Phenotype of WN/D2-SL RNA.
DEN2 and WN viruses are members of different groups among mosquito-borne flaviviruses, based on serologic and genetic relatedness (6
). The nucleotide sequences of the 3′SL for the genome of WN virus strain 956 (41
) and that for DEN2 strain New Guinea C (NGC) (42
) are shown in Fig. . Computer analysis revealed that there was about 65% similarity between these two sequences. In a previous study of DEN2 virus replication (42
) in which DEN2 genome RNAs containing DEN2/WN chimeric 3′SL nucleotide sequences were assessed for replication competence, results showed that an 11-bp segment, comprising the topmost part of the bottom portion of the long stem in the DEN2 3′SL nucleotide sequence (Fig. ), the dengue-required sequence [DRS], was essential for replication of DEN2 virus. Here, we conducted the converse investigation to determine the minimal requirement for WN virus 3′SL-specific nucleotide sequences for replication of WN virus mutant RNAs containing WN/DEN2 chimeric 3′SLs. The wt WN virus strain 956 infectious DNA used as the basis for constructing all mutant viruses described in this study was reported previously (41
FIG. 1. The 3′-terminal 93-nt sequence of the DEN2 strain NGC 3′SL is shown on the left, and the 95-nt sequence of the WN virus strain 956 3′SL is shown on the right. Nucleotides are numbered in 3′-to-5′ direction from (more ...)
Replication of wt and mutant RNAs was assessed by an immune fluorescence assay (IFA) for WN virus antigens in transfected cells with murine polyclonal anti-WN antibodies on days 3, 5, 10, 15, and 20 postelectroporation. All but one mutant RNA exhibited one of two distinctly different phenotypes in the IFA. Viable mutant RNAs gave positive results by IFA within 5 days after transfection and 100% WN virus antigen-positive cells at or prior to the 10-day time point after transfection. Mutant RNAs that gave negative results by IFA after 20 days of observation were said to display a lethal phenotype.
We initially created a cloned mutant WN virus DNA (WN/D2-SL) representing the wt WN virus strain 956 genome sequence, except that the last 95 nt of the WN virus genome (representing the entire WN virus 3′SL) was replaced by the 3′-terminal 93 nt of the wt DEN2 sequence (representing the entire DEN2 3′SL [Fig. ]). RNAs derived from transcription of WN/D2-SL mutant and WN virus wt DNAs were electroporated into both Vero and BHK cells. (Both wt parent viruses, DEN2 NGC and WN virus strain 956, replicate vigorously in either cell line.) For mutant WN/D2-SL RNA, no WN virus-specific antigens were detected in transfected cells for the entire duration of this experiment; it therefore displayed the lethal phenotype (data not shown). In contrast, WN virus wt RNA-transfected cells were positive for WN virus antigens by IFA after 24 h, and nearly 100% of cells in the monolayer were positive by day 5 (Fig. ). The replication phenotypes of all wt and mutant RNAs were essentially identical after transfection of either BHK or Vero cells. Therefore, only the results of the IFA on BHK cells are shown.
FIG. 2. Indirect IFAs after transfection of mutant WN virus RNAs. RNAs were derived by transfection of wt and mutant WN virus genome-length DNAs and used to transfect hamster kidney cells (BHK-21). On the days indicated, cells were replated on a chamber slide, (more ...)
To exclude the possibility that the lethal phenotype of WN/D2-SL RNA was due to an occult mutation upstream from the 3′SL in WN/D2-SL DNA, we first generated a revertant wt WN virus DNA from WN/D2-SL DNA, by substituting a fragment containing the 3′-terminal 1.1 kb of the wt WN DNA (spanning the WN nucleotide sequence between a unique BclI restriction site at nt 9833 and the 3′-terminal XbaI restriction site at nt 10962) for the analogous mutant fragment in the WN/D2-SL DNA. RNA transcribed from this rescued WN/D2-SL DNA was infectious, and the resulting virus exhibited growth kinetics analogous to that of the parental WN virus (data not shown). This indicated that WN/D2-SL DNA did not contain an occult lethal mutation upstream from the BclI site and that the PCR-synthesized wt 1.1-kb BclI/XbaI fragment was able to support virus replication.
