The Epstein-Barr Virus SM Protein Is Functionally Similar to ICP27 from Herpes Simplex Virus in Viral Infections

Abstract

The herpes simplex virus type 1 (HSV-1) ICP27 protein is an essential RNA-binding protein that shuttles between the nucleus and cytoplasm to increase the cytoplasmic accumulation of viral late mRNAs. ICP27 homologs have been identified in each of the herpesvirus subfamilies, and accumulating evidence indicates that homologs from the gammaherpesvirus subfamily function similarly to ICP27. In particular, the Epstein-Barr virus (EBV) SM protein posttranscriptionally regulates gene expression, binds RNA in vitro and in vivo, and shuttles between the nucleus and cytoplasm. To determine if these two proteins function through a common mechanism, the ability of EBV SM to complement the growth defect of an HSV-1 ICP27-null virus was examined in a transient-expression assay. ICP27 stimulated the growth of the null mutant more efficiently than did SM, but the ability of SM to compensate for the ICP27 defects suggests conservation of common functions. To assay for complementation in the context of a viral infection, the growth properties of an HSV recombinant expressing SM in an ICP27-null background were analyzed. SM stimulated growth of the recombinant, although this growth was reduced by comparison to that of an ICP27-expressing virus. By contrast, an HSV recombinant expressing an SM mutant allele defective for transactivation activity and nucleocytoplasmic shuttling did not grow at all. These results suggest that SM and ICP27 may regulate gene expression through a common pathway that is evolutionarily conserved in herpesviruses.

The infectious cycle of herpes simplex virus type 1 (HSV-1) is characterized by a tightly regulated cascade of gene expression. On the basis of their temporal expression, HSV-1 genes have been classified as immediate early (α), early (β), and late (γ) genes (33, 34). Following penetration of the virus into the host cell, the α genes are transcribed in the absence of de novo protein synthesis. The α genes have an important role in the regulation of viral gene expression; ICP0, ICP4, ICP22, and ICP27 coordinately regulate the expression of all classes of viral genes (18, 25, 48, 57, 60, 64, 66, 73, 90, 94). Early gene expression is dependent on α gene expression, and the β genes encode proteins necessary for viral DNA replication. Late genes primarily encode components of the virion and can be further subdivided into γ₁ (leaky late) genes, which are expressed prior to the onset of viral DNA replication, and γ₂ (true late) genes, which require replicated viral DNA templates for expression.

During the course of a productive HSV-1 infection, ICP27 has many activities that involve the regulation of both host and viral gene expression. Viral mutants with deletions of the ICP27 gene are completely defective for growth on standard cell lines, demonstrating that this protein is essential for viral replication (73). ICP27 regulates both early and late viral gene expression at the level of transcription (36, 80) and has a well-defined role in the posttranscriptional regulation of viral late-gene expression (43, 62, 63, 75, 80, 81). In the absence of functional ICP27, viral late-gene expression is severely reduced (67, 73). However, in cells infected with a virus expressing a temperature-sensitive allele of ICP27, the transcription rates of viral late genes are unaffected after a shift up to the restrictive temperature, indicating that ICP27 acts posttranscriptionally (80).

Previous studies suggest that ICP27 is an RNA-binding protein that shuttles between the nucleus and cytoplasm and increases the cytoplasmic accumulation of viral late mRNAs (43, 51, 62, 63, 75, 81). Nucleocytoplasmic shuttling occurs at late times postinfection and in the presence of particular late mRNAs (43, 62, 81); therefore, the data support a model in which ICP27 facilitates viral late-gene expression by mediating the nuclear export of viral late mRNAs.

ICP27 is the only HSV-1 α protein that has a homolog in each of the herpesvirus subfamilies. These homologs include the varicella-zoster virus (VZV) ORF4 (13, 35) and the equine herpesvirus type 1 (EHV-1) UL3 (86, 96) proteins from the alphaherpesvirus subfamily; the human cytomegalovirus (CMV) UL69 (10) protein from the betaherpesvirus subfamily; and the Epstein-Barr virus (EBV) SM (11), the herpesvirus saimiri (HVS) ORF57 (54), and the Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 (1, 29, 70) proteins from the gammaherpesvirus subfamily. This conservation suggests that certain regulatory functions of ICP27 are maintained throughout the herpesvirus family.

