Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK1
Author for correspondence: Nigel Dimmock. Fax +44 24 76523568. e-mail ndimmock{at}bio.warwick.ac.uk
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Abstract |
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Introduction |
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Defective RNAs have been isolated from all eight segments, but those from segments 1 and 2 (respectively encoding proteins PB2 and PB1) are found in the greatest abundance (Davis & Nayak, 1979 ; Davis et al., 1980
; Duhaut & Dimmock, 1998
; Jennings et al., 1983
). Studies have shown that all the information necessary for replication, transcription and packaging is present within the terminal 125 nt from the 5' and 3' ends (Luytjes et al., 1989
). However, almost all defective RNAs analysed to date possess considerably more sequence than this. Jennings et al. (1983)
isolated a number of defective RNAs, largely from segment 1, from egg-grown PR8 virus and found that all possessed at least 8090 nt from each terminus and most had in the region of 200 nt. In a more recent study of 50 segment 1, 2 and 3 defective RNAs isolated from WSN- or EQV-infected eggs or mouse lung tissue, almost all were found to possess at least 178 nt from the 5' end of the virion (v) RNA (Duhaut & Dimmock, 1998
). The length of the 3' end varied much more, and one segment 2 defective RNA possessed just 25 nt from the 3' end.
At least some defective RNAs are able to interfere with the multiplication of infectious virus (Nayak et al., 1978 , 1985
; von Magnus, 1954
). Defective interfering RNAs have also been shown to protect from both homologous and heterologous influenza A virus subtypes in vivo (Dimmock, 1996
; Dimmock et al., 1986
; McLain et al., 1992
; Morgan & Dimmock, 1992
; Morgan et al., 1993
). However, to date it has not been possible to study the mechanism of this interference in detail or determine at what stage in the virus life cycle the interference occurs.
The advent of a plasmid-based transfection system (Pleschka et al., 1996 ) has enabled more detailed study of the requirements for the propagation of defective RNAs from cell culture to cell culture in the form of defective virus particles. We isolated a defective RNA from influenza virus-infected mouse lung, cloned it into a suitable vector and constructed further clones that possessed less of the 5' end and more of the 3' end, such that all were of the same overall size and segment of origin. On transfection and subsequent passage, those defective RNAs that possessed at least 150 nt from the 5' end proved stable on passage in two different cell lines and with three different subtypes of helper virus (Duhaut & Dimmock, 2000
). Those with less 5' sequence were unstable, and survived only a few passages. More recent technical developments have permitted the construction of infectious virus entirely from cloned RNAs (Fodor et al., 1999
; Neumann et al., 1999
), and these have enabled us to analyse interference by plasmid-encoded defective RNAs and hence to take these studies further.
Here, using an entirely plasmid-based system, we have investigated the 5'-sequence requirements for interference by a panel of defective segment 1 RNAs. We demonstrate that increasing amounts of transfected defective DNA reduced virus infectivity titres progressively and that the behaviour of the cloned defective virus was similar to that of conventionally produced, authentic defective virus. Interference required a critical length of 5'-end sequence in the defective RNA, which we estimated to be between 150 and 220 nt. A mechanism of interference was suggested by the observed reduction in the amount of full-length segment 1 RNA in released progeny virions, coupled with the appearance of defective segment 1 RNA in the same preparation. In all, these data demonstrate the importance of 5'-end internal sequences of vRNA to the interference phenomenon, and the means to manipulate it.
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Methods |
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Plasmids.
The 17 plasmids required for the generation of infectious WSN were kindly donated by Y. Kawaoka (Neumann et al., 1999 ). The construction of the EQV (A/equine/Newmarket/7339/79; H3N8) defective RNAs has been described previously (Duhaut & Dimmock, 2000
). Briefly, a defective 445 nt RNA, isolated from infected mouse lungs, was cloned into a POL I vector between a truncated human polymerase I promoter and a hepatitis delta virus ribozyme (Pleschka et al., 1996
). This comprised 225 nt of 3' sequence and 220 nt of 5' sequence of vRNA (POLI-220). Three further defective RNAs were derived from this with 295 nt of 3' sequence and 150 of 5' sequence (POLI-150), 365 nt of 3' sequence and 80 nt of 5' sequence (POLI-80) and 415 nt of 3' sequence and 30 nt of 5' sequence (POLI-30) (Fig. 1
). A second EQV defective RNA of 436 nt (POLI-d136) was also isolated from infected mouse lung and cloned as before (Duhaut & Dimmock, 2000
). This is identical to POLI-220 except for an additional 9 nt deletion at positions 136144 from the 5' end. A defective segment 1 RNA of 585 nt was isolated from cells infected with A/chicken/Dobson/27; H7N7) and cloned as before (POLI-317). This is identical to RNA 1.2 (Duhaut & McCauley, 1996
).