We also recreated the wt WN and WN/D2-SL DNAs as full-length PCR products and demonstrated that wt RNA transcribed from the PCR template was infectious while WN/D2-SL RNA derived in the same manner was not (data not shown). This additionally confirmed that the failure of WN/D2-SL RNA to replicate was due to the substitution of the DEN2 3′SL nucleotide sequence for that of the WN virus 3′SL, despite the similarity between the two 3′SLs in secondary structure (Fig. ).
Nucleotide sequence elements of the WN virus 3′SL required for WN virus replication.
We next sought to determine what portions of the wt WN-specific 3′SL nucleotide sequence were required to restore efficient WN virus replication. To perturb the nucleotide sequence of the 3′SL without altering its predicted stem-loop structure, portions of the 3′ terminal 79 nt of the DEN2 3′SL sequence were substituted for analogous segments of the 3′ terminal 79 nt of the WN virus 3′SL sequence (Fig. ) in the context of the WN virus infectious DNA. Substitution mutations did not extend into the small stem-loop structure formed by WN virus nt 79 to 95, because the WN virus and DEN2 sequences in this region were shown to be freely exchangeable in the previous study centered on DEN2 virus replication (42
). We defined the top and bottom of the WN virus and DEN2 3′SL in accordance with previous studies (3
). Computer analyses (25
) of all resulting chimeric WN/DEN2 3′SL nucleotide sequences suggested that each would form the stem-loop structures shown in Fig. -.
FIG. 3. (A) Nucleotide sequences of mutant 3′SLs in WNmutC1, -A1, and -A1L RNAs, excluding that of the small stem and loop defined by wt WN virus nt 80 to 95 (Fig. ) are shown. Nucleotides native to the DEN2 3′SL are shown in roman (more ...)
FIG. 8. The nucleotide sequence of the long stem-loop structure in the WNmutF1 3′SL is depicted. Boldface type indicates nucleotides native to the wt WN 3′SL. Roman type indicates nucleotides native to the wt DEN2 3′SL. The horizontal (more ...)
Initially, two mutant WN virus DNAs containing chimeric WN/DEN2 3′SL nucleotide sequences were cloned (Fig. ). WNmutA1 DNA contained a substitution of the top half of the WN virus 3′SL long stem (nt 16 to 65, numbering in the upstream direction from the 3′-terminal nucleotide of the genome [Fig. ]) by the analogous segment of the DEN2 3′SL (nt 18 to 62). WNmutC1 DNA contained the converse substitution; the bottom half of the WN virus long stem-and-loop structure sequence, nt 1 to 15 and 66 to 79, was replaced by DEN2 nt 1 to 17 and 63 to 79, respectively. RNAs derived from WNmutA1 and WNmutC1 DNAs were transfected into BHK or Vero cells in separate experiments. For WNmutA1 RNA, IFA was negative up to day 20 posttransfection (data not shown), whereas for WNmutC1 RNA, 40 to 60% of cells were positive by day 5 after transfection, and 100% were positive by day 10, as shown for BHK cells (Fig. ). As a positive control, wt WN virus RNA-transfected cells were 100% positive by IFA within 5 days posttransfection in the same experiment. Thus, substitution of the top half of the WN virus 3′SL by the nucleotide sequence of the top half of the DEN2 3′SL was lethal, whereas substitution of the bottom half of the long stem in the WN virus 3′SL nucleotide sequence with the analogous DEN2 3′SL nucleotide sequences was well tolerated and gave rise to a viable mutant virus.
