Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, UK1
Author for correspondence: E. A. Gould Fax +44 1865 281696. e-mail eag{at}ceh.ac.uk
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Abstract |
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Introduction |
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The Vs strain within the tick-borne encephalitis (TBE) virus complex was isolated from a patient with a latent form of TBE. In experiments on Syrian hamsters and monkeys, Vs virus demonstrated a tendency to produce chronic disease and even latent infections that could be reactivated in the animals following immunosuppression 2 years after the initial infection (Frolova et al., 1982a , b
). Genome sequence alignment of the Vs virus with other isolates of TBE virus demonstrated multiple nucleotide and amino acid substitutions, and phylogenetic analysis identified this virus as a separate Siberian isolate of the TBE complex viruses (Gritsun et al., 1995
, 1997
; Zanotto et al., 1995
).
An infectious clone of Vs virus, pGGVs, was constructed using a long, high-fidelity PCR (Gritsun & Gould, 1998 ; Gritsun et al., 1997
). Characterization of pGGVs revealed two features distinct from the Vs parental virus. Firstly, it was found that the recovered pGGVs virus had a slightly smaller plaque phenotype compared with the Vs parental virus. Secondly, parental Vs and pGGVs viruses had five nucleotide differences, four of which resulted in amino acid substitutions. Two mutations were identified as possibly being responsible for the reduced plaque phenotype of pGGVs. One of them, H496
R (at amino acid position 496 within the polyprotein and corresponding to nucleotide substitution A1619
G), had occurred in a highly conserved domain within the E protein. The other nucleotide substitution, T10884
C (nucleotide position 10884 of the genome), was located in a highly conserved domain of the 3' untranslated region (3'UTR), a proposed recognition signal for viral RNA polymerases (Gritsun et al., 1997
; Proutski et al., 1997
; Rauscher et al., 1997
).
Three other amino acid differences between pGGVs and Vs viruses mapped to less conserved regions of the virus genome, one in the C protein (I45F, corresponding to nucleotide substitution A265
T) and two others in the NS5 protein (T2688
A and M3385
I with corresponding nucleotide substitutions A8194
G and G10287
T).
In this study, the biological significance of these mutations was evaluated by site-directed mutagenesis of the infectious clone and comparative examination of engineered mutants by different biological methods. It was demonstrated that the substitution H496R in the E protein was solely responsible for the reduction of plaque size in cell culture. Virus mutants with R496 also showed reduced virus yield in growth cycles, reduced cytotoxic effect in cell culture and reduced neuroinvasiveness in mice. The biological effect of the substitution C10884
T was not as obvious as that of the substitution R496
H, but was reproducible by cytopathic effect (CPE), cytotoxicity and neuroinvasiveness testing. The effect of mutations mapping within the C (I45
F) and NS5 (T2688
A and M3385
I) proteins was detectable only in neuroinvasiveness tests. The pGGVs virus recovered from the infectious clone was the most attenuated of the mutant viruses when tested in animals, thus demonstrating the cumulative effect of the five mutations.
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Methods |
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Reverse transcription of viral RNA.
First-strand cDNA was synthesized essentially as described previously (Gritsun & Gould, 1995 ). A sample of 11 µl RNA and 5 µl of the appropriate primer (50 pM) were mixed and heated for 2 min at 95 °C. The mixture was then chilled and 3 µl dNTPs (10 mM), 3 µl DTT (0·1 mM), 1 µl RNasin (40 U), 6 µl 5xbuffer and 1 µl Superscript II reverse transcriptase (Gibco BRL) were added. The mixture was incubated at 42 °C for 1 h.
Derivation of PCR products, cloning and sequencing.
PCR amplification was carried out in a volume of 50 µl using thermostable DNA polymerase purchased from Sigma. Reaction conditions were as follows: 30 cycles of 1 min at 95 °C, 1 min at 50 °C and 1 min at 72 °C.
