjected to three cycles of freezing and thawing and the viruses were aliquoted. If no CPE was observed after 5 days, the cultures were subjected to three cycles of freezing and thawing, and passaged to fresh BGM monolayer cells, which were grown for another 5 days.
Single cycle-growth analysis.
100% confluent BGM monolayer cells were infected with virus at an m.o.i. of 1 TCID50 per cell for 30 min at room temperature. The cells were washed three times with PBS, supplied with medium, and grown at 36 °C. At the indicated times post-infection, cells were disrupted by three cycles of freezing and thawing. Virus titres were determined by endpoint titration.
Plaque assay.
Plaque assays were performed with 100% confluent BGM cell monolayers grown on 10 cm2 dishes in six-well plates. Cells were infected with different virus dilutions for 30 min at room temperature. Cells were washed three times with PBS and overlaid with culture medium containing 1% plaque agarose (Gibco) and 25 mM MgCl2. The cells were grown at 36 °C. After 4 days, individual plaques were picked and inoculated to fresh BGM cell monolayers. These cells were grown at 36 °C until CPE was complete.
Analysis of viral RNA synthesis.
BGM cell monolayers were transfected with 1 µg of T7 RNA polymerase-generated RNA transcripts of SalI-linearized pCB3/T7-LUC plasmids as described above. At the indicated times post-transfection, the cells were lysed and the luciferase production was measured as described previously (van Kuppeveld et al., 1995 ).
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Results |
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Sequence analysis of the viral RNA genome
To search for the possible presence of a second-site suppression mutation outside the 2B coding region, the RNA genome of the mutant virus stock was sequenced. For this purpose, three large RTPCR products were generated. These RTPCR products covered the 5' NTR, the P2 coding region, and the P3 protein coding region plus the 3' UTR, respectively. The P1 capsid coding region was not analysed because it has been shown previously that this region is dispensable for vRNA replication (van Kuppeveld et al., 1995 ), making the presence of suppressing mutations in this region very unlikely.
Sequence analysis confirmed the presence of the lysine-40 residue (AAG) in the 2B protein. Furthermore, the replacement of adenine-3335 with guanine, causing the mutation of the 2A residue asparagine-14 (AAC) into a serine residue (AGC), was noted. This mutation was observed upon sequencing in both directions, and was found consistently in three independently generated RTPCR products. Sequence analysis of wild-type CBV3 and the parental pCB3/T7 plasmid confirmed the presence of adenine-3335, indicating that the observed guanine-3335 is not due to a mistake in the published sequence. We also noted the insertion of a guanine at nt position 33. This mutation, however, was also observed in the wild-type CBV3 and the pCB3/T7 plasmid, indicating an error in the published pCB3/T7 sequence (Klump et al., 1990 ). No other mutations were observed.
No evidence for a second-site mutation that rescues the 2B-E[40]K mutation
To investigate whether the 2A-N[14]S mutation could compensate for the defect caused by mutation 2B-E[40]K, in vitro mutagenesis was performed to construct the single mutants pCB3/T7-2A-N[14]S and pCB3/T7-2B-E[40]K, and the double mutant pCB3/T7-2A-N[14]S/2B-E[40]K. For each mutation, two independently generated clones were constructed. Each clone was transfected in quadruplicate. The outcome of the transfections is summarized in Fig. 2(B). Transfection of BGM cells with RNA transcripts derived from the wild-type pCB3/T7 plasmid and the 2A-N[14]S mutant resulted in complete CPE within 3 days in all eight cultures. Amplification and sequence analysis of the 2A coding region of the obtained virus demonstrated that the introduced mutation was retained in the viral genome. No CPE was observed in cells transfected with RNA from the 2A-N[14]S/2B-E[40]K mutant. Passage of cytoplasmic extracts to fresh BGM cells also failed to reveal virus growth. Upon transfection of cells with RNA from the 2B-E[40]K mutant, CPE was observed in one of the eight cell cultures. Amplification and sequence analysis of the 2B coding region of the obtained virus revealed the reversion of the introduced lysine-40 (AAG) to the original glutamic acid (GAG). This wild-type revertant virus still contained the introduced AUU codon at isoleucine-38 (Fig. 2B
), indicating that this obtained virus is a true revertant and not merely a wild-type virus contamination. No CPE was observed in the remaining seven cultures. Passage of cytoplasmic extracts to fresh BGM cells also failed to reveal virus growth.
These results clearly demonstrate that the N[14]S mutation in the 2A protein does not suppress the defect caused by the 2B-E[40]K mutation. Viruses carrying mutation 2A-N[14]S mutation exhibited wild-type growth characteristics in single-cycle growth experiments (Fig. 3A). The non-deleterious effect of the 2A-N[14]S mutation is in agreement with the occurrence of serine-14 in the 2A protein of some enteroviruses (e.g. enterovirus type 70 and bovine enterovirus type 1). Most likely, the observed 2A-N[14]S mutation is an accidental mutation that arose early in the RNA replication process and that survived the genetic selection pressure because of the wild-type activity of the 2A-N[14]S mutant protein.