The 3′ termini of genomic RNAs derived from WNmutC1 virus and that of all other viable mutant viruses were sequenced to determine whether spontaneous mutations had occurred within the 3′SL after transfection. For WNmutC1 RNA, results showed that there were heterogeneities at certain nucleotide positions in the 3′SL. (We assayed for heterogeneity of the average nucleotide sequence by a visual search for peaks coexisting at a single site on the computer-generated graph of the nucleotide sequence that constitutes the output of the automated sequencers; see Materials and Methods. Unpublished data suggested that we could detect heterogeneity at a given site in the nucleotide sequence at the level of one substitution mutation per 5 to 10 molecules by this method.) Therefore, PCR products representing the C1 3′SL in C1 virus RNA were cloned, and six cloned DNA fragments were sequenced. All six DNAs contained spontaneous mutations of the bottom portion of the long stem in the C1 3′SL (Fig. ; sequences a to c), but all spontaneous mutations conserved the general predicted secondary structure of the C1 3′SL. Sequence b was detected in three of six clones, and after three additional passages of WNmutC1 virus in Vero cells, sequence b was dominant in C1 genome RNAs. In the C1b 3′SL, an A was inserted between U at position 3 (U3) and U4, and a U was inserted between U81 and A82 of the predicted C1 nucleotide sequence. The G6-C81 base pair was deleted. These mutations had the net effect of shifting the U4-U81 bulge in the predicted C1 3′SL upward by one (U/A) base pair from the bottom of the long stem. The apparent deletion of one or two 3′ terminal nucleotide s from C1 spontaneous mutants a and c could have been an artifact, due to the activity of enzymes used to circularize viral RNA prior to PCR. The occurrence of second-site mutations in the C1 3′SL was unique for viable genomes containing WN/DEN2 chimeric 3′SLs; in all other cases, the respective mutant 3′SL nucleotide sequences were stable in replicating viral genomes (data not shown).
We next generated WNmutA1L DNA as a full-length PCR product, using wt WN DNA as a template and a 3′ negative-sense primer encoding the A1L mutations, to identify more specifically the nucleotides in the top half of the WN virus 3′SL that were required for WN virus replication. In A1L DNA, only the double-loop structure atop the long stem (WN nucleotides 29 to 52) (Fig. and ) was replaced by the analogous nucleotide sequence of the DEN2 3′SL. WNmutA1L RNA was infectious in both BHK and Vero cell monolayers. About 50% of cells in either transfected monolayer were positive for WN virus antigens by day 5 after transfection, and cells were 100% positive by day 10, as shown for BHK cells (Fig. ). This result suggested that the lethal phenotype of WNmutA1 RNA was due to the absence in that construct of the 14-bp top portion of the long stem in the wt WN virus 3′SL.
This finding was not consistent with the previously mentioned study (42
), in which part of the bottom portion of the DEN2 3′SL nucleotide sequence was shown to be required for replication of DEN2 RNAs containing chimeric 3′SLs (Fig. ). One possible explanation for the disparate results was that bulges in the top and bottom halves of the WN virus and DEN2 long stems, respectively, were required for replication of both virus species. The lower portion of the top half of the long stem in the WN virus 3′SL contained a bulge formed by the apposition of noncomplementary nucleotides G20 and A61, whereas no analogous bulge occurs in the top half of the DEN2 3′SL. Conversely, the dengue-required segment DRS contained two bulges that were not precisely represented in the analogous region of the WN virus 3′SL. We therefore focused further attention on a 5-bp double-stranded (ds) segment (WN virus nt 16 to 20 and 61 to 65) (Fig. ) that included both the bulge formed by nt A61 and G20 and the major in vitro eF1-α-binding site (4
) in the WN virus long stem (the sequence 5′CACA3′; WN virus nt 61 to 64) (Fig. ). For the latter reason, we referred to the segment as the translation elongation factor (TEF)-binding domain.
To demonstrate a requirement for the TEF-binding domain in WN virus replication, we next generated WNmutC2 and WNmutA2 DNA templates as cloned recombinant plasmids (Fig. ). WNmutC2 DNA was identical in nucleotide sequence to WNmutC1 DNA, except the TEF-binding domain was deleted. Conversely, WNmutA2 DNA was derived from WNmutA1 DNA by the insertion of the TEF-binding domain into the WNmutA1 3′SL nucleotide sequence at the boundary between the bottom and top parts of the A1 3′SL.
