Department of Molecular Virology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany
Correspondence
Wolfgang J. Neubert
neubert{at}biochem.mpg.de
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ABSTRACT |
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INTRODUCTION |
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Sendai virus (SeV), consisting of a single-stranded, negative-sense RNA genome, belongs to the genus Respirovirus within the family Paramyxoviridae and can cause severe respiratory illness in rodents. As shown for many other viruses within this family, such as respiratory syncytial virus (O'Donnell et al., 1999), canine distemper virus (Moro et al., 2003
), measles virus (Servet-Delprat et al., 2000
) and Newcastle disease virus (Lam, 1996
), SeV acts intracellularly by inducing apoptosis (Bitzer et al., 1999
, 2002
; Tropea et al., 1995
).
Little is known to date about how apoptosis is induced during SeV infection. Experiments with a recombinant SeV strain Z (SeV Z) mutant, rSeVGP48, that contains an exchange of the first 48 nt of the genomic leader with the equivalent antigenomic complementary trailer sequence (ctr), result in complete abrogation of programmed cell death during infection of various cell lines (Iseni et al., 2002). Interaction between a short viral RNA expressed from the mutated leader and the cellular T-cell-activated intracellular antigen-related (TIAR) protein was identified as causing this effect. Cells infected with wild-type SeV Z or overexpressing TIAR still died through apoptosis, as demonstrated by phosphatidylserine exposure on the outer leaflet of the plasma membrane (Garcin et al., 1998
; Iseni et al., 2002
).
As well as this involvement of TIAR, data from SeV strain F (SeV F) infection, another laboratory strain, have described the activation of certain caspases involved in pathways that lead to apoptosis (Bitzer et al., 1999, 2002
). Surprisingly, during immunohistochemical experiments SeV F seemed to induce cell death much faster and to a greater extent than SeV Z (R. Sedlmeier, personal communication).
The apoptotic cascade involving various caspases at defined steps has been relatively well described. However, whether and where TIAR is involved in these pathways, and to what extent, still has to be determined. While induction of apoptosis during SeV Z infection has been found to involve TIAR, caspases have been detected during infection with SeV F. Whether these observations are SeV strain-specific characteristics or whether cellular reactions during virus infection can vary depending on cell type has yet to be elucidated. Thus, apoptosis induction by SeV Z or F could be restricted to only one mechanism in a cell type-dependent manner, or could occur simultaneously via two mechanisms (TIAR involvement and caspases), which might somehow be connected with each other.
In this report, we investigated the cytotoxic effect of a specifically designed SeV mutant (Fctr48z) that combined the TIAR-inactivating leader region with the caspase-activating genomic backbone from SeV F. Based on these data, we determined that induction of cell death during infection with wild-type SeV F occurred via two processes that were simultaneously activated. We demonstrated a cell type-dependent interference between both mechanisms by infection of cells with mutant Fctr48z. This interference seemed to be based on the suppressive action of ctr RNA transcribed from the mutated leader region of Fctr48z and led, in the case of LLC-MK2 cells, to a complete abrogation of cell destruction.
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METHODS |
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SeV Z was obtained from M. F. G. Schmidt (Berlin, Germany). SeV F was recovered from a cDNA clone derived from SeV D52 (ATCC).
For construction of the mutant virus Fctr48z cDNA containing the mutated leader sequence, two single PCR reactions were first performed whose products also included the sequence complementary to nt 148 from the SeV Z trailer (antigenomic). Template for the reactions was a pUC plasmid containing the entire SeV genome (strain F). The following primer pairs were used: (i) standard pUC/M13 R 5'-AGCGGATAACAATTTCACACAGG-3' (forward) and 5'-AGACAAGAAAATTTAAAAGGATACATATCTCTTAAACTCTTGTCTGGTCCCTATAGTGAGTCGTATTACG-3' (reverse), and (ii) 5'-GTATCCTTTTAAATTTTCTTGTCTGGATTTTAGGGTCAAAGTATCCAC-3' (forward) and 5'-CCATGAGAGATACAAGGC-3' (reverse) from inside the N gene. A fusion PCR was then performed to join both fragments using the forward primer of the first reaction and the reverse primer of the second. The resulting PCR product contained a unique restriction enzyme recognition site at each end for introduction into the viral cDNA.