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RNA extraction.
RNA was extracted from infected cells and from clarified, virus-containing tissue culture fluids by using the hot-phenol method (Duhaut & McCauley, 1996 ). RNA concentrations were estimated spectrophotometrically and adjusted to 1 µg/µl.
RTPCR.
Forward primers were complementary to the first 25 nt of the 3' end of all eight WSN RNA segments. We reverse-transcribed 0·5 µg RNA, extracted from transfected dishes at 72 h, in 20 µl reaction buffer at 37 °C for 60 min. Reaction buffer was 50 mM TrisHCl (pH 8·3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 1 mM each dNTP, 10 U ribonuclease inhibitor (Amersham Life Sciences), 13 U Moloney murine leukaemia virus reverse transcriptase (Gibco BRL) and 200 pmol complementary primer. Second-strand synthesis used primers complementary to the first 25 nt of the 5' end of all eight segments of WSN RNA. PCR was carried out in 20 mM TrisHCl (pH 8·4), 50 mM KCl, 2 mM MgCl2, 0·2 mM each dNTP, 2·5 U Taq DNA polymerase (Gibco BRL), 20 pmol primer and 20 µl first-strand synthesis reaction product. Reactions were performed in a Hybaid touchdown PCR block for one cycle of 94 °C for 120 s, 30 cycles of 94 °C for 60 s, 50 °C for 60 s and 72 °C for 120 s and a final cycle of 72 °C for 8 min. PCR products were separated on a 1·5% agarose gel.
Northern blotting.
Cell RNA (2 µg) or all the RNA extracted from the medium of one 3 cm diameter dish of Vero cells was electrophoresed through agarose. Northern blotting was carried out as described previously (Duhaut & McCauley, 1996 ) except that run-off riboprobe transcripts (Promega Transcription Protocols) were labelled using digoxigeninUTP. Hybridization probes were nt 1225 complementary to the 3' end of WSN vRNA segments 1 and 5. These were used in excess as determined empirically on filters with known amounts of bound vRNA. Filters were prehybridized for 2 h at 65 °C in 50% formamide, 1% blocking reagent (Roche Diagnostics), 5x SSC, 45 mM sodium phosphate (pH 5) and 0·3 mg/ml calf intestinal DNA. The RNA probe was boiled with calf intestinal DNA for 5 min and cooled on ice prior to incubation overnight in hybridization buffer, constituted as above, except with 2% blocking reagent and 0·07 mg/ml boiled calf intestinal DNA. Conditions were optimized for blotting of segment 1 RNAs from H1, H3 and H7 viruses so that binding of the probe was equalized. Filters were then washed at room temperature and 68 °C (Duhaut & McCauley, 1996
) and reacted according to the manufacturers instructions (Roche DIG luminescent detection kit) except that the amount of blocking reagent was increased to 2·5% and three washes of 20 min each were used. Filters were autoradiographed with Fuji X-ray film for 30 min and bands were quantified by densitometry.
Statistical analysis.
Interference data were expressed graphically and analysed to determine whether plots deviated from linearity (the runs test) and whether they deviated from the horizontal (the F-test) (Sokal & James, 1995 ).
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Results |
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Defective influenza virus RNA expressed from pPOLI-317 DNA interferes with the production of infectious WSN
Vero cell monolayers were transfected with all 17 infectious WSN plasmids and increasing amounts (0·12 µg) of pPOLI-317 plasmid DNA. Samples of medium were removed at 24, 48 and 72 h post-transfection for plaque assay. Fig. 2 shows that 2 µg pPOLI-317 DNA caused a drop in yield of infectious virus of over 90% at 24 and 48 h post-transfection. However, little reduction in infectivity titre was seen at 72 h post-transfection. Statistical analysis using an F-test (Sokal & James, 1995
) on log-transformed data showed that the departure of the slopes from zero at 24 and 48 h (i.e. the influence of the defective RNAs on virus titres, as opposed to purely random effects) was significant (P=0·0001) (Table 1
). A runs test (Sokal & James, 1995
) on log-transformed data determined that the 24 and 48 h plots did not deviate significantly from linearity (Table 1
). At 72 h, while the F-test showed that defective RNA was not having a significant effect on titres at this time-point, the runs test showed that the resulting line was linear. The combined data from six separate experiments and two statistical analyses mean that it is unlikely that the data resulted from any random transfection or toxic effects. We conclude that RNA expressed from transfected pPOLI-317 (from an H7N7 avian virus) can interfere with the production of cloned infectious human H1N1 WSN, in a concentration-dependent manner.
