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The Kaposi's sarcoma-associated herpesvirus (KSHV) SOX protein, encoded by ORF37, promotes shutoff of host cell gene expression during lytic viral replication by dramatically impairing mRNA accumulation. SOX is the KSHV homolog of the alkaline exonuclease of other herpesviruses, which has been shown to function as a DNase involved in processing and packaging the viral genome. Although the exonuclease activity of these proteins is widely conserved across all herpesviruses, the host shutoff activity observed for KSHV SOX is not. We show here that SOX expression sharply reduces the half-life of target mRNAs. Extensive mutational analysis reveals that the DNase and host shutoff activities of SOX are genetically separable. Lesions affecting the DNase activity cluster in conserved regions of the protein, but residues critical for mRNA degradation are not conserved across the viral family. Additionally, we present evidence suggesting that the two different functions of SOX occur within distinct cellular compartments—DNase activity in the nucleus and host shutoff activity in the cytoplasm.
Kaposi's sarcoma-associated herpesvirus (KSHV) is the most recently discovered human herpesvirus and the etiologic agent of Kaposi's sarcoma (KS), a neoplasm frequently associated with untreated AIDS patients (4). In addition to KS, KSHV is associated with two lymphoproliferative disorders, primary effusion lymphoma and multicentric Castleman's disease (3, 5, 17). As with other herpesviruses, KSHV exists in one of two forms in infected cells: either a latent stage where there is no virion production and viral gene expression is minimal or a lytic phase characterized by large-scale viral gene expression and virus replication (2, 12, 18). We have recently demonstrated that lytic KSHV infection results in a rapid and global shutoff of host cellular gene expression at the level of mRNA accumulation (6, 7). The primary effector of this shutoff is the KSHV SOX (shutoff and exonuclease) protein, encoded by ORF37 (7). SOX is the KSHV homolog of the viral alkaline exonuclease (AE), encoded by a gene conserved across the herpesvirus family but which has not been implicated in host shutoff in any of the other herpesviruses.
Herpes simplex virus (HSV) AE has been shown to possess both alkaline exonuclease and resolvase activity on DNA (9, 11, 14, 15); these functions are believed to be important for processing and packaging the viral genome in lytically infected cells. KSHV SOX and HSV-1 AE proteins exhibit a 26% overall identity and 67% identity within a set of domains that are highly conserved across all the herpesvirus homologs (7), suggesting that KSHV SOX likely retains many functions ascribed to the other family members. Indeed, SOX possesses an in vitro DNase activity similar to that of HSV-1 AE (7). However, in addition to its DNase activity, SOX can clearly influence host mRNA metabolism. Although the DNase activity of SOX could not account for its mRNA shutoff function (7), we had not previously been able to determine whether the two functions are genetically separable. To further explore the relationship between DNase activity and shutoff, we have attempted to separate these two functions by extensive mutational analysis. Here we show that these two key activities are indeed separable and that residues critical for the shutoff function of SOX are not evolutionarily conserved across other herpesviruses. This supports the contention that SOX has evolved a novel function unique to KSHV. Finally, we provide evidence that SOX-induced mRNA degradation may occur within the cytoplasm, suggesting that the different functions of SOX take place in distinct cellular compartments.