To sequence the substitutions I45F (C), H496
R (E), T2688
A (NS5), M3385
I (NS5) and T10884
C (3'UTR), five regions of the Vs viral genome, corresponding to nucleotides 1344, 22962313, 80648983, 1000010518 and 1041510928, respectively, were re-amplified by RTPCR using Vs viral RNA and the appropriate primers. PCR products for each gene were amplified in five different tubes, pooled and purified using the QIAGEN DNA purification kit. PCR-derived DNA containing the E protein gene and the 3'UTR were then cloned using the pGEM-T vector system (Promega) to create pGEMT-E and pGEMT-3'UTR, respectively (Fig. 1
). Three clones of each pGEMT-E and pGEMT-3'UTR were selected and sequenced. For the substitutions I45
F, T2688
A and M3385
I, PCR products were sequenced directly without cloning.
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Construction of plasmids and mutagenesis of the infectious clone.
The construction of the infectious clone, pGGVs, and the basic strategy for the introduction of mutations were described previously (Gritsun & Gould, 1998 ). The plasmid pGGVs was used to construct the cassette of intermediate plasmids (Fig. 1
) to produce mutant viruses (Fig. 2
).
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Introduction of single substitutions into the E gene and the 3'UTR of the infectious clone.
To restore the wild-type Vs virus E protein containing H496, the DNA fragment of pGGVs6601982 between the restriction sites BsrGI and Sse8387I (nt 13891982 of the genome) was substituted with the corresponding fragment from pGEMT-E (Fig. 1) to create pGGVs6601982H496. To restore the wild-type Vs virus 3'UTR containing T10884, the DNA fragment within pGGVs6601982del between the restriction sites BssHII and PspAI (nt 1050110927 of the genome) was substituted with the corresponding fragment from pGEMT-3'UTR (Fig. 1
) to create pGGVs6601982delT10884. The resulting plasmids were sequenced completely to verify the introduction of mutations.
Construction and recovery of mutant viruses.
To re-create the full-length infectious clone, the plasmids pGGVs6601982 and pGGVs6601982H496 (15 µg) were digested with NotI and dephosphorylated by incubation with shrimp alkaline phosphatase (USB) for 30 min at 37 °C (Gritsun & Gould, 1998 ). Plasmids pGGVs6601982 and pGGVs6601982H496, and pGGVs6601982del and pGGVs6601982delT10884 were then digested with AgeI/Sse8387I and the short excised DNA linker fragments were removed using MicroSpin S-400 HR Columns (Pharmacia). Both pGGVs6601982 and pGGVs6601982H496 were ligated in vitro with pGGVs6601982del to generate full-length Vs virus cDNA. Similarly, plasmids pGGVs6601982 and pGGVs6601982H496 were ligated with pGGVs6601982delT10884. Each mutant plasmid was sequenced completely. Plasmids were then digested with SmaI (to linearize the plasmid) and SP6 polymerase was used for transcription, as described previously (Gritsun & Gould, 1998
). SP6-transcribed RNA was inoculated intracerebrally into suckling mice to recover the mutant viruses (Gritsun et al., 1995
). The presence of mutations in each mutant virus was validated by sequencing the RTPCR products amplified from the appropriate genomic regions of virus mutants.
Plaque assays.
Plaque morphology and initial titres of the virus mutants were evaluated, essentially as described previously (Gritsun & Gould, 1998 ). Briefly, PS cells on 6-well plates were inoculated with tenfold dilutions of a 10% suspension of virus-infected mouse brain. After 1 h of virus adsorption, virus inoculum was removed and cell monolayers were overlaid with 1% low-melt-agarose (Flowgen). After incubation for 35 days at 37 °C, agarose was removed and cell monolayers were stained with naphthalene black.
Plaque assay for the estimation of virus titres in growth cycle experiments (see below) was performed on 96-well tissue culture plates. The supernatant medium from each time-point for different mutants was diluted tenfold in 100 µl Eagles MEM (EMEM). A 100 µl sample of PS cell suspension in EMEM containing 4% foetal calf serum (FCS) was then added to each well. After incubation for 4 h at 37 °C, an overlay containing 2% carboxymethylcellulose with 2% FCS was added to each well (30 µl) and the plates were incubated at 37 °C for 4 days. Plaques were visualized by staining monolayers with naphthalene black.
Virus growth cycles.
