KSHV SOX enhances degradation of RNA.
We previously demonstrated that the KSHV SOX expression decreased the levels of a reporter mRNA while affecting neither its DNA template nor its rate of transcription; from this we inferred that SOX promotes enhanced mRNA turnover (7
). To directly demonstrate that SOX reduces mRNA stability, we examined the half-life of GFP mRNA in cells in the presence or absence of SOX using the transcriptional inhibitor actinomycin D. When expressed in 293T cells alone, GFP mRNA was quite stable, exhibiting a half-life of approximately 12 h (Fig. ). By contrast, in the presence of SOX, the GFP message half-life decreased to less than 1 h (Fig. ), confirming that SOX promotes mRNA degradation.
FIG. 1. SOX promotes enhanced mRNA turnover. 293T cells were transfected with either empty vector or a plasmid expressing GFP or cotransfected with plasmids expressing GFP and SOX at a 1:4 ratio. Forty-eight hours posttransfection, cells were treated with actinomycin (more ...) The DNase and shutoff functions of SOX are separable.
To explore the relationship between the DNase activity and shutoff function of SOX, we attempted to separate the two functions by mutagenesis. In preliminary studies, we observed that SOX is extremely sensitive to mutation; even relatively small deletions or insertions in the protein usually rendered it unstable (data not shown). We therefore elected to perform a random PCR-mediated mutagenesis protocol using conditions to achieve a mutation rate of ~1 mutation/kb, generating a pool of mutants containing one to three missense mutations throughout the 1.46-kb SOX gene. Approximately 250 SOX mutants were then screened by immunoblotting for their ability to be expressed to levels comparable to that of the wild-type protein in 293T cells by Western blotting; only approximately one-third of the mutants met this criteria (data not shown). These remaining stable mutants were then tested for their ability to degrade GFP mRNA when cotransfected with a GFP plasmid into 293T cells and to degrade linearized DNA in an in vitro DNase assay. Mutants that retained only one of the two functions were then sequenced to identify mutated residues, and in cases where multiple mutations were present, each mutation was recloned to isolate single-amino-acid mutants and rescreened in the RNA and DNA turnover assays. We did not pursue mutants that were negative in both assays, since lack of any functional activity could simply be the result of a misfolded protein.
Using this system, we successfully identified seven mutants that in at least five independent experiments for each assay exhibited either DNase or shutoff activity but not both (Fig. and ). One mutant (Q129H) retained wild-type shutoff activity but was completely inactive as a DNase, whereas the other six mutants (T24I, A61T, P176S, V369I, D474N, and Y477* [the asterisk indicates a stop codon]) were capable of degrading DNA but were defective at promoting mRNA turnover. Although we occasionally noted some partial retention of shutoff activity for the A61T and D474N mutants (Fig. ), this was not consistently observed over the course of multiple experiments.
FIG. 2. Analysis of the host shutoff activity of SOX mutants. 293T cells were transfected in duplicate with either empty vector or a plasmid expressing GFP or cotransfected with plasmids expressing GFP and either wild-type or the indicated mutant SOX at a 1:4 (more ...)
FIG. 3. Analysis of the DNase activity of SOX mutants. Linearized pCDNA3 plasmid DNA was incubated with aliquots of the indicated IVT protein for 1 or 15 min in degradation assay buffer at 37°C. The DNA was then extracted, resolved by agarose gel electrophoresis, (more ...)
In order to determine whether the functional defects observed in the mutants were a result of aberrant protein localization, we examined the expression pattern of each mutant in SLK cells by immunofluorescence. These experiments were performed using HA-tagged constructs, since the antibody we raised to recombinant SOX does not recognize the native protein. Unlike HSV-1 AE, which was exclusively nuclear, wild-type SOX, although predominantly nuclear, also showed additional cytoplasmic staining; the same was true of all of the SOX mutants (Fig. ). Identical results were obtained in 293T cells (data not shown). Thus, the inability of these SOX mutants to function in the DNase or RNA turnover assays is not caused by mislocalization within the cell.
FIG. 4. SOX single-function mutants do not display aberrant subcellular localization. SLK cells were transfected with either empty vector or plasmids expressing HA-tagged HSV-1 AE or HA-tagged wild-type or mutant SOX. Forty-eight hours posttransfection, cells (more ...) Residues required for the SOX shutoff function are not conserved.
