1 Department of Virology and WHO National Influenza Center, Erasmus Medical Center, PO Box 1738, 3000 DR Rotterdam, The Netherlands
2 Department of Zoology, Cambridge University, Cambridge, UK
Correspondence
G. F. Rimmelzwaan
g.rimmelzwaan{at}erasmusmc.nl
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ABSTRACT |
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MAIN TEXT |
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It is a general property of RNA virus quasispecies to adapt to changing environments and select for successful variants that have, for example, increased resistance to antiviral agents or altered interferon-inducing capacity, or have enabled escape from antibodies and also from virus-specific CTLs (Domingo, 2000; Domingo & Holland, 1997
).
Amino acid substitutions at the anchor residue of a CTL epitope may prevent binding of the epitope to its corresponding major histocompatibility complex class I molecule, resulting in the loss of the epitope. Variation in T-cell receptor-contact residues may prevent recognition by specific T-cell receptors present on virus-specific T cells. Variation in CTL epitopes can thus result in effective immune evasion from CTLs and has been shown to play an important role in the pathogenesis of various virus infections. The selective pressure exerted by CTLs must be significant, as it has been demonstrated that escape can occur at the cost of viral fitness. This was shown recently for HIV escape mutants, which reverted upon transmission to new susceptible hosts in which HIV CTL immunity was not present (Friedrich et al., 2004a; Leslie et al., 2004
). In addition, it has been demonstrated that, in some cases, viruses need to accumulate amino acid substitutions outside the epitope in order to restore viral fitness as a result of functional constraints imposed by the amino acid sequence in the epitope (Friedrich et al., 2004a
, b
; Kelleher et al., 2001
; Leslie et al., 2004
; Peyerl et al., 2003
). Apparently, viruses can overcome these functional constraints, as there are examples of virus variants that have escaped from CTLs, but retained their viral fitness (Friedrich et al., 2004b
; Peyerl et al., 2003
). Amino acid substitutions in the influenza virus nucleoprotein (NP) have been described to be associated with escape from virus-specific CTLs (Berkhoff et al., 2004
; Boon et al., 2002b
; Gog et al., 2003
; Rimmelzwaan et al., 2004a
, b
; Voeten et al., 2000
) and these mutations can become fixed rapidly, even when there are only small selective advantages potentially due to population-dynamic effects (Gog et al., 2003
). The R384G mutation in the HLA-B*2705- and HLA-B*0801-restricted CTL epitopes NP383391 and NP380388, respectively, resulted in a marked reduction in the human influenza virus-specific CTL response in vitro (Berkhoff et al., 2004
; Gog et al., 2003
; Rimmelzwaan et al., 2004a
; Voeten et al., 2000
). Thus, the immunodominant nature of the epitope (Boon et al., 2002a
), prolonged viral shedding in the absence of these epitopes in HLA-B*2705- and HLA-B*0801-positive individuals and population dynamics could have contributed to the emergence of these CTL-escape variants. For these epitopes, it has also recently been demonstrated that an extra-epitopic co-mutation was required to compensate functionally for the detrimental effect of the R384G substitution. It was shown that the naturally occurring E-to-G substitution at position 375 partially restored viral fitness in viruses in which the detrimental R384G mutation was introduced by site-directed mutagenesis. Position 384 is the anchor residue of the epitopes and loss of the arginine resulted in loss of the anchor residue for binding to HLA-B*2705 and HLA-B*0801 (Rimmelzwaan et al., 2004b
; Voeten et al., 2000
). As the co-mutation at position 375 only partially compensated for the loss of viral fitness and multiple co-mutations can be identified associated with the mutation at position 384 (Macken et al., 2001
; Rimmelzwaan et al., 2004a
), we wished to study the contribution of these additional mutations (R65K, D127E, I197V and M239V) to viral fitness in viruses that had escaped from NP383391- and NP380388-specific CTLs.