Monolayers of PS cells in 24-well plates were infected with mutant viruses at an estimated m.o.i. of 10 p.f.u. per cell. Each experiment was performed in quadruplicate. The inoculum (0·1 ml) was removed after 1 h of incubation and monolayers were washed thoroughly with serum-free medium. Fresh medium (1 ml) containing 2% FCS was then added to each well. The supernatant medium from appropriate wells was collected at different time-points (from 0 to 53 h post-infection) and frozen at -70 °C. Virus titres were estimated by plaque assay using PS cells in 96-well plates.
Estimation of CPE.
PS cells in 24-well plates were infected with mutant viruses at an estimated m.o.i. of 10 p.f.u. per cell. After 4872 h, cell destruction was observed by light microscopy. Additionally, cells were stained with naphthalene black and the intensity of cell destruction was estimated visually.
CPE caused by each mutant virus was also compared by estimating the amount of lactate dehydrogenase (LDH) retained in infected PS cell monolayers using the CytoTox 96 Non-Radioactive Cytotoxicity assay (Promega), with some modifications. PS cells in 24-well plates were infected with mutant viruses (five wells for each mutant) at an estimated m.o.i. of 10 p.f.u. per cell. The virus inoculum was removed after 1 h adsorption and substituted with 200 µl EMEM containing 2% FCS. After incubation at 37 °C for 7080 h, cell monolayers were washed three times with PBS and frozen at -70 °C. Lysis buffer (100 µl) was added to each well and plates were incubated at 37 °C for 45 min. Cell lysate (25 µl) from each well was transferred to 96-well assay plates and 25 µl of reconstituted substrate mixture was added. After incubation of the plates at room temperature in the dark for 30 min, 25 µl of stop solution was added and absorbance was measured at 492 nm.
Neuroinvasiveness test.
In preliminary tests, viruses were titrated by the estimation of p.f.u./ml and intracerebral inoculation of newborn mice to obtain comparative ratios. Subsequently, in four separate experiments, 34-week-old mice (five in each group) were injected intraperitoneally with 2000 p.f.u. of mutant virus. Mice were observed daily for up to 4 weeks. Results were evaluated by mortality rate and average survival time (AST) of infected animals.
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Results |
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Viral RNA was extracted from a 10% suspension of virus-infected mouse brain and the five regions of the Vs virus genome were amplified by RTPCR. To eliminate possible errors produced by reverse transcriptase and Taq polymerase, we analysed the pooled PCR products for each region of the Vs virus genome (see Methods). Nucleotide sequences of the three clones of the Vs E protein gene and three clones of the 3'UTR were determined and the amino acid sequences for the E protein were deduced. PCR products for the C and NS5 genes were sequenced directly without cloning procedures. Results show that the four amino acid substitutions between Vs (accession no. L40361) and pGGVs virus, i.e. H496R in the E protein (numbers show polyprotein position), I45
F in the C protein, T2688
A and M3385
I in the NS5 protein, and one nucleotide substitution, T10884
C, within the 3'UTR were present only in the infectious clone.
Biological properties of the engineered viruses
Plasmids pGGVs6601982 and pGGVs6601982H496 were ligated to plasmids pGGVs6601982del and pGGVs6601982delT10884 (Fig. 1) in order to construct the full-length DNA for four viruses, namely Vs-c (constructed), pGGVs-c (constructed), E(H)3'UTR(C) and E(R)3'UTR(T) (Fig. 2
). Viruses E(H)3'UTR(C) and E(R)3'UTR(T) each contained only one wild-type substitution, H496 in the E protein or T10884 in the 3'UTR, respectively. Virus Vs-c contained both wild-type substitutions, H496 and T10884. In this respect, Vs-c was similar, but not identical, to the Vs parental virus. As shown in Fig. 2
, Vs and Vs-c viruses differed in the C (I45
F) and the NS5 (T2688
A and M3385
I) proteins. Viruses pGGVs and pGGVs-c have identical genotypes, but they were assembled from different plasmids (see Methods, Table 1
). In the text of this manuscript, we shall only refer to pGGVs, although pGGVs-c was included, with identical results in all experiments. These six viruses (Fig. 2
) were compared in different biological assays (see Figs 3
, 4
and Table 1
).