FIG. 4. Nucleotide sequences of the long stem-loop structures of 3′SLs present in WNmutC2 (A) and WNmutA2 (B) RNAs are depicted in comparison to WNmutC1 and WNmutA1 3′SLs, respectively. Boldface type indicates nucleotides derived from the wt WN (more ...)
Neither WNmutC2 RNA nor WNmutA2 RNA was infectious in BHK or Vero cells. The phenotype of WNmutC2 RNA (lethal) compared to that of WNmutC1 RNA (viable) was consistent with a requirement for the TEF-binding domain for WN virus replication, but the lethal phenotype of WNmutA2 RNA was in conflict with that hypothesis. We postulated that failure of WNmutA2 RNA to replicate was related to the possibly excessive length of the long stem in the A2 3′SL (Fig. ) and/or to the resulting perturbation of the spatial relationship of the bulge contained in the TEF-binding domain to other predicted bulge or loop structures within the 3′SL. Therefore, WNmutA3 and WNmutA4 DNAs were generated as PCR products, as described for WNmutA1L (Fig. ). WNmutA3 PCR-product DNA contained a deletion of a single DEN2-specific base pair (nucleotides G21 and C63) (Fig. and ) at the lowermost boundary of the top of the WNmutA2 3′SL, and WNmutA4 PCR-product DNA contained a deletion of three additional DEN2-specific base pairs from the same region in the WNmutA1 3′SL (nt 21 to 24 and 62 to 65) (Fig. ). WNmutA3 RNA exhibited a viable phenotype; 30 to 60% of cells were positive by IFA 3 to 5 days after transfection, and 100% of cells were positive at day 10 posttransfection (Fig. ). The A4 mutation was lethal for WN virus replication. The results for mutants WNmutC2 and WNmutA3 were consistent with the hypothesis that the WN virus-specific TEF-binding domain was necessary for the viability of mutant WN virus RNAs containing chimeric 3′SLs, but failure of WNmutA2 and -A4 RNAs to replicate suggested that there were additional constraints on the 3′SL secondary structure or nucleotide sequence that rendered these latter genomic RNAs nonviable. In addition, the results for WNmutA3 RNA demonstrated that WN nt 21 to 29 and 52 to 60 (Fig. ) were not necessary per se for WN genome replication.
FIG. 5. Derivation of the 3′SLs in WNmutA3 and WNmutA4 RNAs from the A2 3′SL nucleotide sequence is shown. Boldface type, nucleotides derived from the wt WN virus 3′SL nucleotide sequence. Roman type, nucleotides derived from the wt DEN2 (more ...)
We next sought to determine whether the DRS (Fig. ) (42
) could substitute for the TEF-binding domain, if it were inserted into the WN virus 3′SL in an analogous locus in the long stem. WNmutE DNA was cloned to address this question; it contained a substitution of DEN2 3′SL nt 12 to 18 and 61 to 68 (Fig. ) for nt 14 to 20 and 61 to 66, respectively, of the wt WN 3′SL nucleotide sequence (Fig. ). WNmutE RNA exhibited a viable phenotype that was nearly indistinguishable from that of wt WN RNA; 100% of cells in the transfected monolayers were positive by IFA within 5 days posttransfection (Fig. ). Thus, our question was answered in the affirmative. Since the TEF binding domain and the substituted DEN2-specific ds segment of the DRS had little nucleotide sequence homology but both introduced a bulge into the top portion of the long stem of the WN virus 3′SL, this suggested that the bulge itself and its location within the long stem were critical determinants of RNA replication competence independent of nucleotide sequence, at least in the context of mutant RNAs containing chimeric 3′SLs.
FIG. 6. The nucleotide sequence of the long stem-loop structure of the WNmutE 3′SL is depicted. Boldface type, nucleotides native to the wt WN virus 3′SL. Roman type, nucleotides native to the wt DEN2 3′SL. Horizontal dashed lines labeled (more ...) The bulge in the TEF-binding domain.