Generation of recombinant SeV from cDNA was carried out and all viruses were propagated in Vero cells as documented previously (Leyrer et al., 1998).
Virus replication studies.
The number of virus particles released from infected cells was quantified using a haemagglutination (HA) test and the infectious fraction by TCID50 assays, as described previously (Bitzer et al., 1997; Neubert & Hofschneider, 1983
). Under our conditions, 40 HA U ml1 was equivalent to 107 virus particles ml1 and 1 TCID50 ml1 was equivalent to 5x103 cell infectious units (CIU) ml1.
Cell viability assay.
A modified version of the MTT assay (Mosmann, 1983) was used for determining the viability of infected cells. After incubation for 72 h, medium from infected cells grown in 96-well plates (20 000 cells per well) was replaced with medium containing MTT [tetrazolium salt of 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide; Sigma-Aldrich] at a final concentration of 5 mg ml1. After incubation for 3·5 h at 37 °C, the formazan crystals formed were dissolved by the addition of 2-propanol with 0·04 M HCl and thoroughly resuspended to homogeneity. The absorbance of formazan at 590 nm was measured using an ELISA reader (Dynatech MR7000). Each value was determined from three parallel infection experiments and absorption by the medium (blank) was subtracted each time.
When the caspase inhibitor z-VAD-fmk [N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethylketone; Merck Biosciences] was added to the medium of infected cells, a final concentration of 50 µM was used and renewed every 24 h. The remaining MTT assay was performed as described.
Sequence determination of the mutated leader.
The original 3'-terminal sequence of the virus genome from nt 1 to 55 was determined using a 5'-RACE kit (Invitrogen).
Total RNA was extracted from infected cells using TRIzol reagent (Invitrogen). Only the antigenomic SeV RNA strand was reverse transcribed to cDNA with Superscript II (Invitrogen). The primer used for this reaction started from inside the N gene towards the antigenomic 5' end. Next, the 3' end of the generated cDNA was elongated by adding guanosine residues with the enzyme terminal transferase (Roche). Subsequent amplification of the fragments containing the viral sequence from nt 1 to 55 was performed according to the manufacturer's recommendations. The 5'-RACE products were analysed directly and also after subcloning into pUC29. DNA sequencing was performed by Medigenomix.
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RESULTS |
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Generation of mutant Fctr48z and analysis of virus replication
After selection of strain F, we constructed the SeV mutant Fctr48z consisting of two portions: the first part from nt 1 to 48, schematically shown in Fig. 2, corresponded to the complementary sequence of the last 48 nt from the genomic 5' end (trailer) of SeV Z and was designated ctr. This sequence is reported to cause abrogation of apoptosis during infection with an analogous mutant derived from strain Z (Garcin et al., 1998
). The second part of the mutant, from nt 49 to 15384, represented the wild-type sequence of SeV F. This part of the genome is thought to harbour elicitors of caspase activation, as demonstrated previously (Bitzer et al., 1999
, 2002
). Generation of recombinant Fctr48z was performed as described in Methods. Despite attenuated replication early after infection, growth of Fctr48z in Vero cells was comparable to SeV Z and F at 48 h post-infection (p.i.) (Fig. 1c
).
In order to ensure the genetic stability of the mutant under selective conditions, we analysed viral genomes after 10 passages on Vero cells. The 5'-end sequence of the viral antigenomic RNA was reverse transcribed and amplified using a 5'-RACE reaction including the outermost nucleotides of the leader. In all viral RNA samples, the correct sequence of the mutated leader could be confirmed except for a nucleotide reversion from C to U (original F strain leader) at position 42 that occurred in approximately 50 % of the genome population.
Cell type-dependent reduced CPE during infection with mutant Fctr48z
Next, we investigated whether the exchange of the leader sequence combined with the SeV F genome also caused abrogation of CPE, as reported for the Z strain (Garcin et al., 1998).
