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. 1998 Feb;72(2):1452-61.
doi: 10.1128/JVI.72.2.1452-1461.1998.

The NS1 protein of human respiratory syncytial virus is a potent inhibitor of minigenome transcription and RNA replication

P L Atreya  1 , M E Peeples, P L Collins
Affiliations

The NS1 protein of human respiratory syncytial virus is a potent inhibitor of minigenome transcription and RNA replication

P L Atreya et al. J Virol. 1998 Feb.

Abstract

The NS1 protein (139 amino acids) is one of the two nonstructural proteins of human respiratory syncytial virus (RSV) and is encoded by a very abundant mRNA transcribed from the promoter-proximal RSV gene. The function of NS1 was unknown and was investigated here by using a reconstituted transcription and RNA replication system that involves a minireplicon and viral proteins (N, P, L and M2-1) expressed from separate cotransfected plasmids. Coexpression of the NS1 cDNA strongly inhibited transcription and RNA replication mediated by the RSV polymerase, even when the level of expressed NS1 protein was substantially below that observed in RSV-infected cells. The effect depended on synthesis of NS1 protein rather than NS1 RNA alone. Transcription and both steps of RNA replication, namely, synthesis of the antigenome and the genome, appeared to be equally sensitive to inhibition. The efficiency of encapsidation of the plasmid-derived minigenome was not altered by coexpression of NS1, indicating that the inhibition occurs at a later step. In two different dicistronic minigenomes, transcription of each gene was equally sensitive to inhibition by NS1. This suggested that the gradient of transcriptional polarity was unaffected and that the effect of NS1 instead probably involves an early event such as polymerase entry on the genome. NS1-mediated inhibition of transcription and RNA replication was not affected by coexpression of the M2 mRNA, which has two open reading frames encoding the transcriptional elongation factor M2-1 and the putative negative regulatory factor M2-2. The potent nature of the NS1-mediated inhibition suggests that negative regulation is an authentic function of the NS1 protein, albeit not necessarily the only one.

