Respiratory Syncytial Virus Can Tolerate an Intergenic Sequence of at Least 160 Nucleotides with Little Effect on Transcription or Replication In Vitro and In Vivo

Construction and structure of recombinant RSVs with a modified M-G IGS.

To facilitate cDNA manipulations, the IGS mutations were made in an antigenomic cDNA from which the SH gene had been deleted. Deletion of SH from wild-type recombinant strain A2 (wt rRSV) was described previously (37). That antigenomic backbone contained several additional mutations for vaccine development purposes that were not desirable here; we therefore imported this SH deletion into the previously described wt rRSV backbone (6) by restriction fragment replacement (not shown). As summarized in Fig. 1A and described in detail previously (37), the deletion involved replacing a ScaI-PacI restriction fragment, bearing most of the SH gene, with a small synthetic double-stranded DNA. This restored the M gene noncoding region in its entirety and recreated the GE signal (Fig. 1A). The reconstruction was designed to introduce a single nucleotide change into the M GE signal (AGTTAATAAAAAA to AGTTAATTAAAAA, with the change underlined), which created a PacI site (boldface) and made the signal identical to the original SH GE signal. Thus, in the SH deletion virus, the M-G gene junction is identical to the naturally occurring SH-G junction, consisting of the authentic SH GE signal (now attached to the M gene) followed the authentic 44-nucleotide SH-G IGS, followed by the authentic G GS. In this SH deletion virus, this gene junction will be referred to as M-G.

To manipulate the M-G IGS, we subcloned this region as a PacI-StuI fragment (Fig. 1A) and used PacI and AflIII restriction sites to make additions, deletions, or substitutions by restriction fragment replacement using small double-stranded cDNAs made either by annealing synthetic oligonucleotides or by PCR using mutagenic primers, with sticky ends made by restriction enzyme digestion of the PCR product. All of these manipulations employed conventional methods and will not be described in detail. All structures were confirmed by dideoxynucleotide sequencing. The resulting mutant IGS (Fig. 1B and C) had lengths of 16, 30, 44, 58, 72, 86, 100, 120, 140, and 160 nucleotides. As indicated in Fig. 1B and C, assembly of the various mutant IGS involved sequence derived from the naturally occurring SH-G and G-F IGS of strain A2.

The one exception to the construction scheme described above is rRSV/M-G-65, which was constructed in previous work (4). This virus does not contain the point mutation and resulting PacI site in the M GE signal mentioned above. The structure of its IGS is shown in Fig. 1B.

Recovery and propagation of recombinant RSV.

Recombinant RSV was recovered by cotransfection of each antigenomic cDNA with support plasmids encoding the N, P, M2-1, and L proteins into HEp-2 cells infected with vaccinia virus recombinant MVA encoding the bacteriophage T7 RNA polymerase (6). The MVA recombinant was kindly supplied by Linda Wyatt and Bernie Moss. Each recombinant virus was designated according to the length of its M-G IGS, i.e., rRSV/M-G-16, -30, -44, and so forth. The modified viruses were amplified by four to five sequential passages in HEp-2 cells. Opti-MEM (Life Technologies) containing 2% fetal bovine serum (Summit Biotechnologies) was used in all infections. After the last passage, the cells and overlying medium were harvested and centrifuged at 500 × g, the supernatants were taken and adjusted to contain 100 mM MgSO₄ and 50 mM HEPES sodium salt, and the viruses were aliquoted and frozen. wt rRSV was used as one of the controls; however, this was for descriptive purposes rather than direct comparison because it contains the SH gene.

Reverse transcription-PCR (RT-PCR) analysis of the modified viral IGS.

Total RNA was isolated 48 h postinfection from virus-infected HEp-2 cells, using Trizol reagent (Life Technologies) according manufacturer's recommendations with addition of two phenol-chloroform extractions followed by ethanol precipitation. Reverse transcription was performed using SuperScript II reverse transcriptase (Life Technologies) and the positive-sense oligonucleotide primer 5′-TCATCCCAAGTCATTGTT-3′ (nucleotides 4158 to 4175 of wt rRSV antigenome) upstream of the M-G gene junction. An aliquot of the cDNA product was used as the template in PCR using the above-mentioned primer together with the negative-sense primer 5′-TATATAAGCACGATGATATG-3′, corresponding to nucleotides 4763 to 4782 of the antigenome, downstream of the M-G gene junction. An initial 2-min denaturation step was performed during which the Taq DNA polymerase was added, and then 25 cycles were performed (denaturation, 1 min at 94°C; annealing, 1 min at 40°C; elongation, 2 min at 72°C). The products were analyzed on a 2.5% agarose gel.