Therefore, CPE was monitored during infection of seven different cell lines (Vero, LLC-MK2, BHK, 293, A549, HepG2 and HeLa) with Fctr48z. SeV F, which causes a severe CPE (as shown above), was used as a positive control. The CPE in cells infected with either virus was analysed for 72 h (Fig. 3a). This analysis of Fctr48z- or SeV F-infected cells revealed a widely varying CPE. Almost all cells infected with SeV F were dead by 72 h. Fctr48z-infected cells, however, exhibited a cell type-dependent, variable CPE that could be classified into two categories: a strong and quickly developing CPE, similar to SeV F infection, was observed in BHK, A549 and HeLa cells, while a slightly attenuated and more slowly developing CPE was seen in 293, HepG2, Vero and LLC-MK2 cells (data not shown). Incubation of infected cells with serum-free or FCS-containing medium did not result in significant alterations to the CPE.
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In order to test whether this was also the case in the other cell lines, virus replication was monitored in these lines over a period of 4 days. The number of virus particles produced during SeV F or Fctr48z infection was found to differ depending on the cell type, as shown in Fig. 3(b). However, comparison of the CPE in these replication studies (Fig. 3
) clearly showed that there was no general correlation between the efficiency of virus replication and the observed CPE. This was most obvious in infection of LLC-MK2 cells with Fctr48z: by far the highest production of viral progeny was seen for this cell line, but a CPE was barely detectable (Fig. 3
). BHK cells, in contrast, showed a strong CPE after Fctr48z infection and still produced a large number of virus particles. During Fctr48z infection of HeLa, 293 and A549 cells, however, a low level of virus replication coincided with a massive CPE (Fig. 3a and b
). Western blot analysis of Vero and LLC-MK2 cells, which both produced large numbers of virus particles, also showed a high level of viral protein synthesis during wt and Fctr48z infections, verifying a high cellular burden (data not shown).
The above results indicating that the CPE of mutant virus-infected cells varied with cell type over a broad range (Fig. 3a) and did not correspond to the burden resulting from viral propagation (Fig. 3b
) was confirmed by evaluating cell viability. The viability of Vero and LLC-MK2 cells was examined during infections with the mutant Fctr48z or with SeV F as representative cell lines able to survive infection with the mutant virus for a long time, while still producing high numbers of virus particles (Fig. 4
). While almost all Vero cells infected with wild-type SeV F had died by 72 h p.i., approximately one-third of those infected with the mutant were still alive. Viability of LLC-MK2 cells infected with SeV F clearly decreased up to 72 h p.i. When infected with Fctr48z, however, only a slight decline in viability could be observed during the first 2 days of infection, and there was a full recovery by 72 h p.i.
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Viability of HeLa and LLC-MK2 cells, representative of cells showing a strong or no CPE (Figs 3 and 4), respectively, was determined by MTT assays during infection with Fctr48z in the presence or absence of the pan-caspase inhibitor z-VAD-fmk. SeV F (wild-type) was used for comparison (Fig. 5
). SeV F infection without inhibitor reduced the viability of LLC-MK2 cells by about one-third and the viability of HeLa cells almost completely. When z-VAD-fmk was present, viability clearly increased, but was still diminished for LLC-MK2 (by one-fifth) and HeLa cells (by one-third). Remarkably, infection with Fctr48z led to different results: viability of HeLa cells was reduced by two-thirds in the absence of z-VAD-fmk and almost completely retained in the presence of z-VAD-fmk, resembling an accumulative effect of blocking TIAR and caspases. Most strikingly, the viability of Fctr48z-infected LLC-MK2 cells was not impaired, regardless of the presence or absence of z-VAD-fmk. Here, synthesis of ctr RNA was obviously sufficient to prevent CPE or apoptosis induction, including activation of caspases. Thus, the expressed ctr RNA appeared also to act as an apoptosis blocker in the context of SeV F infection, and not only with strain Z, as indicated by Iseni et al. (2002)
.