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Figures

FIG. 1
FIG. 1
Schematic representation (not to scale) of the cDNA-encoded, negative-sense RSV-CAT C2 minigenome RNA and its modified derivatives, MP13 and MP123. The C2 minigenome contains the 3′-terminal 75 nt and 5′-terminal 179 nt of the genome of RSV strain A2, including the leader, GS, non-protein-coding (NC), GE, and trailer regions (open boxes). These sequences flank a negative-sense copy of the CAT ORF (shaded box). MP13 is a version of C2 in which a 35-nt sequence containing a GE signal, a single-nucleotide intergenic region (IG), and GS signal has been inserted in the CAT ORF at its BspEI site. This converts the minigenome into one which is dicistronic, encoding two subgenic CAT mRNAs of 271 and 495 nt, exclusive of poly(A). MP123 is a version of C2 in which the first 34 nt of the leader region have been replaced with a sequence that is complementary to the 5′-terminal 57 nt of the trailer region (Tc) and thus contains the antigenome promoter. In addition, MP13 and MP123 differ from C2 in each having a single-nucleotide substitution in the trailer region that greatly inhibits amplification by the RSV polymerase: in MP13 nt 5 from the 5′ end has been changed from G to A (TrG5A), and in MP123 nt 7 from the 5′ end has been changed from A to C (TrA7C). Nucleotide lengths of the various segments are indicated.
FIG. 2
FIG. 2
Western blot analysis of proteins synthesized in 293 cells which had been infected with 10 PFU of RSV per cell (lane 1) or infected with the vaccinia virus recombinant vTF7-3 (lanes 2 to 6) and mock transfected (lane 2) and then transfected with 200 ng of N plasmid and 100 ng of P plasmid (lane 3) or the same amounts of N and P plasmids supplemented with 30 ng (lane 4), 60 ng (lane 5), or 100 ng (lane 6) of NS1 plasmid. Cells were harvested 18 h (lane 1) or 40 h (lanes 2 to 6) postinfection, and lysates were electrophoresed on a 4 to 20% gradient gel and transferred to nitrocellulose. The upper blot was probed with a polyclonal antiserum against gradient-purified, disrupted RSV virions (17), which is reactive with most of the RSV structural proteins including G, N, P, M, and SH. The lower blot was probed with an antibody against a C-terminal peptide of the NS2 protein, which recognizes both the NS1 and NS2 proteins in RSV-infected cells (lane 1) and NS1 in the plasmid-transfected cells (lanes 4 to 6).
FIG. 3
FIG. 3
Northern blot (A and C) and PhosphorImager (B and D) analysis showing the effects of increasing expression of NS1 plasmid on the synthesis of positive-sense RNA from the C2 minigenome (A and B) and the synthesis of negative-sense RNA from the C4 mini-antigenome (C and D). 293 cells were infected with vaccinia virus vTF7-3 and transfected with 50 ng of C2 minigenome (A) or C4 mini-antigenome (C) plasmid, 200 ng of N plasmid, 100 ng of P plasmid, 0 ng (lane 1) or 25 ng (lanes 2 to 10) of L plasmid, 0 ng (top panel) or 10 ng (bottom panel) of M2 plasmid containing both ORFs, and the indicated amount (0 to 30 ng [lanes 2 to 10]) of NS1 plasmid. Intracellular RNA was harvested 40 h postinfection and analyzed by Northern blot hybridization with a negative-sense (A) or positive-sense (C) RSV-CAT riboprobe. Hybridized radioactivity was quantitated by PhosphorImager analysis: quantitation of panels A and C is shown in panels B and D, respectively.
FIG. 4
FIG. 4
Northern blot analysis showing the synthesis and encapsidation of the mini-antigenome from the plasmid-supplied C2 minigenome in the absence (−NS1) or presence (+NS1) of 50 ng of NS1 plasmid. 293 cells were infected with vTF7-3 and transfected with C2 plasmid together with the support plasmids indicated at the top, added in the same amounts as in Fig. 3. The M2 plasmid was that containing both ORFs. Lysates were prepared 40 h postinfection and were either mock treated (lanes 1, 3, and 5) or treated (lanes 2, 4, and 6) with micrococcal nuclease (MCN), after which the remaining RNA was isolated and analyzed by Northern blot hybridization with a negative-sense RSV-CAT riboprobe. The positions of the mini-antigenome and mRNA are shown.
FIG. 5
FIG. 5
The inhibitory effect of the NS1 plasmid is ablated by a frameshift in the NS1 ORF. (A) The NS1 cDNA was mutagenized to delete 2 nt from codon eight, which shifts the reading frame and terminates the ORF three codons later. This is illustrated in the sequence of codons 1 to 11 of the NS1 ORF. This mutation would preclude translation from either the first or eighth codons, which are AUG. The next AUG in the 139-codon ORF is at codon 80. (B) Western blot analysis showing the expression of the NS1 protein from the wild-type (lanes 1 to 5) or mutant (lanes 6 to 9) plasmid. The indicated amounts of plasmid were transfected into 293 cells infected with vTF7-3. Cell lysates were prepared 40 h later and analyzed with a Western blot probed with an antiserum against a synthetic peptide of the NS2 protein. (C) Expression of CAT by the C2 minigenome complemented by the N, P, L, and M2 (both ORFs) plasmids together with the indicated amount of wild-type or mutant NS1 plasmid. Cells were harvested 40 h postinfection, and lysates were assayed for the ability to acetylate [14C]chloramphenicol as visualized by thin-layer chromatography and autoradiography (the unreacted chloramphenicol comprises the bottom row of spots, and the acetylated forms comprise the two higher rows).
FIG. 6
FIG. 6
The NS1 protein does not affect encapsidation of plasmid-supplied or RSV-amplified C2 minigenome. 293 cells were infected with vTF7-3 and transfected with 50 ng of C2 minigenome, 0 (−NS1) or 50 ng (+NS1) of NS1, and the indicated support plasmids in the same amounts as in Fig. 3. The M2 plasmid was that containing both ORFs. Lysates were prepared 40 h postinfection and were either mock treated (lanes 1, 3, 5 and 7, both panels) or treated (lanes 2, 4, 6, and 8, both panels) with micrococcal nuclease (MCN), after which the remaining RNA was isolated. The RNAs were analyzed by Northern blot hybridization with a positive-sense RSV-CAT riboprobe, which would detect minigenome that is plasmid encoded as well as that which is generated by RSV-mediated replication. Some of the plasmid-encoded minigenome accumulates in a form (labeled minigenome w/ ribozyme) that is slightly larger than the minigenome because it contains uncleaved ribozyme at its 3′ end.
FIG. 7
FIG. 7
The NS1 protein inhibits the synthesis of positive-sense RNA from the minigenome MP13. MP13 is an analog of genomic RNA that encodes two short mRNAs from the upstream and downstream ends of the CAT ORF (mRNAs 1 and 2, which are 271 and 495 nt exclusive of poly[A]), as illustrated in Fig. 1. In addition, MP13 is restricted from amplification by the RSV polymerase due to a point mutation (TrG5A) in the trailer region. (A) 293 cells were infected with vTF7-3 and transfected with MP13 minigenome and the N, P, and L support plasmids in the same amounts as in Fig. 3 and the indicated amount (0 to 50 ng) of NS1 plasmid. L plasmid was omitted from lane 1 as a negative control. RNA was isolated 40 h postinfection and analyzed by Northern blot hybridization with negative-sense RSV-CAT riboprobe. (B) The hybridized radioactivity was quantitated on a PhosphorImager (B). Each RNA species was normalized separately with respect to the sample that received L, N, P, but no NS1 (lane 2) as 100%.
FIG. 8
FIG. 8
The NS1 protein inhibits RNA synthesis from the antigenome promoter. MP123, a copyback-type minigenome in which the 3′-leader region was replaced by the complement of the last 57 nucleotides of the trailer region, which corresponds to the 3′ end of the antigenome, was constructed (Fig. 1). The TrA7C trailer mutation was included to block minigenome amplification by the RSV polymerase. MP123 was analyzed in parallel with MP96, which is a version of the prototype C2 minigenome which also contains the TrA7C point mutation. HEp-2 cells were infected with vTF7-3 and transfected with 100 ng of the MP96 (A and B) or MP123 (C and D) minigenome; the N, P, and L support plasmids in the amounts as described in the legend to Fig. 3; 50 ng of M2-1 plasmid; and the indicated amount (0 to 100 ng [lanes 2 to 6]) of NS1 plasmid. Lane 1 is a negative control in which the L plasmid was omitted. Cells were harvested 40 h postinfection, and lysates were prepared and processed for RNA purification directly (A and C) or following treatment with micrococcal nuclease (B and D). RNA was analyzed by Northern blot hybridization with a negative-sense CAT riboprobe.
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