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Growth curve assay
To assess the effect of these mutations on virus replication, the growth kinetics of mutant viruses were compared with Vs virus (Fig. 3). The results show that viruses could be grouped into two categories, those producing high virus yield (
107·5 p.f.u./ml) with a short lag phase of 12 h and those producing relatively low virus yield (
107·2 p.f.u./ml) with a long lag phase of 16 h. The viruses with a short lag phase, Vs, Vs-c and E(H)3'UTR (C), share the wild-type mutation H496 in the E protein, whereas viruses with R496 in the E protein produced growth curves with a long lag phase.
Cytopathic effect
The cytopathogenicity of each virus was estimated using several different methods. Firstly, we used conventional visualization of cells under the light microscope and comparative intensity of cell monolayers stained by naphthalene black after infection for 7280 h. The results are presented in Fig. 4 and Table 1
. Viruses with H496 in the E protein [Vs, Vs-c and E(H)3'UTR(C)] produced more intensive CPE than viruses with R496 in the E protein [pGGVs and E(R)3'UTR(T)]. However, in this assay, small but reproducible differences were visualized between E(R)3'UTR(T), with only one wild-type substitution, T10884, in the 3'UTR, and two reference viruses pGGVs (or pGGVs-c) and E(H)3'UTR(C). CPE produced by virus E(R)3'UTR(T) was considered to be intermediate between viruses pGGVs and E(H)3'UTR(C) (Fig. 4
and Table 1
).
The CPE caused by each mutant virus was also compared by estimating the level of retained LDH in infected PS cell monolayers using a CytoTox 96 Non-Radioactive Cytotoxicity assay. The amount of retained LDH in the infected cells was measured using an appropriate substrate and absorbance was measured at 492 nm 72 h after infection. These experiments were repeated several times. The results of this test (Fig. 5) showed that cell monolayers infected with viruses containing H496 in the E gene retained reduced levels of LDH (i.e. more cell destruction) in comparison with non-infected cells and cells infected by viruses with R496 in the E protein. The level of LDH in cell monolayers infected with E(R)3'UTR(T) was higher than that produced by pGGVs but lower than that produced by E(H)3'UTR(C), confirming the intermediate CPE produced by E(R)3'UTR(T).
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Discussion |
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Sequence analysis identified five mutations, any one or all of which could have been responsible for the reduced plaque phenotype. In the present work, we repeatedly sequenced five regions of Vs virus and demonstrated that all five substitutions were restricted to the infectious clone and were, therefore, acquired during its construction.
In order to determine whether these mutations, either individually or together, are indeed responsible for the altered plaque phenotype, the reverse substitutions from conserved regions of the genome, R496H in E gene and C10884
T in the 3'UTR, were introduced, both separately and together, into pGGVs and their biological effect on virus phenotype was evaluated. The five recovered back-mutated viruses (Fig. 2
) were compared with each other and with the Vs parental virus in different biological tests (Figs 35
and Table 1
).
Plaque assay demonstrated that the presence of only one amino acid mutation, R496 in place of H496 as in wild-type Vs virus, caused a reduction in plaque size. Three viruses with the wild-type mutation H496 in the E protein, including E(H)3'UTR(C) with only one wild-type mutation, formed plaques that were slightly larger under agarose than viruses with the R496 substitution (Table 1).
Growth curves for the six viruses demonstrated the same principle. Virions of parent Vs and the two mutants Vs-c and E(H)3'UTR(C) with H496 in the E gene accumulated in the extracellular medium more rapidly than virions of mutants with the R496 replacement (Fig. 2).
It is worth noting that the positively charged amino acid H496 is highly conserved among all tick-borne and nearly all mosquito-borne flaviviruses (Gritsun et al., 1995 ; Marin et al., 1995
) and maps closely to the tick-specific peptide EHLPTA (Shiu et al., 1991
). The exceptions are yellow fever virus and St Louis encephalitis virus, which have the amino acids D (negatively charged) and N (polar) in this position, respectively. In terms of the three-dimensional structure of the E protein, this amino acid maps within the dimerization domain at the beginning of the
-helix,
-A, situated deeply in the E protein (Rey et al., 1995
). It was suggested that this region could be involved in low pH-induced fusion of the E protein with endosomal membranes during virion entry into cells. Several point mutations in this conformational region affect the virulence of different flaviviruses, including mutations influencing the threshold pH for fusion leading to conformational changes (Holzmann et al., 1997
; Rey et al., 1995
). The substitution H496
R probably became possible as H and R are similar in size and both are positively charged, making this substitution look conserved, although it obviously affects the virulence characteristics of the virus.
