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Journal of Virology, March 2008, p. 2218-2229, Vol. 82, No. 5
0022-538X/08/$08.00+0     doi:10.1128/JVI.02116-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Functional Analysis of Potential Carboxy-Terminal Cleavage Sites of Tick-Borne Encephalitis Virus Capsid Protein

Sabrina Schrauf,¹ Petra Schlick,^1, Tim Skern,² and Christian W. Mandl^1*

Clinical Institute of Virology,¹ Max F. Perutz Laboratories, Medical University of Vienna, Vienna, Austria²

Received 25 September 2007/ Accepted 15 December 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The mature capsid protein C of flaviviruses is generated through the proteolytic cleavage of the precursor polyprotein by the viral NS2B/3 protease. This cleavage is a prerequisite for the subsequent processing of the viral surface protein prM, and the concerted progression of these events plays a key role in the process of the assembly of infectious virions. Protein C of tick-borne encephalitis virus (TBEV) contains two amino acid sequence motifs within the carboxy-terminal region that match the canonical NS2B/3 recognition site. Site-specific mutagenesis in the context of the full-length TBEV genome was used to investigate the in vivo cleavage specificity of the viral protease in this functionally important domain. The results indicate that the downstream site is necessary and sufficient for efficient cleavage and virion assembly; in contrast, the upstream site is dispensable and placed in a structural context that renders it largely inaccessible to the viral protease. Mutants with impaired C-prM cleavage generally exhibited a significantly increased cytotoxicity. In spite of the clear preference of the protease for only one of the two naturally occurring motifs, the enzyme was unexpectedly tolerant to both the presence of a noncanonical threonine residue at position P2 and the position of cleavage relative to the adjacent internal prM signal sequence. The insertion of three amino acid residues downstream of the cleavage site did not change the viral phenotype. Thus, this study further illuminates the specificity of the TBEV protease and reveals that the carboxy-terminal region of protein C has a remarkable functional flexibility in its role in the assembly of infectious virions.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The genus Flavivirus, within the family Flaviviridae, comprises several important arthropod-transmitted pathogens, such as tick-borne encephalitis virus (TBEV) and the mosquito-borne flaviviruses yellow fever virus, Japanese encephalitis virus, West Nile virus, and the dengue viruses (52). Flaviviruses are small, spherical, enveloped viruses. The virions are composed of only three structural proteins: the capsid protein C and the two membrane-anchored surface proteins, M (derived from a precursor, prM) and E (29). Protein C associates with the viral genome, an unsegmented positive-stranded RNA molecule of approximately 11.5 kb in length, to form the nucleocapsid, which, in contrast to the strictly icosahedral arrangement of the viral surface proteins, does not appear to be regularly ordered (24). All of the viral proteins, the three structural proteins and seven nonstructural proteins, are encoded as a single polyprotein that is cotranslationally and posttranslationally processed by cellular proteases and a viral protease (NS2B/3) to form the individual proteins, which are arranged in the order C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5 (29).

Virion assembly involves a process in which the nucleocapsid buds through a region of the membrane of the endoplasmic reticulum (ER) containing heterodimers of proteins prM and E (33). However, due to the absence of any apparent interaction sites between the nucleocapsid and the surrounding viral envelope, it is difficult to understand which determinants provide specificity and efficiency to the assembly process. Preformed nucleocapsids usually are not detected in infected cells, suggesting that the maturation of protein C, the packaging of the genome, and budding occur (almost) simultaneously and are functionally closely interrelated processes (40). Two different cleavage events, which take place on opposite sides of the ER membrane, separate mature protein C from the adjacent protein prM. In the polyprotein, proteins C and prM are connected by an internal hydrophobic signal sequence that spans the ER membrane and is responsible for the translocation of protein prM into the ER lumen (42). There is substantial evidence indicating that the concerted progression of the two cleavage events on both ends of this signal sequence plays a key role in virion assembly (2, 25, 31, 51). The first cleavage is accomplished by the viral NS2B/3 protease, which resides on the cytoplasmic side of the ER membrane and separates mature protein C from its carboxy-terminal anchor (1, 53). This cleavage causes, presumably by allowing the signal sequence to move in the membrane toward the luminal side, a recognition sequence for the host protease signalase to become accessible to this enzyme, which subsequently liberates the amino terminus of protein prM by cleaving off the signal sequence (30, 50). Thus, the NS2B/3 cleavage at the carboxy terminus of protein C is fundamental to the entire assembly process. It controls not only the generation of anchorless, mature capsid protein but also the liberation of protein prM, which in turn is essential for the proper processing and export of protein E (23, 32).

The catalytic activity of the flaviviral protease resides in the amino-terminal domain of protein NS3, which shares homology with the protease trypsin (3, 8, 15). In contrast, protein NS2B functions as a cofactor and anchors the protease complex to the ER membrane via its hydrophobic domains (6, 9). The atomic structures of two flaviviral proteases, from dengue virus and West Nile virus, have been determined (12). Sequence comparisons reveal that the sequence motif recognized by flaviviral proteases is highly conserved throughout the genus and consists of a pair of basic amino acid residues (Lys-Arg or Arg-Arg) at the P2 and P1 positions (47) followed by a small nonbranched residue at the P1' position, such as Gly or Ser or, in some cases, Ala or Thr (5, 46). The functional importance of these conserved residues for cleavage has been demonstrated by studies both in vitro and in vivo (7, 27, 28, 41).

The carboxy-terminal NS2B/3 cleavage site in protein C of most mosquito-transmitted flaviviruses can be identified unambiguously by an inspection of the amino acid sequence (4). For yellow fever virus, the site also has been confirmed experimentally by site-directed mutagenesis (2). In contrast, the amino acid sequences of TBEV and other members of the tick-borne encephalitis serocomplex exhibit in the carboxy-terminal region of protein C the sequence motif Lys/Arg-Arg-Gly-Lys-Arg-Arg-Ser, which contains two potential NS2B/3 cleavage sites (underlined) separated by one amino acid (39). In this study, we investigated whether each of these sites is used during the assembly of TBEV virions and whether they can functionally substitute for each other.

