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. 1997 Dec 9;94(25):13961-6.
doi: 10.1073/pnas.94.25.13961.

Respiratory syncytial virus (RSV) SH and G proteins are not essential for viral replication in vitro: clinical evaluation and molecular characterization of a cold-passaged, attenuated RSV subgroup B mutant

R A Karron  1 , D A Buonagurio, A F Georgiu, S S Whitehead, J E Adamus, M L Clements-Mann, D O Harris, V B Randolph, S A Udem, B R Murphy, M S Sidhu
Affiliations

Respiratory syncytial virus (RSV) SH and G proteins are not essential for viral replication in vitro: clinical evaluation and molecular characterization of a cold-passaged, attenuated RSV subgroup B mutant

R A Karron et al. Proc Natl Acad Sci U S A. .

Abstract

A live, cold-passaged (cp) candidate vaccine virus, designated respiratory syncytial virus (RSV) B1 cp-52/2B5 (cp-52), replicated efficiently in Vero cells, but was found to be overattenuated for RSV-seronegative infants and children. Sequence analysis of reverse-transcription-PCR-amplified fragments of this mutant revealed a large deletion spanning most of the coding sequences for the small hydrophobic (SH) and attachment (G) proteins. Northern blot analysis of cp-52 detected multiple unique read-through mRNAs containing SH and G sequences, consistent with a deletion mutation spanning the SH:G gene junction. Immunological studies confirmed that an intact G glycoprotein was not produced by the cp-52 virus. Nonetheless, cp-52 was infectious and replicated to high titer in tissue culture despite the absence of the viral surface SH and G glycoproteins. Thus, our characterization of this negative-strand RNA virus identified a novel replication-competent deletion mutant lacking two of its three surface glycoproteins. The requirement of SH and G for efficient replication in vivo suggests that selective deletion of one or both of these RSV genes may provide an alternative or additive strategy for developing an optimally attenuated vaccine candidate.

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Figures

Figure 1
Figure 1
Genetic map of the RSV B1 parental strain (15,225 nts) and its deletion mutant, cp-52 (13,933 nts). Genes are listed on top according to encoded proteins: NS1 and NS2, nonstructural proteins; N, nucleocapsid protein; P, phosphoprotein; M, matrix protein; SH, small hydrophobic protein; G, attachment protein; F, fusion protein; M2, second matrix protein; L, large polymerase protein. The numbers in boxes are gene lengths and numbers below are the length of the intergenic regions with the exception of M2:L, which has a 68-nt overlap rather than an intergenic region. Map is not to scale. (A) Genetic map of wt B1. Noncoding 3′-leader (44 nt) and 5′-trailer (145 nt) are the potential genomic and antigenomic promoters. A primer pair (MSS509/MSS562) used for amplification of fragment IIa across the deleted region of cp-52 virus is depicted by arrows. Genomic location of the RNA probes used for Northern analysis is depicted by solid bars under the RSV B1 genome. (B) Genetic map of cp-52 with deleted SH and G gene regions. Two monocistronic gene products, M and SHΔG, detectable by M and Gsm gene probes, respectively, and several polycistronic transcription products identically detectable by both of these probes are depicted at the bottom. (C) Ethidium bromide-stained 1% agarose gel showing reverse transcription–PCR amplification products generated by primers MSS509 and MSS562 that used RSV B1 or cp-52 genomic RNAs. PCR product amplified from cp-52 RNA (lane 3) was found to be ≈1.3 kb smaller than that from B1 RNA (lane 2). Lane 1 is a reagent control and lane 4 shows size markers (1-kb ladder, Life Technologies).
Figure 2
Figure 2
Northern blot hybridization of total intracellular RNA extracted from B1 and cp-52 virus-infected Vero cells. Replicate RNA samples (5 μg) were fractionated by electrophoresis for 3.5 hr at 90 V in a 1.2% agarose-2.2 M formaldehyde gel in 1× Mops buffer (pH 7.0). RNA was transferred in 20× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate) to 0.2 μm Nytran nylon membrane with a TurboBlotter system (Schleicher & Schuell), and then was fixed by UV crosslinking. Negative-sense riboprobes (≈300 to 400 nt) labeled with [α-32P]UTP were prepared by in vitro transcription of B1 virus-specific PCR products (containing T7 promoter sequence) by using a MAXIscript T7 kit (Ambion, Austin, TX). Probe map positions on the RSV B1 genome are shown in Fig. 1A; 1.25 × 107 cpm of each probe were used for hybridization at 65°C in Rapid-hyb buffer (Amersham). Stringency washes of 15 min each were done twice in 2× SSC, 0.1% SDS at room temperature and twice in 0.2× SSC, 0.1% SDS at 65°C. The 65°C washes were separated by room temperature treatment with 1 μg/ml RNase A in 2× SSC for 15 min to remove nonspecifically bound probe (Promega). The blot was exposed to x-ray film for 6.5 hr. B1, lanes 1, 3, 5, 7, and 9; cp-52, lanes 2, 4, 6, 8, and 10. RSV B1-specific monocistronic mRNA transcripts corresponding to M, SH, G, and F genes are indicated by filled arrowheads. Identical polytranscripts unique for cp-52 virus that were detected independently by the M and Gsm probes are marked by open triangles in lanes 2 and 8 (see Fig. 1B for identification). The predicted SHΔG transcript is identified by the long arrow in lane 8. It should be noted that a short exposure revealed that the cp-52 M-specific signal in lane 2 (marked by the open triangle closest to the bottom) consists of two RNA species of similar size: a monocistronic M mRNA that is identical to the RNA identified by the filled arrowhead in B1 lane 1, and an M:SHΔG read-through transcript. The weak monocistronic M signal for B1 virus (lane 1) that was consistently observed in independent experiments indicates inefficient transcription termination and/or mRNA instability in RSV B1 virus.
Figure 3
Figure 3
Multistep growth curves for RSV B1 wt and cp-52 viruses. Vero cell monolayer cultures were infected with either B1 or cp-52 at an moi of 0.01. Cultures were maintained at 25°C, and aliquots of supernatant were removed daily for 14 days postinfection, snap-frozen, and stored at −70°C. Thawed aliquots were titered by plaque assay on Vero cell monolayer cultures maintained under a semisolid overlay at 32°C as previously described, and results were expressed as log10 pfu/ml (8). Each point represent the mean ± SE of three experiments. The titers of the input viruses were 2.5 × 104 pfu/ml for RSV B1 wt and 2.3 × 104 pfu/ml for cp-52.
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