Reverse genetics system for Uukuniemi virus (Bunyaviridae): RNA polymerase I-catalyzed expression of chimeric viral RNAs - PubMed (original) (raw)
Reverse genetics system for Uukuniemi virus (Bunyaviridae): RNA polymerase I-catalyzed expression of chimeric viral RNAs
R Flick et al. J Virol. 2001 Feb.
Abstract
We describe here the development of a reverse genetics system for the phlebovirus Uukuniemi virus, a member of the Bunyaviridae family, by using RNA polymerase I (pol I)-mediated transcription. Complementary DNAs containing the coding sequence for either chloramphenicol acetyltransferase (CAT) or green fluorescent protein (GFP) (both in antisense orientation) were flanked by the 5'- and 3'-terminal untranslated regions of the Uukuniemi virus sense or complementary RNA derived from the medium-sized (M) RNA segment. This chimeric cDNA (pol I expression cassette) was cloned between the murine pol I promoter and terminator and the plasmid transfected into BHK-21 cells. When such cells were either superinfected with Uukuniemi virus or cotransfected with expression plasmids encoding the L (RNA polymerase), N (nucleoprotein), and NSs (nonstructural protein) viral proteins, strong CAT activity or GFP expression was observed. CAT activity was consistently stronger in cells expressing L plus N than following superinfection. No activity was seen without superinfection, nor was activity detected when either the L or N expression plasmid was omitted. Omitting NSs expression had no effect on CAT activity or GFP expression, indicating that this protein is not needed for viral RNA replication or transcription. CAT activity could be serially passaged to fresh cultures by transferring medium from CAT-expressing cells, indicating that recombinant virus containing the reporter construct had been produced. In summary, we demonstrate that the RNA pol I system, originally developed for influenza virus, which replicates in the nucleus, has strong potential for the development of an efficient reverse genetics system also for Bunyaviridae members, which replicate in the cytoplasm.
Figures
FIG. 1
Schematic diagram of the cloning strategy for constructing chimeric UUK virus reporter plasmids. Two PCR fragments, one containing the murine pol I promoter and the UUK M vRNA 5′ UTR (RF10/4B) and the other containing the CAT (RF5/6) or GFP (RF7B/8) ORF, were ligated with the large _Apa_I-_Nco_I fragment from plasmid pRF7 containing the UUK M vRNA 3′ UTR and the pol I terminator. This gave plasmids pRF33 (UUK M-CAT) and pRF31 (UUK M-GFP). Plasmid pRF20 was constructed by inserting multiple cloning sites immediately downstream of the UUK M vRNA 5′ UTR, using the two PCR fragments RF10/30 and RF5/6, respectively (see also Materials and Methods).
FIG. 2
Schematic diagram of the RNA polymerase I transcription plasmid (pRF42) and the generation of chimeric UUK virus-reporter RNA segments. To construct reporter plasmids, PCR-amplified expression cassettes are inserted between the _Bbs_I sites (shaded boxes) in the RNA pol I-driven expression plasmid pRF42. The cassettes are flanked by the murine RNA pol I promoter (pM1) and terminator (tM1) (shown in bold). The system allows for the transcription of any expression cassette by RNA pol I in rodent cell lines. The transcript starts exactly at the first position of the expression cassette and terminates at the last position before tM1, generating the correct 5′ and 3′ termini of the insert. _Bbs_I recognition sites are shaded; primers I and II used to amplify the expression cassette are shown in bold; UUK virus-specific 5′ and 3′ sequences are in italics. TAC, complementary to the ATG initiation codon in the reporter cDNA, is underlined.
FIG. 3
CAT activity in BHK-21 cells transfected with pRF33 or pRF19 expressing UUK M-CAT RNA chimeras is dependent on UUK virus proteins. BHK-21 cells were transfected with plasmids pRF33, producing vRNA sense transcripts (lanes 1 to 4), or pRF19, producing cRNA sense transcripts (lanes 5 to 8). Viral helper proteins were supplied either by cotransfection with plasmids expressing the L (pCMV-UUK-L), N (pCMV-UUK-N), and NSs (pCMV-UUK-NSs) proteins (lanes 3 and 7), by superinfection with UUK virus 24 h posttransfection (lanes 2 and 6), or by the combination of superinfection and protein expression (lanes 4 and 8). CAT activity was assayed 20 h postinfection (lanes 2 and 6), 20 h posttransfection (lanes 3 and 7), or 44 h posttransfection (lanes 4 and 8). As shown by transfection with the reporter plasmids alone (lanes 1 and 5), CAT activity was found to be completely dependent on viral protein expression. CAT activity measured from each lysate is expressed as percentage of the activity obtained from superinfected cells, which was arbitrarily set at 100 (lane 2).