Protein C shares considerably less sequence homology with different flaviviruses than the two surface proteins prM and E (37). Nevertheless, it still appears to have a conserved overall structural organization (35). In the absence of genomic RNA, it associates into homodimers in solution (16, 18). Atomic structures have been determined by nuclear magnetic resonance and crystallography for the C proteins of dengue 2 virus and Kunjin virus, respectively (10, 34). In both cases, the structure of most of the protein could be determined, and only an amino-terminal peptide was missing. These studies, in agreement with secondary structure predictions for the protein C sequences of different flaviviruses (20), indicated that each monomer contains four helices. The protein C dimer has a hydrophilic and highly positively charged surface that presumably interacts with the RNA. On the opposite side, a hydrophobic groove is found that has been proposed to interact with the viral membrane (34). Deletion analysis performed with TBEV revealed a striking functional flexibility of this protein. Large deletions could be functionally compensated for by single point mutations or sequence duplications (20, 22). In one case, infectious virus progeny was obtained even though almost a third of the mature protein C amino acid sequence had been deleted. A similar flexibility also was recently observed for the capsid proteins of other flaviviruses (45, 55). In general, the constraints of the flaviviral capsid protein to function in genome packaging and particle assembly appear to be remarkably relaxed.

This study extends the functional characterization of TBEV protein C by investigating the sequence requirements in the carboxy-terminal domain containing the two potential NS2B/3 cleavage sites. This domain, which presumably plays a crucial role in the assembly of infectious virions, had not been scrutinized in any of the previous mutagenesis studies. Here, we used site-specific mutagenesis in the context of the full-length infectious genome to clarify the role of the two potential NS2B/3 cleavage sites in the formation of virions in vivo. Clear evidence was obtained for the functional superiority of the downstream site. Mutations disabling the upstream site were fully tolerated; in contrast, mutations in the downstream site provoked spontaneously emerging second-site reversion mutations. The analysis of such revertants revealed a remarkable variability of sequences and locations that could be utilized by the viral protease to yield infectious virus progeny. The analysis of these mutations together with additional point mutations and sequence insertions by reverse genetics has now allowed us to further define the sequence requirements for the functional cleavage of protein C.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Virus and cells. Western subtype TBEV strain Neudoerfl, which has been characterized in detail, including the determination of its entire genomic sequence (37, 38) (GenBank accession no. U27495), was used as the wild-type control in all experiments, and all mutants described in this study were derived from this strain.

BHK-21 cells were grown under standard conditions in Eagle's minimal essential medium supplemented with 5% fetal calf serum (FCS), 1% glutamine, and 0.5% neomycin (growth medium) and were maintained in Eagle's minimal essential medium supplemented with 1% FCS, 1% glutamine, 0.5% neomycin, and 15 mM HEPES, pH 7.4 (maintenance medium).

Plasmids and cloning procedures. Plasmid pTNd/c contains a full-length genomic cDNA insert of TBEV strain Neudoerfl (36). RNA transcribed from this plasmid was used as the wild-type control in quantitative experiments. Plasmids pTNd/5' and pTNd/3' contain cDNAs corresponding to the 5' one-third and 3' two-thirds of the genome, respectively (36), and were used to generate infectious full-length RNA after in vitro ligation. In addition, pTNd/5' was utilized for the construction of all mutants generated for the present study. The mutant C(28-89)-S, which is deficient in the production of infectious virus particles due to a large deletion within the capsid protein (19), was used as another control in some experiments. Unlike the originally described clones, the clones pTNd/c (and derivatives) and C(28-89)-S carried a hepatitis delta ribozyme sequence (49) at the 3' end of the viral cDNA insert, which was added to facilitate the proper formation of the 3' end of the in vitro-transcribed runoff RNA.

All mutations were introduced into plasmid pTNd/5' by using the GeneTailor site-directed mutagenesis system (Invitrogen). Amino acid changes generally were engineered by mutating two nucleotides within a single codon to reduce the likelihood of spontaneous back mutations to the wild-type sequence. The mutated 5' cDNA clones were further ligated in vitro with the supplementary cDNA clone pTNd/3' to obtain full-length cDNAs, which were used for functional and qualitative analyses of the mutants. For a more detailed characterization (that is, a quantitative comparison between the mutants and the wild type), the mutations were transferred into the full-length cDNA clone pTNd/c by taking advantage of two unique restriction sites, namely, SalI, located upstream of the TBEV 5' end, and SnaBI, at position 1880 in the TBEV genome sequence.

All plasmids were amplified in Escherichia coli strain HB101, and small- and large-scale plasmid preparations were made by using Qiagen purification systems. Sequence analysis of the region coding for the structural proteins up to the unique ClaI site was performed with an automated DNA sequencing system (ABI) to confirm that only the desired mutations were present in the plasmids.

RNA transcription and transfection. In vitro RNA transcription and transfection of BHK-21 cells by electroporation were performed as described previously (21, 36, 43). RNA was synthesized from full-length cDNA clones or from in vitro-ligated templates by using reagents of the T7 Megascript kit (Ambion) according to the manufacturer's protocol. The template DNA was digested by DNase I incubation, and the quality of the RNA was checked by agarose gel electrophoresis. For quantitative analysis, RNA derived from full-length cDNA clones was purified using an RNeasy Mini kit (Qiagen) and was quantified spectrophotometrically. Equimolar amounts of RNA then were introduced into BHK-21 cells by electroporation using a Bio-Rad Gene Pulser with previously described settings (11, 43).

Detection of viral proteins. The expression of viral proteins was analyzed by immunofluorescence staining and Western blotting.

For immunofluorescence staining, RNA-transfected BHK-21 cells were seeded in 24-well tissue culture plates containing microscope coverslips, incubated under standard conditions, and permeabilized by acetone-methanol fixation (1:1) 24 and 48 h posttransfection. Intracellular protein E expression was visualized by successive incubation with a rabbit polyclonal anti-TBEV serum predominantly recognizing the structural protein E and fluorescein-isothiocyanate-conjugated anti-rabbit antibody (Jackson Immune Research Laboratory). For passaging experiments, fresh BHK-21 cells were inoculated with virus-containing supernatants, and viral protein expression was detected as described above 24 and 48 h postinfection.