Analysis of each of the single-function SOX mutants revealed that the mutations do not appear to cluster within discrete domains (Fig. ), suggesting that the shutoff activity may reside in conformational rather than linear determinants. Each of the alkaline exonuclease homologs contains seven domains that are conserved across the herpesvirus family and believed to be important for their common DNase function (8
). Interestingly, the mutation whose protein lacks DNase activity (Q129H) is located within one of these conserved domains, whereas five of the six mutations whose proteins lack shutoff activity are located outside of the conserved regions (Fig. and Table ). ClustalW multiple sequence alignments of 14 different herpesvirus alkaline exonuclease homologs revealed that only the Q129H mutation was located at a strictly conserved residue; even the shutoff V369I mutation, located in conserved domain VI, was at a residue not conserved between KSHV and other herpesviruses (Table and data not shown). Thus, the residues required for the common DNase function are conserved across the herpesvirus family, whereas residues required for the shutoff function unique to KSHV SOX are not conserved across herpesviruses. These results are consistent with our model that KSHV SOX has evolutionarily acquired a unique shutoff function, superimposed on the DNase function shared by the entire family.
FIG. 5. Distribution of SOX single-function mutations. SOX mutations whose proteins lack the shutoff function are indicated with black asterisks, while the mutation whose protein lacks DNase activity is indicated by the gray asterisk. Conserved domains are boxed. (more ...)
Summary of location and activity of SOX single-function mutations
SOX promotes degradation of cellular mRNAs in the cytoplasm.
The deviation by SOX from the strict nuclear localization of other alkaline exonucleases (Fig. ) is intriguing given the fact that the majority of cellular mRNA degradation is thought to occur in the cytoplasm. We hypothesized that while the nuclear fraction of SOX is likely required for its DNase-related functions, the cytoplasmic fraction might instead be responsible for host shutoff. We were therefore interested in determining where the SOX-induced mRNA degradation occurred within the cell. To this end, total, nuclear, and cytoplasmic RNA was isolated from 293T cells expressing GFP alone or together with SOX (Fig. ). As expected, Northern blotting with a GFP probe revealed that in the presence of SOX, GFP mRNA levels were decreased in the total RNA fraction. Likewise, the cytoplasmic RNA fraction showed a similar decrease in GFP mRNA in the presence of SOX. However, we observed no reduction of GFP message by SOX in the nuclear fraction, suggesting that SOX enhances mRNA degradation exclusively in the cytoplasm. Furthermore, the fact that there was no accumulation of nuclear GFP mRNA in SOX-expressing cells suggests that SOX is not preventing nuclear export of cellular mRNAs.
FIG. 6. SOX promotes degradation of cytoplasmic mRNA. 293T cells were transfected in duplicate with empty vector or a plasmid expressing GFP or cotransfected with plasmids expressing GFP and SOX at a 1:4 ratio. Forty-eight hours posttransfection, cells were either (more ...)
Analysis of the SOX sequence identified a putative nuclear localization signal (NLS), 315PRKKRK320, located between conserved regions IV and V. However, since our results suggest that the SOX shutoff function instead occurs in the cytoplasm, we hypothesized that the nuclearly localized SOX may be dispensable for the mRNA degradation function. We therefore generated a SOX NLS mutant and tested its localization and activity in 293T cells. In contrast to wild-type SOX, the NLS mutant SOX was almost exclusively cytoplasmic (Fig. ), confirming that amino acids 315 to 320 are required for SOX nuclear localization. Importantly, the NLS mutant displayed wild-type levels of host shutoff activity as measured by GFP mRNA degradation in both 293T and SLK cells (Fig. ). Although we of course cannot formally rule out the possibility that a small fraction of the SOX NLS mutant may remain in the nucleus and promote mRNA degradation from that compartment, these data collectively support the view that the host shutoff function most likely operates within the cytoplasm of cells. Since viral DNA replication and packaging are nuclear events, the DNase activity of the protein presumably operates in this compartment, an inference consistent with the exclusively nuclear locale of HSV alkaline exonuclease (Fig. ).
FIG. 7. Nuclear localization of SOX is not required for shutoff activity. (A) 293T cells were transfected with empty vector or plasmids expressing HA-tagged wild-type (HA-SOX) or NLS mutant (HA-nls mut) SOX. Twenty-four hours posttransfection, cells were stained (more ...)