FIG. 4
Immunofluorescence analysis showing that expression of GFP from pRF31 is dependent on UUK virus proteins. (A) BHK-21 cells transfected with only pHL2823 encoding GFP under the control of the CMV promoter; (B) same cells as in panel A, viewed by regular light microscopy; (C) cells cotransfected with the reporter plasmid pRF31 (UUK M-GFP vRNA) and the expression plasmids pCMV-UUK-L and pCMV-UUK-N; (D) same cells as in panel C, viewed by light microscopy; (E) cells transfected with pRF31 alone, with GFP expression visualized 48 h later by fluorescence microscopy; (F) same cells as in panel E, viewed by light miccropscopy.
FIG. 5
Optimization of reporter gene expression by titration of UUK L, N, and NSs expression plasmids. (A) Titration of pCMV-UUK-NSs. BHK-21 cells (3 × 106) were cotransfected with constant amounts of the reporter plasmid pRF33 (UUK M-CAT vRNA) (1 μg), expression plasmids pCMV-UUK-L and pCMV-UUK-N in a molar ratio of 2:1, and various amounts of pCMV-UUK-NSs as indicated. A sample corresponding to 1/50 of the cell lysate prepared at 20 h posttransfection was used for CAT reactions. In the control experiment (leftmost lane), BHK-21 cells were transfected only with pRF33 (1 μg). (B) Titration of pCMV-UUK-N. BHK-21 cells were transfected with pRF33 (1 μg) and a constant amount of pCMV-UUK-L (2.5 μg) together with various molar amounts of pCMV-UUK-N as indicated. CAT activity was determined as for panel A. (C) Titration of pCMV-UUK-L. BHK-21 cells were transfected with pRF33 (1 μg) and a constant amount of pCMV-UUK-N (0.3 μg) together with various molar amounts of CMV-UUK-L as indicated. To achieve equal transfection conditions in each experiment, the total transfected DNA was adjusted to the same level by adding plasmid pHL2823 (CMV-GFP).
FIG. 6
Optimization of time intervals between transfection with reporter plasmid and viral protein-expressing plasmids, and analysis of CAT activity. (A) Schematic diagram depicting the timetable for transfection with the RNA pol I expression cassette plasmid (pRF33) and the viral expression plasmids (pCMV-UUK-L and pCMV-UUK-N) (solid arrows), as well as the time points for assaying CAT activity (broken arrows). Plasmids were either cotransfected (I) or transfected in either order separated by 6 h (II and III). CAT activity was assayed at indicated times after the last transfection (set as time zero). (B to D) Analysis of CAT activity from the transfection protocols shown in panel A. To be able to determine minor differences in expression levels, cell lysates were diluted 1:50. The number of the experiment (I to III) and time points (hours) when cell lysates were harvested are shown above the CAT signals.
FIG. 7
Optimization of time intervals between plasmid transfections, superinfection with UUK virus, and analysis of CAT activity. (A) Schematic diagram depicting the timetable for (i) cotransfection with the RNA pol I expression cassette plasmid (pRF33) and the two plasmids expressing L and N, (ii) superinfection with UUK virus, and (iii) CAT assay. Plasmids were cotransfected either 32 (I), 24 (II), or 8 (III) h before superinfection (set as time zero). (B to D) Analysis of CAT activity from the transfection/superinfection protocols shown in panel A. To be able to determine minor differences in expression levels, cell lysates were diluted 1:50. The number of the experiment (I to III) and time points when cell lysates were harvested are shown above the CAT signals. The time period between transfection and CAT assay is shown in brackets.
FIG. 8
The CTE from MPMV RNA does not increase CAT activity expressed from reporter plasmids. (A) Schematic diagram showing the insertion of one of two forms (MPMV 566 or MPMV 250) of the CTE (in either sense or antisense orientation) between the 5′ UTR of the UUK M vRNA and the CAT ORF. (B) CAT activity in lysates from BHK-21 cells cotransfected with the CTE-containing reporter plasmids and the UUK L and N expression plasmids. Lane 1, CAT activity in cells transfected without viral expression plasmids. CAT activity in cells cotransfected with pRF33 (CAT-M vRNA) and UUK L and N expression plasmids was arbitrarily set at 100 (lane 2). Plasmid pRF20, containing an additional multiple cloning site downstream of the UUK 5′ UTR, displayed the same CAT activity as the parental pRF33 plasmid. CAT activity measured from the other lysates is expressed as percentage of that obtained in this control.
FIG. 9
Analysis of CAT activity in BHK-21 cells after serial passages of supernatants containing recombinant UUK virus. BHK-21 cells were cotransfected either with pRF33 and UUK L and N expression plasmids (lane 1) or only with pRF33 (lane 4) 24 h prior to superinfection with UUK virus (MOI of 10). Cell lysates were prepared 30 h later and assayed for CAT activity, while the media were collected and undiluted samples were transferred to new BHK-21 cells. This was repeated for another cycle, and CAT activity was assayed after each passage. CAT activities are expressed as percentage of the activity obtained after the first transfection/superinfection (lane 1).
References
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