The green fluorescent protein (GFP) gene was subcloned from pd2EGFP-N1 (Clontech) into the EcoRI/NotI sites of pCDNA3.1(+) (Invitrogen) to generate pCDNA3.1-GFP. pCDEF3-ORF37 (SOX) was described previously (7). A hemagglutinin (HA) tag was introduced onto the 5′ end of SOX by PCR methods and cloned into the EcoRI/NotI sites of pCDEF3 to generate pCDEF3-HASOX. KSHV SOX was randomly mutagenized by PCR with the Genemorph kit (Stratagene) according to the manufacturer's protocol using 7 ng of pCDEF3-SOX template and 30 PCR cycles to generate a pool of random mutants with a mutation frequency of ~1 to 3 mutations/kb. The mutants were then cloned into the EcoRI/NotI sites of pCDEF3. Mutants with separable activities were subsequently 5′ HA tagged using PCR methods and cloned into the EcoRI/NotI sites of pCDEF3 to generate pCDEF3-HAQ129H, -HAT24I, -HAA61T, -HAP176S, -HAV369I, -HAD474N, and -HAY477*. A 5′ HA-tagged SOX mutant with prolines at amino acid residues 317 and 318 within the nuclear localization signal (NLS) was generated by overlapping PCR and cloned into the EcoRI/NotI sites of pCDEF3 to generate pCDEF3-HASOX NLS mut. A 5′ HA-tagged HSV-1 alkaline exonuclease gene was generated by PCR and cloned into the EcoRI/NotI sites of pCDEF3 to generate pCDEF3-HA HSV AE. All plasmids were verified by restriction digest and complete sequence analysis.
293T cells (American Type Culture Collection) and SLK cells (immortalized KSHV-negative spindle cells isolated from a classical KS tumor) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin-streptomycin. Cells were transfected using Fugene 6 (Roche).
Cell extracts used for immunoblotting were prepared in RIPA buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% [vol/vol] Nonidet P-40, 0.5% [wt/vol] sodium deoxycholate, 0.1% [wt/vol] sodium dodecyl sulfate [SDS]) containing protease inhibitors (Roche). Equivalent amounts of each sample were resolved by 7.5% SDS-polyacrylamide gel electrophoresis (PAGE), transferred to polyvinylidene difluoride membranes, and immunoblotted with polyclonal rabbit SOX antisera at a 1:4,000 dilution and secondary horseradish peroxidase-conjugated goat antirabbit antibodies (Southern Biotechnology Associates) at a 1:5,000 dilution. SOX antisera were raised against a purified, bacterially expressed glutathione S-transferase-SOX fusion protein by standard methods (10). For Northern blotting, total RNA was harvested from cells using RNA-BEE (Tel-Test, Inc.), or nuclear and cytoplasmic RNA were fractionated and harvested from cells using the PARIS kit (Ambion) and resolved by agarose-formaldehyde gel electrophoresis. RNAs were transferred to nitrocellulose membranes and probed with 32P-labeled DNA probes generated using the Rediprime II random prime labeling system (Amersham Biosciences, Inc.).
Proteins analyzed for DNase activity were in vitro transcribed and translated (IVT) using the Rabbit Reticulocyte Lysate system (Promega). From each reaction, 8 μl of protein was incubated with 200 ng of EcoRI-linearized pCDNA3.1 plasmid DNA in 42 μl of degradation assay buffer (0.1 M MgCl2, 0.5 M Tris [pH 9.0], 100-μg/ml bovine serum albumin, 5 mM β-mercaptoethanol) at 37°C for 1 or 15 min and then extracted with phenol-chloroform and ethanol precipitated. Pellets were resuspended in 20 μl of water, resolved on 0.8% agarose gels, and visualized by ethidium bromide staining. One-third of each IVT protein reaction was also resolved by SDS-PAGE, and the gels were fixed, dried, and developed by autoradiography to verify equivalent protein expression.
SLK or 293T cells on coverslips were fixed in 4% paraformaldehyde and subjected to immunofluorescence assay as described previously (1) using 12CA5 anti-HA primary antibodies (Roche) at a 1:400 dilution and rhodamine-conjugated goat antimouse secondary antibodies (Santa Cruz) at a 1:300 dilution.
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. (Fig.1).1). By contrast, in the presence of SOX, the GFP message half-life decreased to less than 1 h (Fig. (Fig.1),1), confirming that SOX promotes mRNA degradation.
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. (Fig.22 and and3).3). 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. (Fig.2),2), this was not consistently observed over the course of multiple experiments.
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. (Fig.4).4). 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.