Immunostaining of viral plaques.

Monolayers of HEp-2 cells in six-well plates were infected with virus, covered with Opti-MEM (Life Technologies) containing 2% fetal bovine serum and 0.9% methylcellulose, and incubated at 32°C in a CO₂ incubator. Six days later, the monolayers were fixed with cold 80% methanol and stained with a mixture of three murine monoclonal antibodies against RSV F protein (23). The plaques were visualized using horseradish peroxidase-labeled goat anti-mouse immunoglobulin G (IgG; Kirkegaard & Perry Laboratories). The average plaque diameter was calculated by measurement of 30 plaques for each virus.

Kinetics of viral replication in HEp-2 cells.

Duplicate wells of six-well plates were infected with virus at a multiplicity of infection (MOI) of either 5 or 0.01 PFU in a total volume of 1 ml. After 3 h of adsorption at 37°C, the cells were washed three times and incubated at 37°C. At the indicated time points, 120-μl aliquots of medium were taken and replaced with an equal volume of fresh medium. Magnesium sulfate and HEPES were added to the aliquots as indicated above, and the samples were flash-frozen. Each of the duplicate samples was titrated in duplicates, and the mean concentration of the virus at each time point was determined.

Northern blot hybridization.

Total intracellular RNA was isolated from virus-infected HEp-2 cells using Trizol reagent as described above, electrophoresed in denaturing agarose gel containing formaldehyde (13), transferred onto nitrocellulose membranes, and hybridized with a ³²P-labeled double-stranded cDNA probe or strand-specific RNA probe specific to the RSV N, M, G, F, M2, or L gene, as indicated in the legends to Fig. 5 and 6. The bands corresponding to the individual RSV mRNAs or genomic RNA were quantitated using a Molecular Dynamics PhosphorImager. For each band, the background amount of radioactivity was subtracted.

Virus replication and immunogenicity in mice.

Each of 12-week-old, respiratory-pathogen-free BALB/c mice (Charles River, Wilmington, Mass.) in groups of 20 to 21 were inoculated intranasally under light methoxyflurane anesthesia with 10⁶ PFU of rRSV/M-G/65, rRSV/M-G-140, rRSV/M-G-160, or medium alone. On days 3, 4, and 5 following inoculation, five mice per group were sacrificed (except on day 5 for rRSV/M-G-160, when six mice were sacrificed) by CO₂ asphyxiation, and nasal turbinates and lungs were harvested for quantification of virus titer by plaque assay (23, 24). Serum samples were taken from the remaining mice on day 53 postinfection. Serum IgG antibodies specific to the RSV F protein were quantitated in by enzyme-linked immunosorbent assay (ELISA) using immunoaffinity-purified F protein from cells infected with RSV Long strain as the solid-phase antigen (23).

Construction of rRSVs with M-G IGS of increasing length.

Initially, we sought to manipulate the 44-nucleotide IGS between the SH and G genes in a complete RSV strain A2 antigenomic cDNA. However, the RSV antigenome has a tendency to acquire the insertion of a discrete fragment of 1.4 kb of heterologous sequence into the noncoding region of the SH gene during propagation in Escherichia coli. Therefore, we chose instead to work with a version of the antigenomic cDNA from which the SH gene had been deleted (Materials and Methods; Fig. 1A). As described previously, recombinant RSV lacking the SH gene grows as well as or better than wt rRSV in cell culture (forming larger plaques in HEp-2 cells, as shown in Fig. 2 below) and is slightly attenuated in the upper respiratory tracts of mice and in the upper and lower respiratory tracts of chimpanzees (4, 37). Thus, while the function of the SH protein remains unknown, its gene can be deleted with little apparent effect on RSV gene expression and replication in vitro and only a slight effect in vivo. In this SH deletion virus, the SH GE signal was fused to the M gene, replacing the M GE signal, and was followed by the 44-nucleotide SH-G IGS and the G gene (Fig. 1A). Thus, although we designate this gene junction in the SH deletion virus as M-G, it actually is identical to the naturally occurring SH-G gene junction, and hence all of the subsequent manipulations involved an authentic gene junction structure.

