The M2-2 protein of human respiratory syncytial virus is a regulatory factor involved in the balance between RNA replication and transcription

Abstract

The M2 mRNA of human respiratory syncytial virus (RSV) contains two overlapping ORFs, encoding the transcription antitermination protein (M2-1) and the 90-aa M2-2 protein of unknown function. Viable recombinant RSV was recovered in which expression of M2-2 was ablated, identifying it as an accessory factor dispensable for growth in vitro. Virus lacking M2-2 grew less efficiently than did the wild-type parent in vitro, with titers that were reduced 1, 000-fold during the initial 2-5 days and 10-fold by days 7-8. Compared with wild-type virus, the intracellular accumulation of RNA by M2-2 knockout virus was reduced 3- to 4-fold or more for genomic RNA and increased 2- to 4-fold or more for mRNA. Synthesis of the F and G glycoproteins, the major RSV neutralization and protective antigens, was increased in proportion with that of mRNA. In cells infected with wild-type RSV, mRNA accumulation increased dramatically up to approximately 12-15 hr after infection and then leveled off, whereas accumulation continued to increase in cells infected with the M2-2 knockout viruses. These findings suggest that M2-2 mediates a regulatory "switch" from transcription to RNA replication, one that provides an initial high level of mRNA synthesis followed by a shift in the RNA synthetic program in favor of genomic RNA for virion assembly. With regard to vaccine development, the M2-2 knockout has a highly desirable phenotype in which virus growth is attenuated while gene expression is concomitantly increased.

Figure 1

Construction of the NdeI and K5 mutations, which interrupt M2 ORF2. Nucleotide sequences are in positive sense and blocked in triplets according to amino acid coding in ORF2. Nucleotide positions relative to the complete 15,223-nt recombinant antigenome are in parentheses; the other numbers refer to amino acid positions in the 194-aa M2–1 protein or 90-aa M2–2 protein. (A) Diagram of the two overlapping M2 ORFs. In the sequence at the top, the three potential translational start sites for M2–2 are underlined, and their encoded methionine residues are boxed. The termination codon for ORF1 is also underlined. In the diagram, restriction sites used for mutagenesis and cloning are indicated. (B) Construction of the NdeI mutation. The NdeI site at position 8299 in the middle of the M2–2 ORF was opened, filled in, and religated, which added 2 nt (lower case) to codon 47 of M2–2. This shifted the register to another reading frame, which was open for 18 additional codons encoding non-M2–2 amino acids (underlined). (C) Construction of the K5 mutation. The sequence shows the junction between ORF1 and ORF2, as in A. Potential ORF2 initiation codons in the wt parent are underlined, as is the ORF1 termination codon. Nucleotide changes in K5 are indicated above their wt counterparts. The three potential initiation codons for ORF2, codons 1, 3, and 7, were changed to ACG, which had no effect on amino acid coding in ORF1. The next potential methionyl start site in ORF2 is at codon 30. In addition, stop codons were introduced into all three frames immediately downstream of the M2–1 termination codon. In combination, these mutations had the effect of changing M2–2 amino acid 12 from K to N and terminating at codon 13.

Figure 2

The NdeI and K5 mutations each ablate the inhibitory function of M2–2 in a reconstituted minireplicon system. (A) HEp-2 cells were simultaneously infected with vTF7–3 (five plaque-forming units per cell) and transfected with plasmid encoding the negative-sense C2 minigenome cDNA (200 ng) and support plasmids (N, 400 ng; P, 200 ng; L, 100 ng per well of a six-well dish) and supplemented with pTM constructs (80 ng) expressing neither M2 ORF (lane 2), M2–2 (lane 3), M2–1 (lane 4), M2–1 + 2 (lane 5), or the M2–1 + 2 containing the NdeI (lane 6) or K5 (lane 7) mutations. Lane 1 contains RNA from a reaction that lacked L and is a negative control. Cells were exposed to 2 μg actinomycin D per milliliter from 24–26 hr after infection (21). At 48 hr after infection, total intracellular RNA was isolated and electrophoresed on formaldehyde gels for Northern blot analysis (16). Blots were hybridized to a negative-sense CAT-specific riboprobe to detect both mRNA and miniantigenome. (B) HEp-2 cells were transfected as described above with plasmid encoding positive-sense C4 miniantigenome complemented by the N, P, and L plasmids as in A. The transfection mixtures were supplemented with increasing amounts (0.008, 0.04, and 0.2 times the relative molar ratio of transfected pTM-N) of pTM constructs expressing M2–1 + 2 (lanes 2, 3, and 4), M2–1 (lanes 5, 6, and 7), M2–2 (lanes 8, 9, and 10) or M2–1 + 2 containing the NdeI (lanes 11, 12, and 13) or K5 (lanes 14, 15, and 16) mutation. Total intracellular RNA was analyzed by Northern blots hybridized with a positive-sense CAT-specific riboprobe to detect genomic RNA.

Figure 3

Cytopathogenicity of the rA2-NdeI and rA2-K5 viruses compared with rA2-wt. HEp-2 cells were mock infected or infected at an moi of 1 with the indicated virus, incubated for the indicated time, and photographed (×10). The 48-hr micrographs are darker because of a difference in exposure. Large syncytia are obvious in the two mutant viruses at 48 and 72 hr, and smaller ones are evident at 24 hr and in rA2-wt infected cells at the same three time points.

Figure 4

Kinetics of growth of rA2-wt, rA2-NdeI, and rA2-K5 in cell culture. The dashed horizontal lines indicate the lower limit of detectability. (A) Single-step growth kinetics. HEp-2 cells were infected with rA2-wt, rA2-NdeI, or rA2-K5 at an moi of 5, and the entire medium overlay was harvested at the indicated times after infection and flash frozen. Viral titers were determined by plaque assay. (B) Multicycle growth kinetics. HEp-2 cells were infected in triplicate at an moi of 0.01 plaque-forming units per cell with the above viruses. At the indicated times after infection, the entire medium overlay was removed and flash frozen. Cells were washed twice in PBS and incubated with fresh medium. Mean virus titers determined by plaque assay (with error bars) are shown.

Figure 5

Northern blot analysis of RNA replication and transcription. HEp-2 cells infected with rA2-wt (a, d, and g), rA2-NdeI (b, e, and h) and rA2-K5 (c, f, and i) were harvested at 3-hr intervals (lanes 1–10) from the single-cycle growth curve experiment described in Fig. 4A, and total intracellular RNA was isolated and subjected to Northern blot analysis. Blots were hybridized with a negative-sense N-specific riboprobe (a–c), a negative-sense F-specific riboprobe (d–f) or a positive-sense F specific riboprobe (g–i). Monocistronic mRNA, polycistronic readthrough mRNAs and antigenome and genome RNAs are indicated.

Figure 6

Western blot analysis of the accumulation of F and G glycoproteins in HEp-2 cells infected at an moi of 5 with rA2-wt (A and C) or rA2-NdeI (B and D). Cells were harvested at the indicated time, and total cellular protein was subjected to polyacrylamide gel electrophoresis under denaturing and reducing conditions, transferred to nitrocellulose (5), and reacted with rabbit antipeptide serum against the cytoplasmic domain of the F (A and B) or G (C and D) protein. Bound antibodies were detected with horseradish peroxidase-conjugated goat anti-rabbit IgG and visualized by enhanced chemiluminescence (Amersham Pharmacia).