To this end, recombinant influenza viruses were generated essentially as described previously (de Wit et al., 2004; Pleschka et al., 1996
) by using reverse-genetics technology. For the generation of recombinant influenza viruses with amino acid substitutions at selected positions, the NP gene segment of influenza virus A/Hong Kong/2/68 (A/HK/2/68) was amplified by RT-PCR as described previously (Rimmelzwaan et al., 2004a
) and inserted between the human Pol I promoter and the hepatitis
ribozyme sequence of plasmid pSP72-PhuThep (de Wit et al., 2004
). Site-directed mutagenesis of the NP gene was performed by PCR using a QuikChange Multi site-directed mutagenesis kit (Stratagene) according to the manufacturer's recommendations. Plasmids pHMG-NP, pHMG-PB1, pHMG-PB2 and pHMG-PA, encoding the NP and the polymerase proteins PB1, PB2 and PA of influenza virus A/PR/8/34, respectively, were kindly provided by Dr P. Palese (Pleschka et al., 1996
). The bi-directional reverse-genetics plasmids pHW181pHW188, encoding the viral gene segments of influenza virus A/WSN/33, were kindly provided by Dr R. G. Webster (Hoffmann et al., 2000
). For the generation of recombinant viruses, the NP constructs of A/HK/2/68 were transfected into 293T cells together with pHMG-NP and the remaining genomic constructs of A/WSN/33 as described previously (de Wit et al., 2004
). Twenty-four hours after transfection, virus was passaged in MadinDarby canine kidney (MDCK) cells and the infectious virus titre was determined as described previously (Rimmelzwaan et al., 1998
). The combinations of mutations that were tested are listed in Table 1
. As shown in Fig. 1
, of the single mutants, only the R384G substitution was detrimental to viral fitness. Each of the other amino acid substitutions that were associated with R384G mutation had very modest effects on virus titres compared with viruses containing the wild-type sequence. As we described previously (Rimmelzwaan et al., 2004a
), addition of the E375G substitution to the R384G substitution partially restored virus replication, which was confirmed in the present study. In contrast, none of the other co-mutations compensated functionally for the detrimental effect of the R384G substitution on viral fitness. This indicated that E375G is indeed of crucial importance for viruses with the R384G mutation. Next, we tested whether the other co-mutations played a role in the fitness of viruses with the double mutation E375G/R384G. As shown in Fig. 1(ef)
, addition of the M239V mutation increased virus titres to the level of wild-type virus. Thus, viruses containing 384G in combination with 239V and 375G replicated to titres similar to those of wild-type virus. Addition of the co-mutation D127E had no effect, whereas R65K and I197V influenced the replication of viruses with 375G/384G negatively. These data suggested that positions 239, 375 and 384 are involved in the same function of NP. Surprisingly, however, 239V is not found in all viruses with the 375E and 384G mutations, for which sequences are available in the influenza sequence database at http://www.flu.lanl.gov/ (Macken et al., 2001
; Rimmelzwaan et al., 2004a
). We speculate that these viruses without 239V exist as intermediates. The effect of the respective mutations on viral fitness, exemplified in Fig. 1(g)
, was confirmed by determining multi-step growth kinetics of these viruses after infection of MDCK cells by using an equivalent m.o.i. of 0·001 TCID50 per cell. Introduction of each of the single mutations (with the exception of R384G) did not alter the replication kinetics dramatically (Fig. 1h
) compared with replication of the wild-type virus. In Fig. 1(i)
, the replication of viruses containing three amino acid substitutions was compared with wild-type viruses and the E375G/R384G double mutant. In agreement with the data presented in Fig. 1(ef)
, introduction of 127E in the 375G/384G double mutant was neutral, whereas 65K and 197V had a negative effect on replication rate and 239V increased the replication rate to wild-type level.
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ACKNOWLEDGEMENTS |
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Received 29 December 2004;
accepted 17 February 2005.