Western blot analysis was used to monitor the processing of the polyprotein at the C-prM junction. BHK-21 cells were transfected with in vitro-transcribed RNA, disseminated in 6-well plates, and incubated for about 20 h under standard conditions. The cells then were washed with phosphate-buffered saline and lysed in Frackelton buffer (10 mM Tris, 50 mM NaCl, 30 mM NaPP_i, 50 mM NaF, and 1% Triton X-100) supplemented with 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride, and protease inhibitors at 4°C. Extracts were cleared by centrifugation at 14,000 rpm at 4°C for 5 min and boiled for 10 min at 95°C after the addition of Laemmli sample buffer. The cell lysate proteins were separated under sodium dodecyl sulfate (SDS) denaturating conditions in a 15% polyacrylamide gel and were transferred by being immunoblotted onto a polyvinylidene difluoride membrane with a Bio-Rad Trans-Blot semidry transfer cell. Viral proteins were detected by consecutive incubation with a rabbit polyclonal anti-TBEV serum that recognizes all three structural proteins (C, prM, and E) and anti-rabbit-immunoglobulin G-alkaline peroxidase (Amersham). Protein bands were visualized by an immunoenzymatic reaction using the SigmaFast DAB tablets system.

Focus assay. To quantify the production of infectious virus particles, an immunochemical focus assay was carried out. BHK-21 cells transfected with equal amounts of RNA were seeded into 25-cm² tissue culture flasks, and supernatants were collected at various time points. Various dilutions of supernatants were applied to confluent monolayers of BHK-21 cells. After a 4-h incubation, supernatants were removed and cells were covered with 3% carboxymethyl cellulose overlay dissolved in maintenance medium. Fifty hours postinfection, cells were fixed with acetone-methanol and treated with polyclonal rabbit anti-TBEV serum. Antibody-labeled cells were detected by an immunoenzymatic reaction consisting of successive incubations with goat anti-rabbit immunoglobulin G-alkaline phosphatase and the corresponding enzyme substrate (SigmaFast Red TR/Naphtol AS-MX tablets).

RNA replication and RNA export. BHK-21 cells were transfected with equimolar amounts of RNA (8.7 x 10¹¹ RNA molecules) by electroporation. To get rid of noninternalized RNA, cells were washed four times by suspending them in 20 ml growth medium and collecting them again by low-speed centrifugation. Subsequently, approximately 1 x 10⁶ cells resuspended in growth medium were seeded into 25-cm² tissue culture flasks. At 12 h posttransfection, the medium was replaced by maintenance medium.

RNA replication and export kinetics were analyzed by quantitative real-time PCR (qPCR). For the determination of intracellular RNA copy numbers, cells were collected at individual time points and counted in a Casy TT cell counter (Schärfe Systems). Cytoplasmic RNA was purified from a defined number of cells using an RNeasy Mini kit and subjected to qPCR as described in a previous study (21). For the measurement of viral RNA export, aliquots of supernatants were harvested at the same time points as those used for intracellular RNA levels and were cleared from cell debris and insoluble material by centrifugation. Next, 140 µl of the supernatant was incubated with 35 µl 5x RLN lysis buffer (250 mM Tris-Cl, pH 8.0, 700 mM NaCl, 7.5 mM MgCl₂, 2.5% [vol/vol] Nonidet P-40, 5 mM DTT) for 1 min on ice to break up the viral membrane. Viral RNA was further isolated from one-fifth of each lysate, again using an RNeasy Mini kit by following the protocol utilized for intracellular RNA purification. One-fifth of the isolated RNA then was used as a template for cDNA synthesis using an iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer's protocol. An aliquot corresponding to 2.5 µl of the original cell culture supernatant was subjected to qPCR using the same conditions as those for the quantification of intracellular RNA (21). The amounts of intra- and extracellular RNA were quantified by the comparison of the results to a standard curve. The standard curve was prepared by using a serial 10-fold dilution of spectrophotometrically quantified, purified, in vitro-synthesized RNA. The total amount of RNA present in each culture flask (intra- and extracellular) was further calculated from the measured RNA concentrations to determine the percentage of total RNA exported into the supernatant.

Cytotoxicity assay. The disintegration of cells was quantified by spectrophotometrically measuring the concentration of the enzyme lactate dehydrogenase (LDH) in cell culture supernatants using a commercial cytotoxicity assay (Promega). The obtained absorbance values (at 490 nm) were corrected by subtracting the value obtained from mock-transfected cells. The resulting value, multiplied by 100, was plotted in arbitrary units.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Only mutations in the downstream cleavage site interfere with C-prM processing. The capsid protein sequence of TBEV contains two potential carboxy-terminal NS2B/3 cleavage motifs: the upstream cleavage site K₉₁-R₉₂-G₉₃ and the downstream site R₉₅-R₉₆-S₉₇ (Fig. 1A). Cleavage of protein C by the viral protease is believed to be a crucial step in particle assembly. Therefore, we wanted to determine whether both of the two adjacent sites are functional, whether one of them is principally utilized in the context of in vivo polyprotein processing, and whether they are able to functionally substitute for each other. To this end, mutations of charged residues to alanines (referred to as charged-to-alanine mutations hereafter) were introduced into one or the other of the cleavage motifs of the TBEV genome as depicted in Fig. 1B.

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FIG. 1. Engineered and acquired mutations in the C-terminal region of protein C. (A) Schematic drawing of the TBEV genome (not drawn to scale) and expanded views of the capsid protein (with its four helices and the prM signal sequence) and the amino acid sequences of the two canonical NS2B/3 cleavage motifs (underlined; predicted cleavage positions are indicated by arrows). Numbers at the top of the sequence refer to the amino acid positions within protein C. (B) Engineered mutations are shown together with the corresponding mutant designations. Spontaneous second-site mutations that arose after propagation in BHK-21 cells are shown on the right. (C) Alternative NS2B/3 cleavage sites as generated by engineered (red) and acquired (green) mutations (dashed underline; predicted cleavage positions are indicated by arrows). The corresponding mutant designations are listed on the right. Wt, wild type; NCR, noncoding region.