Using the SH deletion virus as the parent, we prepared and recovered the following panel of recombinant viruses, which are designated according the length of the M-G IGS: rRSV/M-G-16, rRSV/M-G-30, rRSV/M-G-44, rRSV/M-G-58, rRSV/M-G-72, rRSV/M-G-86, rRSV/M-G-100, rRSV/M-G-120, rRSV/M-G-140, and rRSV/M-G-160. The IGS structures of the first six viruses are shown in Fig. 1B, and those of the last four are outlined in Fig. 1C. We also had available the original SH deletion virus prepared previously (4), which has a 65-nucleotide M-G IGS and for the purposes of this report was renamed rRSV/M-G-65 (Fig. 1B). In addition, we made a second version of rRSV/M-G-100 that differed only in the sequence composition of the 100-nucleotide IGS; however, the two different versions were indistinguishable in cell culture, and only the results for the one shown in Fig. 1C will be presented. As indicated in Fig. 1, the artificially long IGS were constructed from segments derived from the naturally occurring SH-G and G-F IGS and did not contain any non-RSV sequence except for the indicated KpnI and XmaI restriction sites. The viruses were indistinguishable with regard to efficiency of recovery.

Increased length of M-G IGS resulted in reduced plaque size.

The plaques formed by the various recombinant viruses in HEp-2 cell monolayers were visualized by staining with monoclonal antibodies against the F protein (Fig. 2); plaque size was measured in several independent experiments. Viruses that had an M-G IGS of 16 to 86 nucleotides were indistinguishable on the basis of plaque size (represented in Fig. 2 by rRSV/M-G-65); wt rRSV also was included, but it contains the SH gene and hence rRSV/M-G-65 rather than wt rRSV should be considered the parental control for the IGS mutants. The viruses with an M-G IGS of 100 nucleotides or more had progressively smaller plaques and also exhibited greater heterogeneity in plaque size compared to rRSV/M-G-65 (Fig. 2). Taking one typical experiment as an example, with the average plaque size of rRSV/M-G-65 normalized to 1.0 (standard deviation [SD] 0.13), the relative plaque sizes were as follows: rRSV/M-G-100, 0.90 (SD 0.13); rRSV/M-G-120, 0.83 (SD 0.16); rRSV/M-G-140, 0.80 (SD 0.26); rRSV/M-G-160, 0.68 (SD 0.28); and wt rRSV, 0.88 (SD 0.17).

The modified IGS are stable.

Preparations of the various viruses were made by amplification by four or five passages in HEp-2 cells. Intracellular RNA from each final passage was isolated and analyzed by RT-PCR using primers that flanked the M-G IGS. The length of the PCR product representing the M-G IGS of each recombinant RSV corresponded to the calculated value, and shorter PCR products that might represent deletions were not detected (Fig. 3). Thus, the modified M-G IGS appeared to be stable.

Effect of increasing IGS length on single-step and multistep replication in vitro.

Analysis of in vitro replication of the viruses with M-G IGS of 86 nucleotides or less revealed no differences between the viruses (results not shown). We therefore focused on the viruses with an M-G IGS of 100 nucleotides or more. To study the kinetics of virus replication in a single-step growth cycle, HEp-2 cell monolayers were infected at an MOI of 5 PFU per cell with various recombinant viruses containing M-G IGS of increasing length. Aliquots from the overlying medium were taken at 8-h intervals and analyzed later in parallel by plaque assay (Fig. 4A). In general, virus growth was delayed and somewhat reduced with increasing IGS length, although in this particular experiment rRSV/M-G-140 grew somewhat better than rRSV/M-G-120. The largest differences between titers of the fastest-growing and most attenuated viruses, rRSV/M-G-65 and rRSV/M-G-160, respectively, were 21-fold at 24 h and 12-fold at 32 h. However, by 48 h postinfection the slower-growing viruses had largely caught up with the rRSV/M-G-65 virus, and the difference in titer was small.