We constructed additional point mutantWN virus DNAs as PCR products in order to determine whether the bulge in the TEF-binding domain was required for replication in the context of the wt WN virus 3′SL nucleotide sequence (Fig. ). A61C (Fig. ) and G20U (Fig. ) RNAs were each predicted to lack the bulge in the TEF-binding domain, but in each mutant one of the two wt nucleotides that constituted the bulge was conserved. Both RNAs were infectious and gave viable virus with kinetics similar to those of wt WN virus RNA in our posttransfection IFA (data not shown). However, nucleotide sequence analysis of RNAs derived from replicating viruses showed that all A61C mutant viral genomes had reverted, such that an A was present at position 61, and the wt A61-G20 bulge was restored. RNA from mutant G20U virus contained the expected substituted U at position 20 in the 3′SL (and therefore lacked the A61-G20 bulge) but was heterogeneous at nt 60 in the wt WN virus 3′SL sequence (Fig. ). Therefore, the G20U virus was passaged one additional time in Vero cells, and five cloned DNAs representing the G20U 3′SL were sequenced. Results showed that a proportion (~1:5) of G20U viral genomes had sustained a novel nucleotide substitution mutation of A at position 60 to C (A60C), thus introducing a new potential bulge by apposition of mutant nucleotide C60 with wt nucleotide U21, displaced by one nucleotide pair compared to the A61-G20 bulge in the wt TEF-binding domain. After three additional passages of G20U mutant virus in Vero cells, the spontaneous A60C mutation and the deliberate G20U mutations had both reverted to wt in all molecules, restoring the wt A61-G20 bulge (data not shown). These results collectively suggested that the bulge in the top portion of the long stem of the wt WN virus 3′SL was not dispensable.
FIG. 7. Nucleotide sequences of the long stem and loop of 3′SLs present in A61C, G20U, and 2U RNAs are depicted. A horizontal line indicates the boundary between the top (t) and bottom (b) portions of the wt WN virus 3′SL (3, 4, 42). The TEF-binding (more ...)
To determine whether nucleotides A61 and/or G20 were per se required for WN virus replication, we generated the WNmut2U DNA by PCR, in which both A61 and G20 were replaced by U (Fig. ). The 2U mutant 3′SL contained a bulge, created by the apposition of the substitution mutations, A61U and G20U. WNmut2U RNA replicated less efficiently than other viable mutant RNAs after transfection, in that transfected cells did not become 100% positive for WN virus antigens until day 15 after transfection (data not shown). However, as a more certain indicator of its replication efficiency, WNmut2U virus recovered from transfected cells was used to infect Vero cells at a low MOI and reached a peak titer of 9 × 106 PFU/ml after 7 days. This peak titer was less than 10-fold different from that of wt WN virus in a similar assay at the same time point (data not shown). Sequencing of the 3′ terminus of the WNmut2U genome revealed that the 2U mutation was completely stable in RNA recovered from virus particles. These results suggested that nt A61 and G20 were not absolutely required for WN virus replication.
Introduction of a bulge into the bottom part of the long stem of the WN virus 3′SL.
One mutant DEN2 virus derived in the previous study of the DEN2 3′SL contained a substitution of the bottom 7 bp of the long stem in the DEN2 3′SL by the analogous 6 bp of the long stem of the WN virus 3′SL (42
). The resulting mutant DEN2mutF virus was host range restricted, in that it was severely retarded for replication in mosquito cells but replicated to wt titers in cultured monkey kidney cells. WNmutF1 DNA (Fig. ) was constructed to determine whether the converse mutation could alter the host range of the WN virus. It contained a replacement of the bottommost 7 bp of the long stem in the WN virus 3′SL (WN virus nt 1 to 7 and 73 to 79) (Fig. ) by the analogous 7 bp of the DEN2 3′SL (DEN2 nt 1 to 7 and 73 to 79). This had the effect of introducing a U-U bulge into the bottom portion of the WN virus 3′SL long stem that was not present in the wt structure, formed by the apposition of U4 and U76 in the DEN2 nucleotide sequence. By IFA, WNmutF1 RNA had a viable phenotype nearly indistinguishable from that of the wt WN virus RNA after transfection of Vero or BHK cells (Fig. ). Data from growth curves indicated that WNmutF1 virus did have an altered host range in C6/36 cells (see below).