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The second defective RNA, encoded by POLI-d136, was isolated from EQV-infected mice (Duhaut & Dimmock, 1998 ). It is identical in sequence to the RNA of POLI-220 and has the same central deletion but differs by virtue of a second, 9 nt deletion (residues 136144) in the 5' sequence and hence has a total of 211 nt of 5' end sequence (Fig. 1
). Cells were transfected with pPOLI-d136 and infectious WSN plasmids. The result presented in Fig. 4
(datum points indicated by asterisks) shows that, at 24 h, POLI-d136 RNA clearly inhibited production of infectious virus, but it did so by an amount equivalent to an RNA with slightly less than 136 nt 5' sequence, rather than an RNA with 211 nt 5' sequence. At 48 and 72 h post-transfection, POLI-d136 RNA interfered as if it had a 5' sequence of 136 nt, and hence was plotted in that position on the x-axis in Fig. 4
. It therefore appears that the 9 nt deletion disrupts and reduces the interfering activity of POLI-d136 RNA, and it may be that interference requires an RNA with a particular 5' region secondary structure rather than a certain length of 5' sequence. All three plots were significant by the F-test and all the plots were linear by the runs test (Table 1
). Northern blot analysis showed that POLI-d136 and POLI-317 defective RNAs were both clearly present in WSN-transfected Vero cells at 72 h post-transfection (Fig. 5b
).
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Discussion |
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In this report, we have shown that transfected POLI-317 DNA gave rise to a defective RNA that was replicated stably in the presence of transfected virus (Fig. 3) and strongly inhibited (by nearly 99%) the production of infectious virus (Fig. 2
). It also inhibited the incorporation of full-length segment 1 into virions (Fig. 3
). This repeats earlier work using conventionally produced infectious virus (Duhaut, 1992
; Duhaut & McCauley, 1996
) and demonstrates that defective RNA derived from the plasmid system behaved authentically.
We investigated the importance of the length of 5'-end sequence to interfering activity by using constructs that encoded a panel of four defective EQV RNAs, all segment 1 and all the same length, but varying in the length of their 5'-end sequence. Data showed that RNAs with 150 or 220 nt of 5' sequence (POLI-150 and POLI-220) interfered with infectious virus production, while those with 30 or 80 nt of 5' sequence (POLI-30 and POLI-80) did not (Fig. 4). Thus, interference required at least 150 nt of 5'-end sequence. Northern blotting of RNA extracted from cultures at 72 h post-transfection detected POLI-150 and POLI-220 RNAs but not POLI-30 and POLI-80 RNAs (Fig. 5a
). Interference is thus consistent with the physical presence of defective RNA, and both properties are apparently dependent on the possession of a critical length of internal 5' sequence. Defective POLI-317 RNA is longer than POLI-220 RNA and has more 5'-end sequence, but it did not interfere significantly more with the production of infectious virus (Fig. 4
). This may be because POLI-220 carries sufficient 5' sequence for interference, and interference is not improved by the presence of additional 5' sequence. These and other data presented above also demonstrate cross-subtype interference, with defective RNAs from an avian H7N7 virus (POLI-317) and from an equine H3N8 virus (POLI-d136, POLI-150 and POLI220) interfering well with a human H1N1 virus.
It is self-evident that a defective RNA that is unstable in infected cells will not interfere significantly. However, detection of a less than optimally stable defective RNA depends on the sensitivity of the technique used. Thus, RTPCR with specific primers detected POLI-80 RNA on passage, albeit faintly and intermittently (Duhaut & Dimmock, 2000 ), while Northern blotting failed to detect it at all (Fig. 5
). Interference with infectious virus production is therefore probably the best measure of the length of the 5' sequence required for the stable expression of defective RNAs.
It is an absolute requirement for interference by defective RNA firstly that it is replicated in the transfected cell and secondly that it is packaged into progeny virions. It begins to look as if the 150 nt of 5' sequence, which is needed for interference and RNA passage stability (Duhaut & Dimmock, 2000
), is also implicated in the packaging of segment 1 defective RNA (Fig. 3
) and by implication in the packaging of full-length segment 1 RNA. However, earlier studies, using RNA that proved unstable on passage, concluded that RNA-packaging signals reside in the first 25 nt of each segment (Luytjes et al., 1989
; Odagiri & Tashiro, 1997
). We also have found that packaging of unstable defective RNAs does occur (Duhaut & Dimmock, 1998
) and we conclude that the earlier data are consistent with the findings reported here. The work of Odagiri & Tashiro (1997)
was limited by the difficulty of studying the role of internal sequences with the technology available at the time. This was also true of other studies, which suggested that the amounts of genomic segments could be reduced by the presence of defective RNAs (Akkina et al., 1984
; Duhaut & McCauley, 1996
; Ueda et al., 1980
). Further work, using plasmid-based systems and more quantification, will establish the relationship between 5'-end sequence and its function(s).