To analyze multistep growth kinetics, HEp-2 monolayers were infected at an MOI of 0.01 PFU per cell, and samples were taken for analysis at 24-h intervals (Fig. 4B). It had been expected that the kinetics of multistep growth with liquid overlay would reflect the plaque size analysis described above. Unexpectedly, this was not the case, and there were no significant differences between the various viruses.

Sequential transcription is not affected by the increased length of the M-G IGS.

Transcription of individual genes of wt rRSV and of the viruses with M-G IGS of increasing length was analyzed by Northern blot hybridization. HEp-2 cells were infected at an MOI of 5 PFU per cell and harvested 48 h later, and total intracellular RNA was isolated and analyzed by Northern blot hybridization with double-stranded cDNA probes. We tested transcription of the genes located upstream of the modified IGS (N and M), as well as ones located downstream (G, F, M2, and L). Typical results are shown in Fig. 5, and the results of quantitation of the bands corresponding to G and F mRNA using phosphorimagery are presented in Table 1. Surprisingly, there was little or no consistent difference between the different viruses in the level of expression of the various mRNAs. In particular, the efficiency of transcription of genes downstream of the M-G IGS, and the frequency of readthrough transcription across the M-G IGS, appeared to be unaffected by its increasing length. The accumulation of genomic and antigenomic RNAs, which migrate as a single band, also did not appear to vary in a consistent manner among the different viruses.

It was possible that differences in transcription or replication occurred that were not apparent by analyzing total RNA harvested late in infection. Therefore, we also investigated the kinetics of accumulation of several mRNAs in HEp-2 cells infected as described above. Total intracellular RNA was isolated 8, 16, 24, and 32 h postinfection and analyzed by Northern blot hybridization. Representative results for rRSV/M-G-44, rRSV/M-G-65, rRSV/M-G-140, and rRSV/M-G-160 are shown in Fig. 6.

The accumulation of mRNA was analyzed by hybridization with a double-stranded cDNA probe specific to the G gene, or with single-stranded RNA probes of negative polarity specific to N, G, F, or M2 mRNA. No significant differences were found between viruses with regard to the overall mRNA patterns or the rates of accumulation of the various mRNAs. Small differences were seen in some experiments, but these were not consistent between experiments. In these particular experiments, antigenomic RNA was not readily detectable.

The accumulation of genomic RNA was analyzed by hybridization with a positive-sense M2-specific (not shown) or F-specific (Fig. 6) RNA probe and also was visualized with the double-stranded G cDNA probe mentioned above. Genomic RNA was undetectable at 8 h postinfection and was barely detectable at 16 h. Surprisingly, at 24 and 32 h, the amounts of detected genomic RNA or genomic/antigenomic RNA appeared to be somewhat increased for the viruses with longer IGS compared to rRSV/M-G-44. Since the transfer and detection of very large RNAs are subject to considerable experimental variability, further work will be necessary to determine whether this increase is a consistent effect. However, it seems clear that the increasing length of the M-G IGS was not associated with a diminution of RNA replication; this is a further sign that sequential transcription was not reduced significantly.

Replication in vivo and immunogenicity of viruses containing M-G IGS of increasing length.

It was of interest to determine whether the decreased plaque size and reduction in single-cycle growth in vitro was reflected by decreased replication of virus in vivo. We compared the abilities of rRSV/M-G-65, rRSV/M-G-140, and rRSV/M-G-160 to replicate in the respiratory tracts of mice. Tissues from BALB/c mice inoculated intranasally were harvested on days 3, 4, and 5 postinfection, and virus titers in the nasal turbinates and lungs were measured (Table 2). In the nasal turbinates, day 3 titers of rRSV/M-G-140 and rRSV/M-G-160 were 2.4-fold (P < 0.02) and 5.0-fold (P < 0.001), respectively, lower than that of rRSV/M-G-65. On the same day, titers of rRSV/M-G-140 and rRSV/M-G160 in the lungs were 25.3-fold (P < 0.05) and 17.6-fold (P < 0.02) respectively, lower than that of rRSV/M-G-65. On days 4 and 5, no significant difference was found among the three viruses.

The immunogenicity of the viruses was compared by taking serum samples on days 53 (Table 2) and 66 (not shown) postinfection and determining the titers of IgG specific to the RSV F protein by ELISA. The viruses were equally and highly immunogenic.