Specific infectivity of mutant RNA.
The results of the IFA suggested that all viable 3′SL mutant RNAs were at least slightly less infectious than wt WN virus RNA, based on the time required for transfected cell monolayers to become 100% positive for WN virus antigens (Fig. ). However, results of the IFA were not necessarily indicative of subtle differences in infectiousness due solely to 3′SL mutations among and between viable mutant RNAs, because some of them were generated by transcription from cloned mutant DNAs and some from full-length PCR products. This variable could have artifactually skewed the kinetics of the IFA. To resolve this issue, an infectious center assay was conducted in which the specific infectivity of mutant RNAs derived from cloned recombinant plasmid DNAs and of mutant RNAs derived from PCR product DNAs was compared to that of wt RNAs derived by each of the two methods. Results of two independent experiments in BHK cells are shown in Table . Wt RNA derived from the WN virus recombinant plasmid DNA (and used in the IFA) had a specific infectivity of 33.4 × 103 and 26.4 × 103 PFU/μg, respectively, in the two experiments, whereas the specific infectivity of wt RNA derived from a full-length wt PCR product DNA was 10.0 × 103 and 12.0 × 103 PFU/μg, respectively, in the same two experiments. For WNmutE RNA, which was also generated by both methods, there was a similar relationship in specific infectivity between RNA derived from cloned plasmid DNA (18.0 × 103 PFU/μg in one experiment) and RNA derived from PCR product DNA (8.0 × 103 and 5.0 × 103 PFU/μg in two experiments). The specific infectivity of WNmutC1 RNA was comparable to that of wt RNA, despite the fact that replication of C1 virus was associated with spontaneous mutation of the C1 3′SL (Fig. ). This suggested that the input C1 3′SL nucleotide sequence was quite functional for recruitment of factors required to initiate replication. RNAs derived from PCR product DNAs might have been slightly less infectious than those derived from cloned DNAs, due to premature termination of transcription off PCR-derived templates or due to random lethal mutations introduced into a subpopulation of DNAs during PCR. In any case, the difference in specific infectivity observed between wt RNAs derived by the two methods and between the respective wt RNAs and relevant mutant RNAs (Table ) was much less than an order of magnitude in all cases and differences did not rise to the level of statistical significance.
Infectious center assays for wt and mutant RNAs in BHK cells
Nucleotide sequence differences between RNA recovered from WN viruses and the WN strain 956 infectious DNA, upstream from the 3′SLa
The infectious center assay was more sensitive for detecting small differences in specific infectivity between mutant RNAs derived from PCR-product DNAs and those derived from cloned DNAs than was the IFA. However, wt and all viable mutant RNAs were markedly contrasted with lethal mutant RNAs (WN/DN-SL, -A1, -A2, -A4, and -C2), all of which failed to induce the synthesis of detectable WN virus antigens after 20 days of observation p.e. This suggested a fundamental difference in functionality of the 3′SLs in wt and viable mutant RNAs compared to that of lethal mutant RNAs, regardless of the method of derivation.
Kinetics of replication of viable mutant viruses in BHK and C6/36 cells.
The kinetics of the replication of wt and viable mutant viruses in BHK and C6/36 cells was determined at an MOI of ~0.01 each case, using amplified and plaque-titered stocks. The 3′ termini of the genomes of viruses used in this assay were analyzed to verify the presence of the respective mutant 3′SL nucleotide sequences shown in Fig. through . WNmutC1 virus contained the C1b 3′SL nucleotide sequence (Fig. .) Plaque titers were determined daily for virus secreted into the medium for 8 or 9 days, and results are shown in Fig. and and Table .
FIG. 9. Replication of viable WN 3′SL mutant viruses in BHK cells. Plaque titers were determined for pools of viruses derived by transfection of BHK cells in Vero cells; these viruses were used to infect confluent monolayers of BHK cells at an MOI of (more ...)
FIG. 10. Replication of viable WN 3′SL mutant viruses in C6/36 cells. Plaque titers were determined for pools of viruses derived by transfection of BHK cells in Vero cells; the viruses were used to infect confluent monolayers of C6/36 cells at an MOI of (more ...)