To test the protein expression of the constructs, full-length RNAs were transcribed from in vitro-ligated partial mutant or wild-type cDNA clones and introduced into BHK-21 cells by electroporation. Intracellular protein E was visualized by immunofluorescence staining 1 and 2 days posttransfection. As illustrated in the left column of Fig. 2, about 80% of the cells transfected with mutant RNAs or control RNA exhibited bright fluorescence due to protein E expression 1 day posttransfection. Previous studies have shown that the visibility of protein E expression by immunofluorescence depends on both RNA replication and protein translation (20). This result therefore indicates that all of the mutants were competent for both of these functions. Moreover, this result confirmed that the BHK-21 cells had been transfected with approximately equal efficiency with all of the mutants and the wild-type control. However, 2 days posttransfection (Fig. 2, middle column), a significantly different picture was observed. In this case, protein E staining was now positive for practically 100% of the cells in the case of wild-type virus and the mutants carrying substitutions in the upstream site (K91A, R92A, and K91A-R92A), but in contrast, only about 30% of the cells transfected with mutants carrying mutations in the downstream site (R95A, R96A, R95A-R96A, and K94A-R95A-R96A) showed protein E expression. An inspection of these cell cultures by phase-contrast microscopy indicated a more severe cytopathic effect (CPE) and increased cell death (data not shown).

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FIG. 2. Immunofluorescence analysis of protein expression and viral infectivity. BHK-21 cells were transfected with wild-type (WT) or mutant RNAs or were mock transfected as indicated on the left. Protein expression was detected with a polyclonal serum 1 and 2 days posttransfection (left and middle columns). Supernatants harvested 6 days posttransfection were transferred onto fresh cells as indicated by arrows, and the infection of the cells was examined by immunofluorescence staining 2 days later (right column).

To directly assess any deficiencies in protein processing caused by the mutations, cleavage at the C-prM junction was examined by Western blotting. Cells transfected with various RNAs were lysed 24 h after transfection, and viral proteins were detected by using a polyclonal serum raised against structural proteins of TBEV. The results shown in Fig. 3A are in good agreement with the previous observations. All of the upstream mutants (K91A, R92A, and K91A-R92A) exhibited prominent bands corresponding to the three structural proteins, E, prM, and C, that were indistinguishable from the pattern seen with the wild-type control. In contrast, the four downstream site mutants (R95A, R96A, R95A-R96A, and K94A-R95A-R96A) exhibited a different pattern, with only faint C and prM bands. Instead, a new band was seen with a size corresponding to that of the C-prM precursor protein. These results indicate that the mutations in the downstream site indeed impaired processing at the C-prM junction. As observed previously, the fractionation of protein C on SDS gels yields an atypical migration due to the high content of basic amino acids. The replacement of charged residues with nonpolar Ala residues resulted in a somewhat faster migration of the mutant proteins (Fig. 3).

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FIG. 3. Western blot analysis of structural protein processing. Cells transfected with wild-type (WT) or mutant RNAs were lysed 24 h posttransfection, and structural proteins were detected using a polyclonal serum. (A) Analysis of the original set of mutants. (B) Analysis of two mutants alongside the corresponding revertants. The positions of the structural proteins E, prM, and C and the uncleaved precursor protein C-prM are marked on the right. Protein C mutants carrying charged-to-alanine mutations migrated slightly faster than the wild-type protein. (The picture was edited with Adobe Photoshop to change the order of the lanes and to exclude unrelated samples. The lanes of each picture were from the same gel, and the relative positions and band intensities were not altered.)

In conclusion, the results indicate that charged-to-alanine mutations in the downstream site R₉₅-R₉₆-S₉₇, but not in the upstream site K₉₁-R₉₂-G₉₃, interfere with cleavage at the C-prM junction of TBEV. Additionally, these data suggest that the upstream cleavage site can substitute only to a very minor degree (or not at all) for the loss of the downstream one. It is therefore reasonable to assume that the NS2B/3 protease predominantly uses the downstream cleavage motif R₉₅-R₉₆-S₉₇ for cleavage.

Compensating mutations emerge in downstream cleavage site mutants. In order to characterize the ability of the mutants to produce infectious progeny, passaging experiments on BHK-21 cells were performed. Supernatants harvested 6 days after the transfection of cells with various mutant or wild-type RNAs were transferred onto fresh BHK-21 cells, and the infection of these cells was monitored by immunofluorescence 2 days posttransfection. From the previous observations, one might have expected to see a clear difference in the infectivity of upstream and downstream site mutants. However, as illustrated in Fig. 2, right column, all of the mutants except R96A infected the cells efficiently and yielded a bright immunofluorescence staining. Moreover, even the downstream mutants caused only a moderate CPE on the cells of the first passage.

To investigate whether any sequence changes had occurred in the course of the passaging experiment that may have restored the infectivity and reduced the cytotoxicity of the downstream mutants, viral RNA was isolated from supernatants 3 days postinfection of the first passage, and the entire structural protein-coding region (C-prM-E) was sequenced by reverse transcription-PCR (RT-PCR). No additional changes were found in the upstream mutants; however, three of the four downstream mutants had acquired single point mutations (Fig. 1C). In one mutant (R95A), the original mutation was found to have changed from Ala to Thr. Thus, the original cleavage site, R₉₅-R₉₆*S₉₇, had become T₉₅-R₉₆*S₉₇. In two other mutants (R95A-R96A and K94A-R95A-R96A), the same mutation (G93R) was identified. This mutation generated new potential cleavage motifs upstream of the destroyed one, namely, R₉₃-K₉₄*A₉₅ and R₉₂-R₉₃*A₉₄ (Fig. 1C). In contrast, no additional mutation was identified for the fourth downstream site mutant, R96A. Sequence analysis of the NS2B/3 region also excluded the occurrence of a compensatory mutation in the protease region. Furthermore, this mutant stayed genetically stable even after several additional rounds of cell culture passages. An inspection of the sequence of this mutant indicated that the R96A mutation by itself generated a potential alternative cleavage site (K₉₄-R₉₅*A₉₆), as depicted in Fig. 1C. The genetic stability of this mutant suggests that although this site may be used by the protease with only low efficiency (Fig. 3), cleavage still suffices to relieve the selection pressure for additional mutations under the chosen experimental conditions.