The functional data that are available for ICP27 homologs indicate that although these proteins show sequence similarity, they exhibit considerable functional diversity. For example, the homologs from CMV (UL69) and VZV (ORF4) primarily stimulate gene expression at the level of transcription (14, 15, 35, 53, 61, 95). In contrast, recent functional studies of ICP27 homologs from the gammaherpesvirus subfamily (EBV SM, HVS ORF57, and KSHV ORF57) indicate that these proteins function in posttranscriptional steps of gene expression and shuttle between the nucleus and cytoplasm (1, 4, 8, 26, 29, 38-40, 42, 72, 78, 92).

The EBV SM protein is the product of a spliced mRNA containing both the BSLF2 and BMLF1 open reading frames (11). At the amino acid level, SM and ICP27 are 30% similar over the entire length of the proteins. SM contains a leucine-rich region that fits the consensus for a leucine-rich nuclear export signal (NES) and shuttles between the nucleus and the cytoplasm in a heterokaryon assay (4, 78). In transient cotransfection experiments, SM activates expression of intronless reporter gene constructs and inhibits expression of intron-containing constructs, including plasmids containing spliced and unspliced EBV genes (38, 39, 45, 72). SM stimulates gene expression by enhancing levels of cytoplasmic and nuclear mRNAs and by promoting mRNA export (42, 72, 78). Collectively, these data suggest that SM, like ICP27, is a regulator of viral mRNA export. However, the detailed mechanism of mRNA transport remains unclear for both proteins.

In this study, we have begun to investigate the functional similarities between the HSV-1 ICP27 protein and the EBV SM protein. The ability of the SM protein to rescue growth of a replication-defective ICP27-null virus was examined in a transient-transfection-based complementation assay. Analyses of SM-complemented functions were facilitated by construction of an HSV-1 recombinant that expresses this protein in an ICP27-null background. The results of these experiments demonstrate that SM can compensate, at least in part, for ICP27-dependent functions and suggest that the regulation of late-gene expression in herpesviruses is an evolutionarily conserved process.

MATERIALS AND METHODS

Cells and viruses.

Vero cells and 2-2 cells (77) were maintained as monolayer cultures in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL, Grand Island, N.Y.) supplemented with 5% bovine calf serum (HyClone Laboratories, Inc., Logan, Utah). The telomerase-immortalized human foreskin fibroblast T18 cell line (5) was provided by T. Shenk (Princeton University, Princeton, N.J.) and was maintained as a monolayer culture in DMEM supplemented with 10% fetal bovine serum (HyClone Laboratories) and 500 μg of puromycin per ml.

HSV-1 KOS1.1A was the wild-type virus strain for all experiments. vBSΔ27 and vBSLG4 (tsR480H) have been described previously (81).

Molecular phylogeny.

Amino acid sequences corresponding to ICP27 and its homologs from the α (HSV-1, HSV-2, bovine herpesvirus [BHV], EHV, pseudorabies virus [PRV], Marek’s disease virus [MDV], and VZV), β (CMV and human herpesvirus 6 [HHV-6]), and γ (EBV, HVS, and KSHV) herpesvirus subgroups were obtained from GenBank. Criteria for inclusion in the analysis were BlastP homology to the HSV-1 strain 17 ICP27 protein (HSV-2, CMV, MDV, PRV, and VZV) or to the CMV UL69 protein (EBV, KSHV, and HVS). Additionally, functional similarity was considered when known for a given protein. Sequences were aligned with MegAlign software using the ClustalW (87) method and PAM250 residue weight table.

The carboxy-terminal conserved portions of the aligned sequences were used for phylogenetic analysis (HSV-1 ICP27 amino acid residues 264 to 508). All positions in the aligned sequences that contained a gap in a majority of the sequences were removed. Phylogenetic trees were generated with software from the PHYLIP package (programs Seqboot, Protdist, Neighbor, and Consense) (20). Distance trees were computed by the neighbor-joining method, and bootstrap scores were calculated from 100 randomly sampled subsets of alignments.

Construction of vSM/TK-Δ27 and v27/TK-Δ27. (i) Transfection and plaque purification: vSM/TK-Δ27.