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DISCUSSION |
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Another way in which CPE could develop during Fctr48z infection is linked to defective interfering (DI) particles. During SeV infection, high levels of non-infectious virus particles are produced that lead to interference with wild-type virus replication (Leppert et al., 1977; Tuffereau & Roux, 1988
) and reduction of CPE has consequently been linked to the diminished virus replication. Thorough consideration of the present results, however, allows exclusion of a comparable situation for Fctr48z infection. Firstly, the CPE caused by Fctr48z infection was reduced in a cell type-dependent manner and only for LLC-MK2 cells was the CPE drastically diminished. Secondly, the production of virus particles was slightly reduced only in some cell lines (from two- to fivefold). However, DI particles are typically reported to diminish viral particle production by up to 100-fold (Tuffereau & Roux, 1988
). Furthermore, while infections with DI particles contribute to the establishment of persistent infections without restrictions on the cell types infected (Perrault, 1981
; Roux & Waldvogel, 1981
), in our experiments only one cell line (LLC-MK2) survived and constantly produced virus particles over a period of at least 13 passages. Fctr48z infection thus did not result in a situation comparable to typical DI particle infections. The enhanced fraction of non-infectious particles (four- to 10-fold) could represent particles containing full-length antigenomic rather than fragmentary genomic RNA (like DI particles). The introduction of a stronger promoter in Fctr48z might initiate synthesis of antigenomic viral RNA more efficiently (see below).
After excluding variable degrees of virus replication as a regulator of the cell type-specific CPE during infections with Fctr48z and SeV F, our investigation focused on cell type-specific apoptotic reactions. Two pathways or factors involved in SeV-induced apoptosis have been described so far.
First, during infection studies with SeV F, induction of apoptosis in MCF-7 and CV-1 cells was shown to coincide with the activation of caspases (Bitzer et al., 1999, 2002
). In our experiments, however, using HeLa and LLC-MK2 cells infected with SeV F, the presence of the pan-caspase inhibitor z-VAD-fmk only partially reduced the CPE (Fig. 5
). Hence, SeV F appeared to trigger apoptosis via at least one additional, caspase-independent mechanism.
Evidence for the induction of apoptosis in a caspase-independent manner has recently been provided. The mitochondrial apoptosis-inducing factor was identified as a possible inducer (Joza et al., 2001; Susin et al., 1999
) and linked to a serine protease able to damage the mitochondrial membrane (Egger et al., 2003
) or to the calcium-dependent proteinase calpain, which cleaves cytoskeletal proteins or the pre-apoptotic protein Bax (Vanags et al., 1996
; Wolf et al., 1999
). However, the exact positions at which these factors could be integrated into the apoptotic pathway have not yet been determined. Similarly, for infections with several viruses such as HIV-1, coxsackie B3 and HSV-1 d120 mutant (Carthy et al., 1998
; Galvan et al., 1999
; Roumier et al., 2002
), the induction of apoptosis and typical signs of a CPE, even in the presence of caspase inhibitors, have been reported. Our results showing that apoptosis could not be completely inhibited by z-VAD-fmk during SeV F infection, combined with the findings that caspase 9 is cleaved (Bitzer et al., 2002
) but subsequently not activated (Wolf et al., 1999
), support the idea of the existence of additional caspase-independent apoptosis induction during SeV F infection.
A second factor has been identified during infection studies with SeV Z mutants. Viral RNA, transcribed from a mutated leader, was found to bind to the cellular protein TIAR, which had been proposed to be involved in apoptosis induction during SeV Z infection (Garcin et al., 1998; Iseni et al., 2002
). Binding of RNA to TIAR seems to influence its activity negatively, thereby diminishing the virus-induced CPE. As a prerequisite for this mechanism to function efficiently, enough molecules of ctr RNA have to be generated during viral transcription (Lamb & Kolakofsky, 1996
), otherwise unbound TIAR proteins could still be functional, contributing to the induction of apoptosis. In the present studies, LLC-MK2 was the only cell line, among seven lines tested, showing no sign of CPE during infection with the mutant Fctr48z (Fig. 3
). Therefore, we infected BHK cells as a representative cell line exhibiting a strong CPE with an enhanced m.o.i. of 10 in order to provide initially more available templates for transcription of ctr RNA. However, no significant reduction in CPE could be observed (data not shown). A loss of functionality of the genetically modified leader sequence within Fctr48z due to mutations could be excluded after monitoring genetic stability for 10 virus passages. We concluded that triggering of an apoptotic response by the virus mutant Fctr48z did not solely depend on the TIAR-induced pathway.