To directly assess whether the selected mutations indeed improved cleavage efficiency at the C-prM junction, the mutants R95T and G93R-K94A-R95A-R96A were generated by reverse genetics and analyzed alongside the original mutants R95A and K94A-R95A-R96A by Western blotting (Fig. 3B). In contrast to the pattern observed for the original mutants, the two revertants exhibited distinct bands for proteins C and prM, indicating that the selected mutations recovered efficient processing at the C-prM junction. With respect to the mutations at position 95, this suggests that the motif T₉₅-R₉₆*S₉₇ is an acceptable substrate for the protease. This contrasts markedly with findings for the originally engineered sequence A₉₅-R₉₆*S₉₇. However, a C-prM precursor band still was clearly discernible in mutant R95T, suggesting that its processing still was less efficient than that for the wild-type sequence R₉₅-R₉₆*S₉₇. Further investigation of the unusual cleavage site with a Thr residue at the P2 position is presented in a separate section below.

The results of the passaging experiments were confirmed by several independent experiments, which again yielded the same spontaneous mutations in three of the four downstream mutants and no mutations in the upstream mutants. Taken together, this indicates that the disruption of the downstream site provoked the emergence of reversion mutations that generated new cleavage sites, albeit at different positions and, in the case of the R95T mutant, with an atypical sequence motif. This suggests that the upstream cleavage motif in these mutants was not sufficient to substitute for the loss of the downstream motif. In contrast, mutations in the upstream motif did not provoke the emergence of any spontaneous mutations, suggesting that these mutations had no significantly negative effect on viral viability.

RNA packaging and virion assembly are impaired by downstream site mutations. Until now, the characterization of our set of mutants had indicated that the downstream cleavage site of TBEV protein C plays a predominant role in processing at the C-prM junction. This cleavage is thought to be crucial for the assembly of infectious particles and viral viability. Therefore, we wanted to quantitatively assess the impact of selected mutations on the packaging and export of viral RNA and the formation of infectious virus progeny. The mutants R95A, R96A, and K94A-R95A-R96A were chosen for these analyses and were compared to wild-type virus as a positive control. As a further control, the previously characterized replicon C(28-89)-S was included in these experiments. This replicon is fully competent for autonomous RNA replication but defective for RNA packaging and the release of infectious virions. Instead, cells transfected with this construct release capsidless subviral particles (19). To avoid experimental variation, which is inevitable when RNAs are transcribed from in vitro-ligated partial cDNA templates (as in the initial experiments), these and all subsequent quantitative experiments were performed exclusively with quantified and standardized RNA preparations transcribed from full-length cDNA clones into which the desired mutations had been engineered.

To assess RNA packaging, BHK-21 cells were transfected with equimolar amounts of RNAs, and the concentrations of viral RNA inside the cells and in the supernatants were monitored separately by qPCR over a time period of 72 h. As shown in Fig. 4A, intracellular RNA concentrations were almost indistinguishable for all of the mutants and the two controls at early time points (3 and 24 h posttransfection), indicating that RNA replication, as expected, was not affected by the mutations in protein C. At later times, somewhat higher levels were measured in cells infected with the wild-type virus than for the C(28-89)-S replicon control. This reflects the ability of the infectious wild-type virus to cause secondary rounds of infection. In contrast, the noninfectious replicon could not spread in the cell culture, and consequently the percentage of cells harboring the replicon decreased over time due to cell death. The three mutants exhibited values between those of the two controls. Figure 4B shows the release of RNA from the cells into the supernatants. The RNA levels released from cells infected with wild-type virus quickly reached a plateau value of approximately 10⁷ RNA equivalents per 2.5 µl, whereas in the case of the packaging-deficient replicon control, RNA levels remained around a background value of approximately 1,000 RNA equivalents per 2.5 µl. This background value probably originates from residual input RNA that could not be completely washed away after the transfection procedure. Furthermore, at later time points the background also may reflect RNA that is released nonspecifically from disintegrating cells.

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FIG. 4. Time course analyses of mutants with mutations in the downstream cleavage site (A to D) and corresponding revertants (E to H) after the transfection of BHK-21 cells with these RNAs. The export-deficient mutant C(28-89)-S was included as a replication-competent but export-deficient control. (A and E) Monitoring of RNA replication by measuring intracellular RNA concentrations by qPCR. (B and F) Monitoring of RNA export by measuring RNA concentrations in the supernatants by qPCR. (C and G) Analysis of RNA export efficiencies. The percentage of total RNA released into the supernatants was calculated from the intracellular and extracellular RNA concentration data. (D and H) Release of infectious particles. Infectivity titers were determined using a focus assay. All data points represent geometric mean values from two independent experiments. The error bars indicate the standard deviations. In one of the experiments shown in panel B, the RNA was not sufficiently removed by the washing steps after transfection, resulting in elevated values and stronger data variation at the 3-h time point (marked by an asterisk). WT, wild type.

The three cleavage site mutants released between four orders of magnitude (at the 24-h time point) and two orders of magnitude (at 72 h) less RNA than the wild-type control. However, RNA release still was clearly above the background level at the later time points. Combining the numbers obtained for intra- and extracellular RNA levels permits the calculation of the export efficiency in terms of the percentage of total RNA exported to the supernatants. This evaluation yields an informative representation of the results, as shown in Fig. 4C, and illustrates a severe packaging deficiency of the cleavage site mutants. For wild-type virus, in agreement with previous studies, between approximately 60% (at 24 h) and 90% (at 72 h) of the total RNA is exported from the infected cell, presumably packaged into virions. The packaging-deficient replicon control exported less than 1% of its RNA at all times. The export of the cleavage mutants was strongly delayed and reduced and finally reached values of only 20 to 25%. The late onset of RNA export may (particularly in the case of mutant R96A) reflect a slow but still active processing of the polyprotein at the mutated cleavage site, or it may be (particularly in the cases of the two other mutants) a sign of the emergence of revertants.