Analysis of each of the single-function SOX mutants revealed that the mutations do not appear to cluster within discrete domains (Fig. (Fig.5),5), 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. (Fig.55 and Table Table1).1). 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 (Table11 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.
The deviation by SOX from the strict nuclear localization of other alkaline exonucleases (Fig. (Fig.4)4) 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. (Fig.6).6). 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.
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. (Fig.7A),7A), 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. (Fig.7B).7B). 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.44).
The fact that the AE gene is conserved across all known herpesviruses strongly suggests an important role for this factor in the virus life cycle. Indeed, there is a growing body of evidence for roles of the HSV-1 AE (UL12) protein as both a DNase responsible for processing and packaging the replicated viral genome into the capsid and a resolvase involved in viral DNA replication (9, 11, 14-16, 19). Recombination stimulated by HSV-1 AE occurs in concert with the single-stranded DNA binding protein ICP8 in a manner analogous to that of the bacteriophage λ Red recombinase (15). The high degree of homology between KSHV SOX and the other herpesvirus alkaline exonuclease genes is likely indicative that in KSHV this factor has retained these DNA-based functions. Indeed, we have observed in SOX a similar in vitro DNase activity, and residues previously shown to be critical for HSV-1 AE enzymatic function are both conserved and required for SOX DNase function (7, 8). Although SOX has yet to be tested for interaction with ORF6, the KSHV single-stranded DNA binding protein homolog, or for resolvase activity, it is anticipated to retain these properties as well.
However, unlike the other AE homologs, KSHV SOX has acquired a novel function: the ability to promote cellular mRNA degradation. SOX-induced mRNA turnover leads to a global host gene shutoff during lytic KSHV infection (7), an event that likely facilitates efficient expression of viral genes and may also impair production of antiviral factors by the infected cell. Our ability to isolate single-function mutants defective for either DNase or shutoff provides strong evidence that these activities are truly distinct; in particular, they provide strong evidence against the notion that the loss of mRNA in SOX-expressing cells is due to DNase-mediated degradation of the DNA template. The fact that mutations inactivating the RNA degradation function do not cluster in conserved regions is consistent with the idea that this function is a recent evolutionary acquisition by the protein, in contrast to the DNase function, which is ancient and conserved across the entire family.
How does SOX function to facilitate RNA turnover? Certainly the simplest model would be that SOX has acquired RNase activity, in keeping with its membership in the nuclease superfamily. While we cannot entirely rule out this possibility, extensive efforts to detect RNase activity with recombinant SOX protein have failed (Glaunsinger and Ganem, unpublished data), despite the fact that the protein retains DNase activity (Fig. (Fig.3)3) and hence must be properly folded. However, it is possible that SOX may indeed possess latent or cryptic RNase activity that requires activation by a cellular cofactor. Alternatively, SOX may act by deregulating one or more components of the normal host mRNA degradation machinery. Recent work indicates that the majority of normal cellular mRNA turnover occurs within the cytoplasm (13), and our results suggest that SOX similarly promotes degradation of mRNAs in this cellular compartment (Fig. (Fig.6).6). It is therefore probably no coincidence that KSHV SOX localizes to both the nucleus and the cytosol, while its HSV counterpart is exclusively nuclear. Indeed, a SOX NLS mutant localized primarily in the cytosol retained wild-type host shutoff activity, suggesting that nuclear localization is not critical for this function. The notion that SOX may deregulate cytosolic mRNA degradation machinery accords well with our findings that the SOX shutoff function acts primarily in the cytoplasm. We are currently searching for host proteins that interact biochemically or functionally with SOX in an effort to identify the direct target of SOX action. If the history of virology is any guide, the identification of SOX targets may be expected to yield new insights into the control of mammalian mRNA turnover, much as the study of human immunodeficiency virus rev informed analysis of the RNA export pathway of the cell.
B.G. was supported by an American Cancer Society fellowship.
We thank Adam Grundhoff for critical reading of the manuscript.