A linearized fragment of p27PSM/TK was cotransfected into 2-2 cells with vBSΔ27 nucleocapsids (79) by the calcium phosphate precipitation method (93). Transfection mixtures contained approximately 10 μg of the linear p27PSM/TK fragment, 5 μg of carrier plasmid DNA, and approximately 100 PFU of vBSΔ27 nucleocapsids. Viruses obtained from transfections were plaque purified in the presence of 30 μg of bromodeoxycytidine (BdC) per ml. Plaques were picked into 50 μl of DMEM containing 1% bovine calf serum and screened by PCR for the presence of the SM ORF in the thymidine kinase (tk) locus and simultaneously for the presence of contaminating tk⁺ viruses. The resuspended plaques were used for further rounds of plaque purification. PCR-positive plaques were purified a total of three times on 2-2 cells in the presence of BdC.

(ii) Transfection and plaque purification: vSMLRRΔ/TK-Δ27.

vSMLRRΔ/TK-Δ27was constructed as above with the following modification: a linearized fragment of pSMLRRΔ/TK was cotransfected into 2-2 cells. Plaques were picked and screened as above.

(iii) Transfection and plaque purification: v27/TK-Δ27.

v27/TK-Δ27 was constructed as above with the following modifications. A linearized fragment of pICP27/TK was cotransfected into Vero cells with vBSΔ27 nucleocapsids. Plaques were screened by PCR for the presence of ICP27 in the tk locus and simultaneously for the presence of contaminating tk⁺ viruses. PCR-positive plaques were purified a total of three times on Vero cells in the presence of BdC.

PCR screening.

Approximately 1/30th of each resuspended plaque was used directly as a template for PCR screening. PCR conditions were 5 min at 95°C, followed by 1 min at 95°C, 1 min at 60°C, and 80 s at 72°C for 35 cycles. Each PCR contained a total of three primers for simultaneous detection of plaques containing recombinant (tk⁻) viruses, plaques containing a mixture of recombinant viruses and contaminating tk⁺ viruses, and plaques containing nonrecombinant (tk⁺) vBSΔ27. Two of the primers were complementary to the tk locus in the region flanking the targeted insertion, TK-29/-19.S (5′-GACGCGTGTGGCCTCGAATA-3′) and TK-29/-19.AS (5′-GCCAGGCGGTCGATGTG3-′). At the wild-type tk locus, these two primers generate a 778-bp product. When the tk locus contains an insertion, this primer pair is predicted to generate a 2,670-bp product that was not detectable with these PCR conditions.

To detect insertion of the SM ORF at the tk locus, a third primer that was complementary to the SM ORF was included in the reaction, TK/SMint.A/S (5′-CTGAGACCGCTTCGAGTTCC-3′). The primer pair TK/SMint.A/S and TK-29/-19.S generates a 595-bp product. To detect insertion of ICP27 coding sequence at the tk locus, a primer complementary to ICP27 was included in the reaction, 27/TKint.S (5′-CGGTGTCATAGTGCCCTTAGGA-3′). TK-29/-19.A/S and 27/TKint.S generate a 686-bp product. All plaques were also screened for retention of the lacZ gene at the ICP27 locus with primers LacZ5′3′ (5′-CTCTATCGTGCGGTGGTTGA-3′) and LacZ3′5′ (5′-CGGCGTTAAAGTTGTTCTGC-3′). This primer pair generates a 237-bp product.

Southern blotting.

Cytoplasmic viral DNAs were prepared from 10⁶ infected cells as previously described (46). Viral DNA was digested with SalI, separated by agarose gel electrophoresis, and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, N.H.) in 6× SSC (20× SSC is 3 M NaCl, 0.3 M sodium citrate, pH 7.0). Following transfer, the membranes were baked for 2 h at 80°C in a vacuum oven and then prehybridized for 3 h at 68°C in 20 ml of prehybridization solution (3× SSC plus 8× Denhardt's solution [50× Denhardt's solution contains 1% Ficoll 400, 1% polyvinylpyrrolidone, and 1% bovine serum albumin]).