To evaluate the ability of the mutants to form infectious virus progeny, the infectious titers of the supernatants were determined using a focus assay. The results shown in Fig. 4D illustrate the impairment of the mutants in the assembly and release of infectious virions. Wild-type virus quickly reached a plateau level between 10⁶ and 10⁷ focus-forming units (FFU) per ml, while the replicon control remained negative. In contrast, the mutants did not export significant amounts of infectious particles before the 48-h time point and reached titers of only 10⁴ FFU per ml at 72 h. Again, the significant lag phase observed for the assembly and release of virions can be interpreted in terms of a slow and inefficient C-prM cleavage or the emergence of revertants. To check for the possibility that revertants arose even within the short time frame of this experiment, RNAs harvested at the 72-h time point were subjected to RT-PCR analysis. Indeed, mixed sequence patterns, clearly indicating the presence of subpopulations with the same sequence changes as those observed before (Fig. 1C), were detected for mutants R95A and K94A-R95A-R96A but not R96A (data not shown).

In summary, these data demonstrate that the impairment of the C-prM cleavage by mutations in the downstream cleavage site resulted in a severe deficiency of those mutants for the packaging of RNA and the release of infectious virus particles.

RNA packaging and virion assembly are restored by second-site mutations. To assess whether the observed reversion mutations by themselves were capable of restoring the packaging and assembly of TBEV, full-length cDNA clones corresponding to the sequences of the revertants R95T and G93R-K94A-R95A-R96A (Fig. 1C) were constructed, and the derived RNAs were subjected to the same quantitative tests as those described in the previous section. The results, summarized in Fig. 4E to H, demonstrate that RNA packaging and virion production were indeed largely restored by both of these mutations. The G93R mutation successfully restored all parameters to levels almost indistinguishable from that of the wild-type control. Furthermore, multistep growth curves using low multiplicities of infection (MOI; 0.1 or 0.01) of this mutant exhibited a wild-type growth capacity (data not shown). The R95T mutation, however, was not as successful in restoring a wild-type phenotype. The R95T revertant still exhibited a small but distinct delay in the export of RNA (Fig. 4F and G) and infectious particles (Fig. 4H). It is reasonable to assume that cleavage at the atypical sequence motif T₉₅-R₉₆*S₉₇ was somewhat less efficient than that at the R₉₃-R₉₄*A₉₅ motif present in mutant G93R-K94A-R95A-R96A. RT-PCR sequence analysis of RNA isolated 96 h posttransfection confirmed the integrity of the nucleotide sequence of the entire structural protein coding region, indicating that both mutants were genetically stable throughout the course of the experiment.

Taken together, these data support the view that the reversion mutations restored the coordinated processing at the C-prM junction and the dependent processes of RNA packaging and assembly of infectious particles.

Thr at position P2 yields a functional but context-dependent cleavage site. The highly conserved consensus motif recognized by the flaviviral NS2B/3 protease has positively charged amino acid residues at positions P1 and P2. Therefore, the emergence of a cleavage site revertant carrying the uncharged residue Thr at the P2 position was unexpected and prompted us to characterize the sequence requirements of this mutant in more detail. First, we wanted to clarify whether the atypical T-R*S motif alone was indeed sufficient for the production of infectious virus progeny or whether the potential upstream cleavage site played a functional role in the particular context of this mutant. Second, the role of the Lys residue in position P3 of the cleavage motif was addressed. Normally, position P3 of flaviviral NS2B/3 cleavage sites is not restricted to specific amino acids. However, a sequence comparison among various flaviviruses reveals a clear preference for basic amino acids at position P3 in the cleavage site at the C-prM junction (data not shown). To address these issues, the set of four mutants shown in Fig. 5A as well as the wild-type and replicon controls were subjected to quantitative testing of RNA export and virion production as described in the previous sections. The mutants included the already described revertant R95T, with its T₉₅-R₉₆*S₉₇ cleavage motif, and mutant K91A-R92A, which has a wild-type downstream cleavage site, but its upstream cleavage site is disabled by two charged-to-Ala mutations. In addition, two new constructs were generated that combined the T₉₅-R₉₆*S₉₇ cleavage motif with the mutations in the upstream site (mutant K91A-R92A-R95T) and, in the case of mutant K91A-R92A-K94A-R95T, had an additional charged-to-Ala mutation in position P3 (Fig. 5A).

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FIG. 5. Characterization of mutants containing threonine at position P2 of the cleavage site. (A) Schematic diagram illustrating the analyzed viruses using the same outline as that used for Fig. 1. The export-deficient mutant C(28-89)-S was included as a replication-competent but export-deficient control. (B) Monitoring of RNA replication by measuring intracellular RNA concentrations by qPCR. (C) Monitoring of RNA export by measuring RNA concentrations in the supernatants by qPCR. (D) Analysis of RNA export efficiencies. The percentage of total RNA released into the supernatants was calculated from the intracellular and extracellular RNA concentration data. (E) Release of infectious particles. Infectivity titers were determined using a focus assay. (F) Specific infectivity determination. The ratio of RNA equivalents per infectious unit was calculated for the 48-h time point. All data points represent geometric mean values from two independent experiments, with error bars indicating standard deviations. (The controls shown in panels B to F are the same as those shown in Fig. 4E to H, as these experiments were done concurrently.) WT, wild type.

All of the mutants were indistinguishable from the wild-type virus control with respect to intracellular RNA levels, indicating no impairment of RNA replication (Fig. 5B). Moreover, all of the mutants showed significant export of RNA (Fig. 5C and D) and infectious virions (Fig. 5E), indicating that the upstream cleavage site was not essential even in the context of the downstream cleavage site mutated to T₉₅-R₉₆*S₉₇. This observation provides evidence that this atypical cleavage site actually is utilized in vivo and is sufficient to permit the production of infectious virions. However, there were clear quantitative differences in packaging and virion release. Mutation of the upstream cleavage site in the context of a wild-type downstream cleavage site (mutant K91A-R92A) did not affect the export of RNA and infectious particles at all (Fig. 5). In addition, growth curve analyses using low MOIs revealed no differences for the wild-type control (data not shown). These results are in good agreement with the observations reported above. In contrast, the same mutation did impair these functions in the context of the T₉₅-R₉₆*S₉₇ downstream cleavage site. As already described above, the export kinetics of mutant R95T were delayed compared to those of the wild-type control. This delay was worsened in the presence of the mutations in the upstream cleavage site. In mutant K91A-R92A-K94A-R95T, which had the additional mutation at position P3, this effect was even more pronounced. The maximum titers for mutants K91A-R92A-R95T and K91A-R92A-K94A-R95T were only 10^5.5 and 10⁴ FFU per ml, respectively, compared to 10⁷ for the wild-type control. Notably, foci formed by these two mutants were significantly smaller than those formed by the wild-type control (data not shown). In Fig. 5F, the ratio of RNA equivalents (as derived from data shown in Fig. 5C) to infectious units (as derived from data shown in Fig. 5E) is plotted for the 48-h time point. According to this evaluation, this ratio is significantly higher in the cases of mutants K91A-R92A-R95T and especially K91A-R92A-K94A-R95T than for the wild-type control and the two other mutants. In other words, the specific infectivity of these two mutants also is impaired, an observation that is compatible with the view of a disturbed assembly process that may lead to the aberrant production of noninfectious virions.