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Taken together, these results support the idea that the virus stock contains a small fraction of viable revertant viruses and pseudorevertant viruses.
Only (pseudo)revertant viruses produce plaques
We reasoned that if viruses carrying mutation 2B-E[40]K can only grow in cells coinfected with (pseudo)revertant viruses, then only these latter viruses will be able to produce individual plaques. A plaque assay on BGM cells was performed to test this hypothesis. Plaques of more or less homogeneous size were observed (Fig. 5A). Five individual plaques were picked from the plates and the viruses were grown on BGM cells. Sequence analysis revealed that three isolates contained the original glutamic acid-40 (GAG). One isolate was found to contain threonine-40 (ACG). The fifth isolate revealed the occurrence of asparagine-40 (AAU), another pseudoreversion mutation. All (pseudo)revertant viruses contained the introduced AUU codon at isoleucine-38 (Fig. 2B
), confirming that these viruses had originated from the 2B-E[40]K mutant (Fig. 5B
).
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Low level of trans-complementation of the 2B-E[40]K mutation by wild-type virus
To obtain further evidence that the defect caused by mutation 2B-E[40]K could be rescued in trans, a genetic complementation assay was performed. For this, the 2B-E[40]K mutation was introduced in the plasmid pCB3/T7-LUC (Fig. 6A), a subgenomic replicon that contains the luciferase gene in place of the P1 capsid coding region (van Kuppeveld et al., 1995
). First, the effects of the 2B-E[40]K mutation on viral plus-strand RNA replication was studied. BGM cells were transfected with RNA transcripts of the wild-type and mutant pCB3/T7-LUC constructs. Fig. 6(B)
shows that the 2B-E[40]K mutation did not affect the initial increase in luciferase activity (between 1 and 4 h post-transfection) that reflects translation of the input RNA, but that the mutation interfered with the second increase in luciferase activity, which occurs from the fifth hour and reflects the replication of the input RNA and subsequent translation of the newly synthesized RNA strands (van Kuppeveld et al., 1995
).
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To exclude the possibility that the increase in the luciferase level upon CVB3 infection was due to trans-activation of the CVB3 IRES in the pCB3/T7-LUC-2B-E[40]K replicon by the superinfecting virus or to increased translational efficiency due to the viral infection, we also tested two mutant pCB3/T7-LUC replicons harbouring coding region deletions that rendered the viral infectious RNA nonviable. One of these replicons contains a 2B protein with an in-frame deletion of the amphipathic -helix (aa 3754). The other replicon, pCB3/T7-LUC-
3D, contains an almost complete in-frame deletion of the 3D polymerase (van Kuppeveld et al., 1995
). BGM cells were transfected with RNA transcripts from pCB3/T7-LUC-2B-E[40]K mutation, pCB3/T7-LUC-2B
3754, or pCB3/T7-LUC-
3D and either mock-infected or infected with wild-type CBV3 at 2 h post-transfection. Luciferase activities were assayed at 10 h post-transfection (i.e. at 8 h post-infection). Again, the luciferase production by replicon pCB3/T7-LUC-2B-E[40]K was increased in the virus-infected cells. No increases in luciferase production were observed upon infection of cells transfected with replicon RNAs carrying either the 2B
3754 or the
3D deletion (Fig. 6D
). These data suggest that the observed increase in luciferase production by replicon pCB3/T7-2B-E[40]K upon CBV3 infection truly reflects a low level of trans-complementation.
We reasoned that if trans-complementation of the defect in the 2B-E[40]K protein is rather inefficient, it will be unlikely that viruses carrying the 2B-E[40]K mutation will be stably maintained in the virus population upon several passages. To investigate this, the virus stock was passaged three times on BGM cells. The 2B coding region was amplified by RTPCR and cut with DraI. Fig. 7 shows that viruses carrying the 2B-E[40]K mutation were gradually deleted from the virus population upon serial passage. After one passage, the amount of viruses carrying the mutation 2B-E[40]K mutation was already decreased. After the second passage, only a very small proportion of the virus population contained the 2B-E[40]K mutation. The RTPCR product obtained after the third passage was completely resistant to DraI cleavage, indicating that viruses carrying the 2B-E[40]K mutation were deleted from the virus population. Sequence analysis of the RTPCR product obtained after the third passage showed the presence of the original glutamic acid-40 (GAG).
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Discussion |
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Genetic complementation of mutants mapping to the 2B protein has not been described before. The defects in vRNA synthesis caused by linker insertions at amino acid positions 28 and 34 of the poliovirus 2B protein were found to be noncomplementable (Bernstein et al., 1986 ; Li & Baltimore, 1988
; Johnson & Sarnow, 1991
). Mutations in a hydrophobic domain (aa 6380) of the CBV3 protein 2B were also found to be cis-dominant (van Kuppeveld et al., 1995
). It should be emphasized that the level of trans-complementation observed in this study was very low. Such a low level of trans-complementation may also be considered as inefficient and interpreted as evidence for a preference for the protein to function in cis. Notwithstanding this fact, the low level of trans-complementation was relevant in supporting the growth of viruses carrying the 2B-E[40]K mutation.