Peak titers of WN 3′SL mutant viruses in BHK and C6/36 cells
The peak titer for wt WN virus in BHK cells was about 8 × 107 PFU/ml, achieved on day 6 postinfection (Fig. and Table ). In the same experiment, WNmutA3 and WNmutF1 viruses were similar in their peak titers compared to wt virus, whereas the titers of WNmutC1 and WNmutE viruses were about 10-fold lower. We noted that the replication of WNmutE virus was markedly retarded compared to wt WN virus at early times after infection, despite the fact that it ultimately attained a peak titer approaching that of the wt. For example, on day 4 the titer was about 100,000-fold lower than that of wt WN virus and most of the other mutant viruses (Fig. ). The kinetics of replication of WNmutA1L virus were similar to those of WNmutE virus in the first 6 days postinfection. However, the peak titer of WNmutA1L virus never exceeded 1.2 × 106 PFU/ml, nearly 100-fold lower than that of the wt (Fig. and Table ). Therefore, substitution of the WN virus nucleotides forming the double-loop structure atop the long stem by analogous DEN2 nucleotides had a slight negative effect on replication competence of the virus that was not obvious from results of the IFA or the infectious center assay performed after transfection to assess the RNA.
The peak titer for wt WN virus in C6/36 cells was about 1.2 × 107 PFU/ml, achieved on day 8 postinfection (Fig. ; Table ). WNmutA3 and WNmutE viruses achieved similar peak titers of 4 × 107 and 2.5 × 107 PFU/ml, respectively, even though WNmutA3 virus replicated more vigorously than wt virus at early times after infection, for example, on days 3 through 6. In contrast, the replication of WNmutE virus was retarded at early times after infection compared to wt virus, as was observed after infection of BHK cells. The kinetics of replication of WNmutA1L virus in C6/36 cells paralleled that of wt virus up to day 4 postinfection, when the titers of both viruses were about 105 PFU/ml. However, the titer of the mutant virus did not increase after day 4, and therefore the peak titer of WNmutA1L virus was almost 100-fold lower than that of the wt virus.
Surprisingly, the peak titers of WNmutC1 and WNmutF1 viruses in C6/36 cells exceeded that of wt WN virus by about 80 and 100 fold, respectively. On day 8 postinfection, WNmutC1 virus reached a peak titer of 8 × 108 PFU/ml, and WNmutF1 virus reached a peak titer of 1.2 × 109 PFU/ml (Table ). In both mutant genome sequences, the lowermost 7 bp of the long stem of the 3′SL was derived from the DEN2 nucleotide sequence (Fig. , , and ). We inferred that the U-U bulge introduced in the context of the 7-bp DEN2-specific segment was mosquito cell growth enhancing for both WNmutC1 and WNmutF1 viruses. Thus, we tentatively identified a second locus in the long stem of the 3′SL where the presence of a bulge resulted in altered growth properties of WN virus.
Complete genome sequences of viable mutant WN viruses.
As mentioned previously, the 3′SLs in all viable mutant viruses were sequenced to determine whether the respective mutant nucleotide sequences were stable in replicating virus. Except for the C1 3′SL (Fig. ) and the A61C and G20U genomes (see above), this was shown to be true. In addition to verifying the stability of all 3′SL nucleotide sequences in viable RNAs, we also sequenced the complete genomes of wt and WNmutA1L, -A3, -C1, -E, and -2U viruses after amplification in Vero cells (wt, WNmutA1L, and WNmut2U viruses) or BHK cells (WNmutA3, -C1, and -E viruses), to determine whether any other second-site mutations might have been required for viability. The sequences of these RNAs were then compared to the sequence of the WN strain 956 infectious DNA (Table ).