In summary, it appears that the basic residues upstream of the T₉₅-R₉₆*S₉₇ motif were not essential but did have an augmenting effect on packaging and assembly, whereas such an effect was not observed in the context of the wild-type R₉₅-R₉₆*S₉₇ downstream cleavage motif.

Insertion mutations downstream of the cleavage site have no effect on viral phenotype. The analysis of the revertants described above suggested that the position of the NS2B/3 cleavage relative to the adjacent signal sequence was somewhat flexible. On the basis of this observation, we wanted to investigate the effects of insertions downstream of the unchanged cleavage site R₉₅-R₉₆*S₉₇. Such insertions (Fig. 6A) would effectively elongate the signal peptide and thus increase the distance between R₉₅-R₉₆*S₉₇ and the signalase cleavage site on the luminal side of the ER membrane. Two mutants carrying insertions of three Ala residues or the motif Arg-Ala-Arg therefore were constructed (Fig. 6A). The strictly hydrophobic insert is predicted to locate inside the membrane; in contrast, the positively charged residues likely are positioned in the water-lipid interface. An analysis of the C-prM cleavage by Western blotting yielded the same cleavage pattern as that of the wild-type virus control, indicating that the insertions did not interfere with NS2B/3 cleavage (data not shown). Similarly, the quantitative analysis of RNA replication (Fig. 6B), RNA export (Fig. 6C), and infectivity titers in the supernatants (Fig. 6D), as well as multistep growth curve analyses using an MOI of 0.1 or 0.01 (not shown), revealed no impairment of these mutants relative to the levels for the wild-type control.

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FIG. 6. Characterization of mutants with sequence insertions. (A) Schematic diagram showing the insertion sites. The position of the insertions between positions 98 and 99 is indicated. The export-deficient mutant C(28-89)-S was included as a replication-competent but export-deficient control. (B) Monitoring of RNA replication by measuring intracellular RNA concentrations by qPCR. (C) Monitoring of RNA export by measuring RNA concentrations in the supernatants by qPCR. In one of the experiments, the RNA was not sufficiently removed by the washing steps after transfection, resulting in elevated values and stronger data variation at the 3-h time point (marked by an asterisk). (D) Release of infectious particles. Infectivity titers were determined using a focus assay. All data points represent geometric mean values from two independent experiments, with error bars indicating standard deviations. (The controls shown in panels B to D are the same as those shown in Fig. 4A to D, as these experiments were done concurrently.) WT, wild type.

Taken together, the insertion mutations had no measurable effect on the viral life cycle, suggesting that the number of amino acids between the capsid cleavage site and the signalase cleavage site can be increased without causing a negative effect on the regulated processing events at the C-prM junction.

Disturbance of C-prM processing correlates with an increase in cytotoxicity. Throughout the course of the analysis of various mutations in the carboxy-terminal region of protein C, the inspection of cells with the light microscope indicated that mutants with an impairment of C-prM processing caused a more distinct CPE than other mutants or the wild-type virus (an example of this observation can be seen in Fig. 2, middle column). We wanted to confirm this and evaluate this correlation with a quantitative test. For this purpose, the concentration of LDH released from disintegrating cells was measured in the supernatants harvested from BHK-21 cells transfected with standardized concentrations of the various RNAs described above. LDH concentrations generally still were similar at 24 h posttransfection (not shown); clear differences, however, were detected at the 48-h time point (Fig. 7) and were in good agreement with the observations from the light microscope. All mutants that in previous experiments had been shown to be impaired for C-prM cleavage or the dependent processes of RNA packaging and virion assembly exhibited an elevated LDH level at this time point, suggesting that viral cytotoxicity was indeed increased by disturbances of the processing events at the C-prM junction.

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FIG. 7. CPE in BHK-21 cells. The concentration of the enzyme LDH, which is released upon cell lysis, was measured in supernatants of cells transfected with wild-type (WT) or mutant RNAs 48 h posttransfection. Absorbance values were corrected by subtracting the background value obtained for mock-transfected cells and were plotted as arbitrary units (a.u.). The graph shows data that are assembled from two typical experiments. The shading of the bars reflects the previously determined C-prM cleavage phenotypes (Fig. 3) of the indicated viruses: black, impaired cleavage; white, efficient cleavage; gray, intermediate phenotype.

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The concerted cleavage events at the junction of proteins C and prM are believed to play an important role in the process of the assembly of flavivirus virions (2, 25, 51). In the absence of a nucleocapsid, the surface proteins prM and E are capable of budding off by themselves into the lumen of the ER and forming empty subviral particles (13). The mechanisms by which an efficient incorporation of nucleocapsids into viral envelopes is ensured are largely enigmatic. It is noteworthy that virion assembly also can take place when protein C and the surface proteins prM and E are translated from different translation units instead of the natural single-polyprotein precursor. This was achieved in experimental setups in which various replicons were packaged into single-round infectious particles by providing the surface proteins in trans (14, 17, 48, 54). An alternative approach used an infectious bicistronic TBEV construct that had the coding regions for proteins prM and E placed into a separate coding unit (43, 44). Nevertheless, there is convincing evidence that, during the natural virus infection, the order of cleavage events at the C-prM junction (i.e., the viral NS2B/3 protease cleaves first, and this enables cleavage by host signalase) plays an important role for achieving a specific and efficient assembly of infectious virions (35). Therefore, we wished to study these processes in the natural context of the full-length genome to obtain relevant information on the correlation between cleavage and the incorporation of the nucleocapsid into infectious virions.

