Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Carretera Majadahonda-Pozuelo Km2, Majadahonda, Madrid 28220, Spain1
Department of Microbiology, BBRB 17/Rm 366, University of Alabama Medical School, Birmingham, AL 3594-2170, USA2
Author for correspondence: Nieves Villanueva. Fax +34 91 5097966. e-mail nvilla{at}isciii.es
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Abstract |
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An alternative means of controlling HRSV infection is to develop specific antiviral compounds, a goal that requires knowledge of the function(s) of specific viral proteins. We have focused on the function of phosphorylation of the phosphoprotein (P).
The P protein (241 amino acids) is found in infected cells and is also a component of the virus ribonucleocapsid (Huang et al., 1985 ; Lambert et al., 1988
), which is the functional template for virus transcription and replication. The P protein of HRSV is mainly phosphorylated in vivo on serines 116, 117, 119 and 232, located in the central and C-terminal regions of the molecule (Navarro et al., 1991
; Villanueva et al., 1994
). However, phosphorylation of serine residues 116, 117 and 119 has been questioned (Mazumder & Barik, 1994
). The enzymatic activity responsible for P protein modification was partially purified from HEp-2 cells and characterized as casein kinase II (CKII) (Mazumder & Barik, 1994
; Mazumder et al., 1994
; Villanueva et al., 1994
). P protein, produced in bacteria, can be phosphorylated on serine 232 or 237, depending on the level of purification of CKII (Barik et al., 1995
), and it has been suggested that each of these CKII-mediated phosphorylations is essential to render unphosphorylated P protein active for viral transcription in vitro (Barik et al., 1995
). However, inhibition of P protein phosphorylation in HRSV Long strain-infected HEp-2 cells does not impair transcription or replication (Villanueva et al., 1991
).
To clarify the function of P protein phosphorylation, P proteins with substitutions at the phosphorylatable serines were analysed in vivo for their ability to support transcription and replication of subgenomic HRSV RNA replicons in a vaccinia virusT7 expression system (Yu et al., 1995 ; Hardy & Wertz, 1998
).
The P gene of the HRSV Long strain, contained in plasmid P20 (López et al., 1988 ), was subcloned under the control of the T7 promoter into the SmaI site of pGEM3 (Promega) as a StuIHpaI fragment. Mutant P genes constructed previously in vaccinia virus recombinants (Sánchez-Seco et al., 1995
) were subcloned into T7 expression plasmids. The pGEM3 expression plasmids were named in the same manner as the corresponding vaccinia virus recombinants from which they were derived: VP (Long strain P protein), VP3, in which the serines at positions 116, 117 and 119 were changed to leucine, arginine and leucine (S116L, S117R, S119L), VP8, in which serine 232 was changed to alanine (S232A), and VP9, in which serine 237 was changed to alanine (S237A). VP3-8 (S116L, S117R, S119L, S232A) was prepared by substitution of the P gene NcoI fragment from plasmid VP3 for that present in plasmid VP8. The nucleotide sequences of the P genes of all recombinant plasmids were determined by sequence analysis.
The levels of phosphorylation of all P protein variants were analysed in HEp-2 cells transfected with wild-type or mutant plasmids (García et al., 1993 ) by using the vaccinia virusT7 RNA polymerase expression system (Fuerst et al., 1987
). The transfected cultures were labelled for 924 h post-transfection with 100 µCi [32P]orthophosphate or 50 µCi [35S]methionine, cellular extracts were prepared and the P protein was immunoprecipitated (Villanueva et al., 1991
). The results of these analyses are shown in Fig. 1(a
, b
). Quantitative analysis to determine the levels of P protein phosphorylation (Fig. 1c
) was performed by using a phosphorimager. The relative activities obtained from the transfected cultures were similar to those found when the different P protein variants were expressed from the corresponding vaccinia virus recombinants, as reported previously (Sánchez-Seco et al., 1995
).
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Unexpectedly, the mutant VP3-8 showed only 2% of the wild-type level of phosphorylation. Thus, it seems that 98% of P protein phosphorylation is due to modification of serine residues 116, 117, 119 (19·6%) and 232 (78·4%), but there is an additional 2% residual phosphorylation. Therefore, we conclude that P protein phosphorylation shows a hierarchy for its modification at different residues, as has been reported for other phosphoproteins (Roach, 1991 ).
The function of the mutant P proteins was first examined by using a subgenomic replicon of HRSV that directs both replication and synthesis of a single mRNA when expressed in cells with the N, P, L and M2 proteins (Hardy & Wertz, 1998 ). The subgenomic replicon RNA, WT5, contained the HRSV leader sequence, the start of the NS1 gene fused to the end of the L gene and the trailer sequence of HRSV A2 strain. Thus, this replicon contains all the signals required for its own replication and for the transcription of a single mRNA (Hardy & Wertz, 1998
) (Fig. 2a
).