The assumption that SeV F induces apoptosis via more than one mechanism, one of which is caspase independent, as recently shown for adenovirus (Zou et al., 2004), was confirmed by results obtained from infection of HeLa cells with our Fctr48z mutant. Compared with SeV F, the apoptotic response in Fctr48z infection was only slightly reduced, but was completely abolished when z-VAD-fmk was added (Fig. 5
). Thus, prevention of apoptosis in HeLa cells was only prevented when both mutated leader RNA transcribed during Fctr48z infection and z-VAD-fmk were present. This lends further support to apoptosis induction via two mechanisms in an additive manner (Fig. 6a
).
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Support for complete abrogation of the apoptotic response via a caspase-independent mechanism comes from studies performed with mutants of SeV Z. This virus has been demonstrated to induce apoptosis with involvement of the cellular protein TIAR (Iseni et al., 2002). The only known characteristics of TIAR so far are its ability to bind RNA, to regulate mRNA translation and to be involved in alternative splicing of several pre-mRNAs (Forch & Valcarcel, 2001
; Gueydan et al., 1999
; Piecyk et al., 2000
). Direct intracellular interaction of TIAR proteins with transcribed viral RNA has been documented during infection with SeV (Iseni et al., 2002
) and the flavivirus West Nile virus (Li et al., 2002
).
Results from previous work with SeV Z and F (Bitzer et al., 1999, 2002
; Garcin et al., 1998
; Iseni et al., 2002
) combined with our data thus lead to the following conclusions: (i) so far unknown virus strain-specific characteristics determine which mechanism for induction of apoptosis is triggered and to what extent; and (ii) variable apoptosis-inducing mechanisms exist simultaneously during SeV infections. For the first time, we have demonstrated a combination of TIAR-induced and caspase-mediated apoptosis within virus-infected cells. During evolution of eukaryotic cells, the development of redundant pathways or mechanisms to trigger programmed cell death does not seem to be surprising: apoptosis marks an essential reaction for multicellular organisms to survive. On the other hand, viruses have to provide an efficient means of shutting off these cell death-inducing pathways.
While most apoptotic reactions involve the participation of caspases, as shown here for SeV infection (Fig. 5), the TIAR-related pathway can also influence virus-induced CPE, albeit to a lesser degree.
Interestingly, repression of the TIAR-related pathway of apoptosis induction alone can (trans)-dominantly block the caspase-related CPE, as shown for LLC-MK2 infection with Fctr48z (Fig. 5). A complete block of apoptosis has previously also been shown for infection of various cell lines with SeV Z mutants (Garcin et al., 1998
; Iseni et al., 2002
). This phenomenon can only be detected if cells are infected with mutants that transcribe ctr RNA in larger amounts, such as Fctr48z, resulting in enhanced interaction with cellular TIAR proteins. During SeV wild-type infection, transcription of trailer RNA, corresponding to the first 48 nt of ctr RNA, starts before new genomes are synthesized. Genome amplification provides more templates for viral transcription enabling high-level protein synthesis, delineating a starting point for the development of a strong CPE. Thus, transcription of trailer RNA may ensure efficient viral amplification by delaying the early onset of an apoptotic reaction.
In conclusion, we have elucidated the co-existence and interrelationships of two different pathways of apoptosis induction using a SeV mutant genetically adapted to allow this investigation. Whether this and possibly other means of preventing or delaying apoptotic reactions play an important role during infections with other viruses remains to be explored.
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ACKNOWLEDGEMENTS |
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Received 10 March 2005;
accepted 13 May 2005.
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