A probe complementary to tk was randomly primed with [α-³²P]dCTP from a 1.9-kb gel-purified AlwNI-Tth111I restriction fragment from pTK-29/-19. This probe contains the entire tk ORF and 486 bp of sequence upstream of the tk ORF. Prior to hybridization, the probe was boiled for 10 min with 1 mg of salmon sperm DNA and then cooled on ice. The hybridization solution contained the boiled probe, 3× SSC, 0.1% sodium dodecyl sulfate (SDS), and 4× Denhardt's solution in a final volume of 10 ml. Following overnight hybridization at 68°C, the blots were washed in 2 liters (total volume) of wash solution (0.6% SDS, 0.1× SSC) at 68°C and visualized by autoradiography.

Plasmids.

pCGNUL69 contains a hemagglutinin (HA)-tagged version of the human CMV (HCMV) UL69 gene and was a kind gift from T. Shenk (37). pVZVORF4 was a generous gift from K. Ampofo. pCMV27 was constructed by subcloning a BamHI-EcoRI fragment from pBS27BAM (82) into the corresponding polylinker sites of pCDNA3. pCMVSM was constructed by subcloning a NotI-SalI fragment from pEW58 into the NotI and XhoI sites of pCDNA3. pEW58 contains a cDNA copy of the EBV SM ORF (72) cloned into pBluescript (Stratagene, La Jolla, Calif.).

pTK-29/-19 contains the entire tk ORF and upstream control region and has been described previously (49). pCPC-CMV4 contains a 4.5-kb fragment of HSV-1 genomic DNA spanning the ICP4 coding sequence and has been described previously (59). pBS27 contains a 2,417-bp BamHI-SacI fragment of HSV-1 genomic DNA spanning the ICP27 gene and has been described previously (81). pBSSM was constructed by replacing the ICP27 ORF from pBS27 with the SM ORF. The ICP27 ORF was removed from pBS27 by digestion with AgeI and EcoNI. The SM ORF was obtained from pEW58 by digestion with XbaI and HindIII. After filling in the ends of this fragment with Klenow, it was cloned into the filled AgeI and EcoNI sites in pBS27. The orientation of the insert was confirmed by restriction enzyme digestion.

p27PSM/TK was generated by replacing the 5′ end of the tk ORF (629 bp) and 58 bp of the tk upstream control region in pTK-29/-19 with the expression cassette containing the SM ORF under the control of the ICP27 promoter from pBSSM. pTK-29/-19 was digested with BglII and Asp718I, and these ends were filled in with Klenow. The SM expression cassette was removed from pBSSM as an EcoRV-PvuII fragment and cloned into the filled BglII and Asp718I sites of pTK-29/-19. The orientation of the insert was confirmed by restriction enzyme digestion. pSMLRRΔ/TK was constructed as for pBSSM. A HindIII-NotI fragment containing the mutant SM allele was isolated from pVR114 (4). After filling in with Klenow, this fragment was subcloned into the filled AgeI and EcoNI sites in pBS27. An EcoRV-PvuII fragment from pBSSMLRRΔ was then subcloned into pTK-29/-19 as described above.

pICP27/TK was constructed by replacing the 5′ end of the tk ORF (629 bp) and 58 bp of the tk upstream control region in pTK-29/-19 with the ICP27 ORF and regulatory regions from pBS27. pTK-29/-19 was digested with BglII and Asp718I, and these ends were filled in with Klenow. The ICP27 expression cassette containing the entire ICP27 gene was removed from pBS27 as an EcoRV-PvuII fragment and cloned into the filled BglII and Asp718I sites of pTK-29/-19. The orientation of the insert was confirmed by restriction enzyme digestion.

Immunoblotting. (i) Complementation assays.

Vero cells (1.75 × 10⁵ cell equivalents) were collected in 1.5× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, resolved through SDS-7.5% PAGE gels, and electrophoretically transferred to nitrocellulose. Membranes were blocked in a solution of 5% nonfat dried milk in phosphate-buffered saline (PBS)-0.1% Tween (PBS-T) for at least 1 h at room temperature. Primary antibody dilutions were also incubated with the membranes for at least 1 h at room temperature. Primary antibodies used for immunoblotting were as follows: anti-HA (α-HA) (Clonetech), α-ICP27 Clu38 (59), α-EBV SM (72), and α-VZV ORF4 (47). Blots were washed twice in 1% nonfat dried milk-PBS-T for 10 min and then once for 5 min. Horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse immunoglobulin G [IgG]-horseradish peroxidase [HRP] or goat anti-rabbit IgG-HRP; Kirkegaard and Perry Laboratories, Gaithersburg, Md.) were diluted in 5% nonfat dried milk-PBS-T and incubated for 45 min at room temperature. Blots were washed as above and developed with an enhanced chemiluminescent substrate system (KPL) for 1 min.