Plaque assay and growth curve experiments did not demonstrate any biological effect of the T10884C10884 substitution in the highly conserved region of the 3'UTR. In both of these tests, mutant virus E(R)3'UTR(T), with only one wild-type substitution T10884 in the 3'UTR, was grouped with viruses containing C10884 in the 3'UTR. Nevertheless the small biological effect of this mutation was noticeable in the other tests, namely CPE, cytotoxicity and neuroinvasiveness tests (Table 1
). In the CPE (Fig. 4
) and cytotoxicity assays (Fig. 5
), virus E(R)3'UTR(T), with only one wild-type substitution T10884 in the 3'UTR, lysed PS cells with slightly less efficiency than its counterpart mutant virus E(H)3'UTR(C), with only one wild-type substitution H496 in the E protein, but more efficiently than pGGVs virus, with C10884 in the 3'UTR. The same intermediate biological effect of virus E(R)3'UTR(T) was revealed in the neuroinvasiveness test (Table 1
). Despite the fact that these effects were quite small, they were reproducible. The effect of the T10884
C substitution in the 3'UTR was similar to that produced by a long deletion in the 3'UTR of TBE virus (Mandl et al., 1998
). In both cases, clear plaques became turbid: plaque turbidity reflects incomplete cell lysis and is probably due to a delay in the rate of virus replication, resulting from poor recognition of the disturbed stemloop structures at the 3'UTR by the virus RNA polymerase complex. The substitution of wild-type T10884 for mutant C10884 in the 3'UTR probably has a similar effect on the rate of virus replication in cell culture.
The other three mutations mapped to less conserved regions of the virus genome, one in the C protein (I45F) and the other two in the NS5 protein (T2688
A and M3385
I). These mutations did not appear to have any effect on the rate of virus replication. Indeed, parental Vs virus and mutant Vs-c, both of which contained wild-type mutations H496 and T10884, produced the same biological effects in plaque assay, growth curve experiments, CPE test and cytotoxicity assay. Nevertheless, the differences between these two viruses were reproducible in neuroinvasiveness tests, where Vs-c virus showed a lower mortality rate and a longer AST. Therefore, these mutations in the C (I45
F) and NS5 (T2688
A and M3385
I) proteins, either individually or together, were also contributing to the delayed replication rate of the virus.
The main problem in this type of research is the recognition of small biological effects produced by point mutations. There are many publications demonstrating the clear effect of point mutations that map within different regions of flavivirus genomes (Gritsun et al., 1995 ; Hall et al., 1999
; Holzmann et al., 1997
; Kawano et al., 1993
; Lee et al., 2000
; Lindenbach & Rice, 1999
; Muylaert et al., 1997
; Pletnev et al., 1993
; Pryor et al., 1998
; Rey et al., 1995
; Stocks & Lobigs, 1998
; Valle & Falgout, 1998
; Xie et al., 1998
). Each of these mutations significantly changes plaque morphology, virus growth cycle, neurovirulence and neuroinvasiveness.
We have demonstrated that it may be possible to attenuate virus by introducing a number of point mutations, each of which causes a small effect on virus virulence. The advantage of using a strategy that utilizes the cumulative effect of these mutations is that reverse mutation would not result in increased virulence. Live attenuated virus vaccine, such as yellow fever 17D, was derived empirically and is known to contain point mutations that, individually, do not necessarily induce distinctive changes in virulence. In fact, it is believed that all available live attenuated virus vaccines are based on these principles, although it has never been formally demonstrated.
This study complements the ongoing research into the molecular basis of flavivirus attenuation and could eventually lead to the development of a safe and effective live attenuated vaccine against the TBE complex viruses.
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Received 9 November 2000;
accepted 14 March 2001.