It was intriguing that the TBEV protein C sequence contained within its carboxy-terminal domain two different sites that perfectly matched the consensus sequence for NS2B/3 cleavage, thus making it difficult to predict which of them actually is used in the context of a natural infection. The results obtained in this study consistently indicate that the downstream site is predominantly used. Although our data cannot rule out the possibility that, to a minor degree, cleavage also occurs at the upstream site, it is still clear that this cleavage is not sufficient to compensate for the loss of the downstream site, as demonstrated by the immediate emergence of reversion mutations generating new cleavage sites. Although the exact cleavage sites were not determined experimentally, the correlation of the emergence of new canonical sequence motifs with the restoration of cleavage and virion assembly provides strong, albeit indirect, evidence for the functionality of these motifs. Remarkably, some of the reversions created cleavage sites two or three residues upstream from the original site, which had been destroyed by the engineered mutations (Fig. 1C). In fact, the predicted position of cleavage in mutant G93R-K94A-R95A-R96A is only one residue downstream from the natural upstream site. It is puzzling that this newly formed site was efficiently used for cleavage and provided the revertant with an almost wild-type phenotype. In contrast, the naturally occurring site, which completely matches the criteria for an NS2B/3 cleavage site and is only a single position upstream, was incapable of doing so. An inspection of various flavivirus sequences reveals the presence of canonical NS2B/3 sequence motifs in some of these viruses that are even further upstream (4, 37). Apparently, these sites also are not utilized to any detectable degree. The explanation for this observation is most likely provided by structural constraints that may determine the accessibility of the protease to the substrate. Indeed, the TBEV natural upstream site, based on sequence alignments with capsid proteins for which atomic structures are available, is adjacent to or may even overlap at its P2 position with helix 4. The structural organization of helix 4 possibly prevents access to the protease. Moreover, the spatial distance from the cleavage site to the membrane may restrict cleavability by the membrane-associated NS2B/3 protease. In fact, the deletion of the downstream cleavage site of TBEV, which presumably moves the upstream site closer to the membrane, generates a viable, albeit strongly impaired, mutant (unpublished observation), suggesting that this modification slightly increases the accessibility of the upstream site to cleavage by the viral protease.

A further aspect of our findings, which is relevant for a better understanding of the requirements for the assembly of flaviviruses in general, concerns the considerable and unexpected variation in the position of the NS2B/3 cleavage site. The signal sequence connecting proteins C and prM, although generally conforming to the requirements for translocation and signal peptidase processing, lacks polarity in its C-terminal region (51). This unusual property is predicted to impose an -helical rather than the extended β-strand conformation required for the interaction of the signal peptidase cleavage domain with the active site of the enzyme (25). As a consequence, the signalase cleavage site is believed to remain inaccessible for signalase cleavage until protein C is cleaved off from the cytoplasmic end of the signal sequence. This event then may permit a structural rearrangement of the signal sequence and a move toward the luminal side, thus exposing the signalase cleavage site, which subsequently can be cleaved by signalase. Based on this hypothesis, one may assume that the position of the NS2B/3 cleavage site, particularly the number and nature of the residues that remain associated with the signal sequence after cleavage, plays an important role in this mechanism. The fact that revertants with cleavage sites shifted upstream were found to have no significant impairments clearly contradicts this assumption. The additional observation that insertions of hydrophobic or charged residues downstream of the cleavage site also did not disturb the assembly of virions at all confirmed the unexpected finding that a considerable degree of variability in the position of the NS2B/3 cleavage site as well as the length of the signal sequence is tolerated.

It is important to define cleavage specificities in a natural environment, because proteolytic cleavages often are highly dependent on the structural context of the substrate, and in vitro studies can easily be misleading. Our experiments provided an opportunity to use selection pressure to gain information about the specificity of the TBEV protease. It was interesting that, in one case, a reversion mutation generated the noncanonical cleavage site T-R*S. Although in vitro studies already had indicated that Thr at the P2 position can be accepted by the flavivirus protease (26), it still was surprising to see this atypical motif arise spontaneously and repeatedly in several independent passaging experiments in vivo. The analysis of further mutants that had the upstream site destroyed provided confirmatory evidence that the T-R*S motif could be utilized in vivo.

In conclusion, our study further demonstrates the importance of the NS2B/3 cleavage site and has allowed the naturally used cleavage site in the TBEV sequence to be identified. However, it also reveals a remarkable flexibility of the carboxy-terminal domain of protein C and demonstrates the ability of the protease to cleave a noncanonical site in vivo. Given the central biological relevance of the cleavage event in this region for virion assembly and the entire viral life cycle, the variability of acceptable sequence motifs and cleavage positions certainly was an unexpected observation. The plasticity of this region is reminiscent of the previously observed astonishing tolerance of protein C for accepting sequence modifications (20, 45). Remarkably, this protein of 96 amino acids can even tolerate deletions of 30 amino acids in its central domain (22). Taken together, these observations illustrate the extraordinary functional elasticity of protein C. This property makes the flaviviral capsid protein an attractive target for the engineering of flavivirus vaccines and vectors.

 
We are grateful to Steve Allison for helpful discussions and his valuable assistance during the preparation of the manuscript. We thank Susanne Soelch for her participation in mutant construction and Paul Breit for expert photographic documentation.

This project was funded by two grants from the Austrian Fonds zur Förderung der wissenschaftlichen Forschung (FWF-P16376 and FWF-P19528).

 
* Corresponding author. Mailing address: Clinical Institute of Virology, Medical University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria. Phone: 43-1-40490, ext. 79502. Fax: 43-1-40490-9795. E-mail: christian.mandl{at}meduniwien.ac.at

Published ahead of print on 26 December 2007.

Present address: Intercell AG, Campus Vienna Biocenter, Vienna, Austria.

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Journal of Virology, March 2008, p. 2218-2229, Vol. 82, No. 5
0022-538X/08/$08.00+0     doi:10.1128/JVI.02116-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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