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The products of RNA synthesis from replicon WT5 are shown diagrammatically in Fig. 2(a) and were identified, as described previously, by annealing of specific oligonucleotides followed by RNase digestion (Hardy & Wertz, 1998
). These products, as indicated in Fig. 2(a)
, are the products of genomic positive- and negative-strand RNA replication (rep), the single mRNA species (mRNA) and a product that resulted from the polymerase failing to terminate at the end of the mRNA and reading through into the trailer to generate a readthrough product (r/t), which migrates faster than the replication product because it lacks the 44 nucleotide leader RNA.
All the labelled RNAs species were synthesized by the viral polymerase formed by the L and P proteins, as none of them appeared when P protein was omitted (Fig. 2b, no P). Additionally, the P protein from the Long strain formed functional nucleocapsids with the N, L and M2 proteins of the A2 strain, as shown by the finding that it was able to support transcription and replication from the WT5 replicon to a similar extent to the wild-type A2 strain P protein (Fig. 2
, A2 and VP). The P protein mutants VP3 and VP8 were also able to replicate and transcribe the HRSV RNA analogue. We also tested the behaviour of the variant VP9 (S237A), since this P protein variant has been reported to have a dominant-negative effect on P protein function (Mazumder et al., 1994
), although in our hands we did not find any modification of this serine residue (Sánchez-Seco et al., 1995
). Again, this P protein mutant was also able to transcribe and replicate the HRSV RNA analogue (Fig. 2
, VP9), although with a lower efficiency than its wild-type counterpart. This could be due to the possible conformational change induced in the variant (Sánchez-Seco et al., 1995
).
Because the P protein variants VP3 and VP8 are phosphorylated at serine 232 and at serines 116, 117 and 119, respectively, the experiment described above only indicates that the simultaneous presence of both phosphorylated domains of P protein is dispensable for transcription and replication. In order to determine the effect of a P protein that was not phosphorylated at either of the two domains on viral transcription and replication, another P protein mutant, VP3-8, was prepared for use in subsequent experiments.
Since the mutant P proteins were all functional when examined by using a subgenomic replicon that expressed one mRNA, we next examined the activities of VP3, VP8 and VP3-8 during RNA replication and transcription by using a subgenomic replicon encoding two mRNAs. This was done to determine whether the P protein might have a separate role in the transit of the polymerase across a gene junction. The RNA synthesis assays were performed as indicated above but using the replicon M/SH, which encodes two mRNAs (see Fig. 3a; Hardy & Wertz, 1998
). The construction of the plasmid, pM/SH, that encodes this replicon has been described previously (Hardy & Wertz, 1998
). This replicon contains the leader and trailer elements surrounding two transcriptional units separated by the M/SH gene junction. The ability of the mutated P proteins to support replication and transcription of the two mRNAs was examined as described above. The results obtained are shown in Fig. 3
. The major RNA products were, as shown in Fig. 3(a)
, the products of genomic RNA replication (rep), the two monocistronic mRNAs, mRNA1 and mRNA2, and the bicistronic products of readthrough at the end of mRNA1 into mRNA2 (r/t B) or mRNA1 into the trailer (r/t C). The abundant readthrough products occurred because of the increased processivity of the polymerase in the presence of the M2 protein and its failure to terminate at the ends of the genes. The identification of each RNA species was made by annealing specific oligonucleotides followed by digestion with RNase H, as described previously (Hardy & Wertz, 1998
).
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The experiments shown in Fig. 3 were also carried out in the absence of the M2 protein. The results obtained were qualitatively the same as those obtained in the presence of M2 protein, in that all the mutants could support replication and transcription although to slightly varying degrees (data not shown). However, since the mRNA levels were reduced in the absence of M2, as reported previously, we have chosen to show analysis of the P mutants in the presence of the M2 protein as this most closely reflects the natural situation.
These results suggest that the bulk of P protein phosphorylation (98%), which occurs at residues 116, 117, 119 and 232, is not essential for transcription or replication of HRSV RNA subgenomic replicons expressing one or two mRNAs. Whether or not the residual phosphorylation (2%) displayed by the P protein variant VP3-8 is essential for viral transcription and replication remains to be determined. However, the level of modification of the P protein modulates RNA transcription and replication.
The results described here are similar to those found in HRSV-infected cells treated with the inhibitor xanthate D609, although in that case, the level of P protein phosphorylation was even less than 2% of that found in the absence of the drug (Villanueva et al., 1991 ). These results indicate that the bulk of phosphorylation, which occurs at serines 116, 117 and 119 (19·6%) and 232 (78·4%) (Sánchez-Seco et al., 1995
), is not essential for viral transcription or replication.
Taking into account that P protein phosphorylation modulates viral RNA transcription and replication, it is possible that completely dephosphorylated P protein or P protein with only 2% of its total phosphorylation level can function in HRSV-infected cells and in the vaccinia-virus based system. However, this low level of phosphorylation may not be sufficient to support transcription in an in vitro system like that used by Barik et al. (1995) .
In summary, the results presented in this paper indicate that the bulk of P protein phosphorylation is not essential for transcription or replication of viral RNA but that the level of phosphorylation can modulate P protein function in these processes.
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Acknowledgments |
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References |
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Received 15 June 1999;
accepted 24 September 1999.