Efficient trans-complementation of genetic defects in the nonstructural proteins has thus far been observed only with mutations that render the RNA genome replication competent, albeit in a defective or conditional-lethal manner. Efficient trans-complementation of non-self-replicating genomes is rare. The only direct example of trans-complementation of a lethal mutation has been reported by Cao & Wimmer (1995) , who were able to rescue a lethal mutation in the poliovirus 3AB protein using an intragenomic complementation procedure. This intragenomic complementation was very inefficient and the intragenomic recombinants displayed only a qi phenotype. Teterina et al. (1995)
examined rescue of lethal 2C mutations in poliovirus and observed up to a tenfold increase of synthesis of the mutated RNA upon cotransfection with wild-type helper RNA. However, the maximal levels of the mutated RNA represented still only a fraction of the level of the wild-type virus RNA. Therefore, they concluded that trans-complementation did occur, but was very inefficient, and that the RNA shows a marked preference for the 2C protein provided in cis. Giachetti et al. (1992)
observed low levels of trans-complementation of nonviable mutations targeted to the hydrophobic domain of the poliovirus 3A protein and concluded that the 3A protein cannot be provided in trans. Unfortunately, in these latter two studies it was not examined whether this low level of trans-complementation was sufficient to enable the emergence of mutant viruses. In this study, we observed that even a low level of trans-complementation of a qi mutation in the 2B protein was sufficient to enable virus growth. Taken together, these observations indicate that although some functions of the enterovirus 2B, 2C (or their precursor 2BC) and 3A proteins exhibit a clear cis-preference, these functions are not absolutely cis-dominant.
The identification of both cis-acting and trans-acting functions of the enterovirus 2B protein most likely reflects the multifunctional nature of this protein (or its precursor 2BC). The identity of these cis-acting and trans-acting functions remains to be established. It is as yet unclear whether the 2B-E[40]K mutation exerts its effect at the level of the precursor 2BC or at the level of the mature cleavage product 2B. The qi phenotype of this mutation and the emergence of (pseudo)revertant viruses argue that this mutation does not abolish the ability of the 2BC protein to induce the proliferation of the membrane vesicles that build the virus replication complex. At present, the function of the 2B protein in the enteroviral life-cycle is unknown. Individual expression of the 2B protein results in alterations in host cell membrane permeability (Doedens & Kirkegaard, 1995 ; Aldabe et al., 1996
; van Kuppeveld et al., 1997a
, b
), the disassembly of the Golgi complex (Sandoval & Carrasco, 1997
) and, possibly as a consequence, the inhibition of protein secretion (Doedens & Kirkegaard, 1995
; van Kuppeveld et al., 1997b
). Through the individual expression of mutant 2B proteins, we found that the activities of protein 2B to modify plasma membrane permeability and to inhibit protein secretion may represent two different functions, rather than that one effect is the consequence of the other (van Kuppeveld et al., 1997b
). Individual expression of the 2B-E[40]K protein revealed that this protein exhibited a wild-type activity in permeabilizing the plasma membrane to hygromycin B, but a reduced activity (about 60% relative to the wild-type 2B protein) in inhibiting protein secretion (F. J. M. van Kuppeveld, W. J. G. Melchers, K. Kirkegaard & J. R. Doedens, unpublished data). The glutamic acid-40 residue is located in the hydrophilic part of a predicted cationic amphipathic
-helix, a well-conserved structural element in the enterovirus 2B protein (van Kuppeveld et al., 1996
). In all enterovirus 2B proteins, this hydrophilic part is formed by three positively charged residues (most often lysines), one glutamic acid residue and a number of polar residues. Cationic amphipathic
-helical peptides have been implicated in the permeabilization and destabilization of membranes (Bernheimer & Rudy, 1986
; Segrest et al., 1990
). Previously, we demonstrated that both the cationic character and the amphipathic character of the
-helix are required for the membrane permeabilization function as well as for the secretion inhibition function of the 2B protein (van Kuppeveld et al., 1997b
). That the E[40]K mutation does not disrupt the membrane permeabilizing function of the 2B protein is not surprising, because this mutation disrupts neither the cationic nature nor the amphipathy of the
-helical domain. The finding that the E[40]K mutation specifically interfered with the ability of the 2B protein to inhibit protein secretion is remarkable and points to an important role of the aa-40 residue in this function. It is tempting to speculate that the secretion inhibition function represents the trans-acting function of the 2B protein. However, this suggestion must be taken with care, as it cannot be excluded that another, yet unidentified, function, which may represent the trans-acting function, is affected by the E[40]K mutation as well.
In summary, we have provided the first evidence for trans-complementation of a genetic defect in the enterovirus 2B protein. The identity of the trans-acting function of the 2B protein may be the secretion inhibition function, but this awaits further investigation. Understanding the cis-acting and trans-acting functions of the 2B protein requires the elucidation of how the activities of the 2B protein to modify membrane permeability and to manipulate the protein secretion machinery contribute to the process of vRNA replication and/or other steps in the virus life-cycle.
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Acknowledgments |
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Received 19 July 2001;
accepted 22 October 2001.