wt WN virus RNA contained two mutations that differentiated it from that of the parent infectious DNA. Both were silent mutations, one in the envelope gene segment (G1968U) and one in NS5 (A9465G), respectively. The G1968U mutation was also detected in the WNmutA1L and WNmutC1 genomes. The A9465G mutation was also detected in the WNmutA3 genome. Since they occurred in the wt genome, these mutations were unlikely to have any relationship to the replication phenotypes of the mutant viruses in question. We similarly discounted the significance of silent mutations detected in the ORFs of the WNmutA3 genome (C309U and U1323C), the WNmutE genome (G7356A and A8076G), and the WNmut2U genome (T6682G). The C1 and E genomes both contained mutations in the premembrane (prM) gene segment that were predicted to result in amino acid changes in prM. In view of the results of the IFA and other data (see below and reference 42
) suggesting that lethal mutations of the 3′SL abrogate translation initiation and/or RNA synthesis and since there is no published information to implicate prM in those processes, we doubted that these mutations were compensatory for the presence of the C1 or E 3′SLs, respectively, in the WN genome. Therefore, it remained possible that the A10684G mutation detected in the 3′NCR of both the A1L and E genomes, the C10502U mutation in the A1L genome, and the A7898U sense mutation of the NS5 gene segment in the A3 genome (Table ) could have been required for replication of these genomes, due to altered function of the respective mutant 3′SLs. There was no correlation between the loci of second-site mutations and the cell type in which the viruses were amplified (Vero versus BHK cells).
Northern blot analysis of negative-strand synthesis.
To evaluate further the defect in replication of lethal mutant RNAs, we conducted a Northern blot analysis of total cellular RNA harvested from cells after transfection with wt and lethal mutant RNAs, using a radiolabeled, positive-sense ssDNA probe (Fig. ). The probe was WN negative-strand RNA specific in that it strongly hybridized to a full-length negative-strand WN RNA synthesized in vitro (lane 1) but did not hybridize at all detectably to positive-strand WN RNA harvested from infectious virus (Fig. , lane 2) or to RNA harvested from mock-transfected BHK cells (lane 4). The probe was able to detect genome-size negative strands in cells that had been transfected with wt WN RNA transcripts (lane 3), but no negative-strand RNA was detected in cells transfected by WNmutA1, WNmutA2, WNmutC2, and WN/DN-SL RNAs (lanes 5 to 8, respectively). rRNAs were visualized in the gel by ethidium bromide staining (Fig. ) to establish that roughly equivalent amounts of total cellular RNA were loaded onto the gel. We could not rule out the possibility that very small amounts of negative-strand RNA beneath the level of sensitivity of our assay were present in cells transfected by mutant RNAs that failed to replicate, but the results were consistent with our hypothesis based on the IFA results and previous work (17
) that lethal mutations of the 3′SL abrogated replication at either the level of translation of input virion RNAs or initiation of RNA synthesis. This was consistent with the hypothesis that at least some of the lethal mutant 3′SLs were unable to recruit factors necessary for one or both processes.
FIG. 11. (A) Northern hybridization analysis of negative-strand WN RNAs in cells transfected by wt and lethal mutant RNAs. Total cellular RNAs were isolated from mock-transfected cells (lane 4) and from cells transfected with wt WN RNA (lane 3) or WNmutA1, WNmutA2, (more ...) Confirmation of the phenotypes of lethal mutations in the WN virus 3′SL.
To reduce the possibility that a technical or procedural error could account for the observed lethal phenotypes of the WNmutC2, WNmutA1, WNmutA2, and WNmutA4 mutations, each transfection experiment was repeated three times with identical results. In addition, to reduce the possibility that we had introduced an occult lethal mutation into the wt WN virus DNA during mutagenesis, we rescued lethal mutants WNmutC2, WNmutA1, and WNmutA2 by replacing the 3′-terminal 508-nt sequence of the respective mutant DNAs with that of wtWN virus, using a technique analogous to that described above for rescuing the prototype WN/D2-SL mutant virus genome. We also recreated C2, A1, and A2 DNAs by PCR, using 3′ primers containing the respective mutant 3′SL nucleotide sequences and WNdl16 DNA as template, as also described previously for confirmation of the phenotype of WN/D2-SL RNA. The results in all cases confirmed that the lethal phenotypes of the mutant RNAs were due only to the mutations introduced into the respective 3′SL nucleotide sequences.