Defective POLI-d136 RNA has the same central deletion as POLI-220 RNA and is identical in sequence except for a second deletion, of 9 nt, situated 136 nt from the 5' end, and was replicated in cells (Fig. 5) and interfered with production of infectious virus (Fig. 4
). However, the level of interference was less than that given by POLI-150 RNA (150 nt of 5'-end sequence) and was consistent with that expected from an RNA with a 5'-end sequence of approximately 136 nt rather than the 211 nt that it possesses. This, together with the stepped nature of the interference response at 24 h derived with the POLI-30, -80, -150 and -220 defective RNAs (Fig. 4
), suggests that the interference may not be determined simply by the total length of 5' sequence present, but rather may involve other properties of the RNA such as secondary structure. However, the 24 h plots of the POLI-30, -80, -150 and -220 defective RNA data or data from all defective RNAs (Fig. 4
) were not significantly non-linear (Table 1
). Thus, there remains some doubt as to their interpretation and more defective RNAs need to be analysed to determine whether secondary structure is indeed important for their interference activity.
Statistical analysis by the F-test of interference data from Figs 2 and 4
indicated that the probability that the defective RNAs had no effect on virus infectivity titres at 24 and 48 h or that effect was random was 0·0001% (Table 1
). This is highly significant and provides strong evidence of the involvement of internal 5' sequences during the virus life cycle. However, there was less interference at 72 h post-transfection, and in the data shown in Fig. 2
this was not significantly different from the virus control that received no transfected defective DNA. We believe this arises because of the successive rounds of infection by infectious and defective progeny virions that take place over 72 h. Initially, a defective RNA is replicated and interferes with infectious virus production if it enters the same cell as infectious virus. As there was strongest interference at early times post-transfection, we conclude that, at a relatively high initial input of defective RNA, the majority of virus was replicating in cells that also contained defective RNA. Thus, little infectious virus was made and released for subsequent rounds of infection. As a result, the number of p.f.u. per cell dropped below 1 and propagation of the defective RNA inevitably followed suit (Palma & Huang, 1974
). This allows infectivity to escape from interference, so that yields of infectivity at 72 h are near maximal across the board.
The runs test, used to determine whether the plots were significantly non-linear, showed that all plots in Figs 2 and 4
were significantly linear with, in some cases, a 9899% chance that they were linear (Table 1
). More data are needed to determine the point at which the plot deviates from linearity, i.e. where no more interference is taking place. This will define the minimum length of 5' sequence required. Our studies have concentrated on the 5' end of vRNA, because no defective RNAs lacking internal sequences from the 5' end have been found. Sequences at the 3' end are more diverse, and can be short one apparently stable segment 2 defective RNA possesses only 25 nt from the 3' end (Duhaut & Dimmock, 1998
). Work to address the significance of the 3'-end sequence is currently ongoing.
Previous studies have identified influenza virus promoter sequences within nt 1 to 15/16 of each segment (Flick & Hobom, 1999 ; Flick et al., 1996
; Fodor et al., 1998
; Kim et al., 1997
). In addition, replication and polyadenylation signals have been identified within nt 1 to 2530 (Luo et al., 1991
; Poon et al., 1998
; Pritlove et al., 1999
; Zheng et al., 1996
). The data presented here take our knowledge further and suggest that
150 nt from the 5' end of defective segment 1 RNAs are essential for interference as well as passage stability (Duhaut & Dimmock, 2000
) and possibly packaging. It remains to be determined how much 5' sequence of virion segments 28 is needed. Our work has implications, too, for understanding reassortment of RNA segments between different virus subtypes, since incompatibilities between 5' sequences may affect the ability of segments to be propagated and hence the formation of recombinants. It is also likely that influenza virus expression systems function most efficiently when RNAs possess enough 5' sequence. In all, the appreciation that
150 nt from the 5' end of segment 1 RNA are essential for various aspects of the virus life cycle other than protein coding represents an important advance in our understanding and provides new insights into the biology of influenza A viruses.
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Acknowledgments |
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References |
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Received 5 September 2001;
accepted 31 October 2001.
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