(ii) Viral protein synthesis.

Infected Vero cells were collected at the indicated times postinfection and resuspended in 1.5× SDS-PAGE sample buffer. For each time point, 10⁵ cell equivalents were resolved through SDS-7.5% PAGE gels and electrophoretically transferred to nitrocellulose. Membranes were immunoblotted under the conditions described above with the following primary antibodies: α-gC (provided by G. Cohen, University of Pennsylvania), α-VP16 (Clontech), α-ICP8 3-83 (provided by D. Knipe, Harvard University), α-ICP4 H1114 (Rumbaugh-Goodwin Institute, Plantation, Fla.), α-ICP27 Clu38 (59), α-EBV SM (72), and α-UL38 (provided by B. Roizman, University of Chicago).

Immunofluorescence. (i) Virus spread assay.

Vero cells (7 × 10⁵ per well) were seeded onto glass coverslips and then infected with vBSΔ27, vSM/TK-Δ27, or v27/TK-Δ27 at a multiplicity of infection (MOI) of 0.01 PFU/cell in DMEM supplemented with 1% bovine calf serum for 1 h at 37°C. At the indicated times postinfection, the coverslips were processed for indirect immunofluorescence. Coverslips were washed three times in PBS, and cells were fixed in 3.7% formaldehyde in PBS for 12 min at room temperature, followed by three washes in PBS. Fixed cells were stored at 4°C or permeabilized directly with ice-cold acetone for 30 s and then washed three times with PBS. Nonspecific binding was blocked by incubation in 10% normal goat serum in PBS for 1 h at room temperature.

Primary antibody was diluted in 5% normal goat serum in PBS (α-ICP4 H1114, 1:100) and incubated with the cells for 1 h at room temperature, followed by three washes with PBS. Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG or goat anti-rabbit IgG secondary antibodies were diluted 1:200 in 5% normal goat serum in PBS and incubated with the cells for 40 min at room temperature, followed by three washes with PBS. After a final rinse with distilled water, coverslips were mounted onto slides with Gel/Mount (Biomeda Corp., Foster City, Calif.). Fluorescence was observed with a Leitz Dialux photomicroscope with epifluorescence illumination and filters for the appropriate fluorophore. Images were captured and cropped with Adobe Photoshop software.

(ii) PFU to FFU ratio.

Vero cells (10⁶ per well) were seeded onto duplicate six-well plates. For one of each replicate, the cells were seeded onto glass coverslips. All cells were infected in duplicate (with and without coverslips) with serial dilutions of vBSΔ27, vSM/TK-Δ27, or v27/TK-Δ27 in DMEM supplemented with 1% bovine calf serum for 1 h at 37°C. At 7 h postinfection, the coverslips were processed for indirect immunofluorescence with an ICP4 antibody as described above. Fluorescent plaques were counted in defined fields, and this number was used to calculate focus-forming units (FFU) per milliliter. A standard plaque assay was employed to determine the PFU per milliliter for the cells plated directly onto the six-well plates.

Complementation assays.

Vero cells (7 × 10⁵ cells) were transiently transfected with expression plasmids encoding HSV ICP27, EBV SM, VZV ORF4, or HCMV UL69 or, as a control, the empty parental plasmid. Cells were transfected with the Lipofectamine Plus reagent (Life Technologies, Gaithersburg, Md.) according to the manufacturer's instructions. At 24 h posttransfection, the cells were infected with vBSΔ27 at an MOI of 5 PFU/cell for 1 h at 37°C in DMEM supplemented with 1% bovine calf serum. To remove unadsorbed virus following infection, cells were treated for exactly 45 s with citrate buffer (40 mM citric acid, 135 mM NaCl, 10 mM KCl, pH 3.0) and then washed three times with PBS. The transfected/infected cells were collected at 24 h postinfection. Transfection efficiencies were monitored between samples in individual experiments by cotransfection of a green fluorescent protein expression plasmid. The number of green fluorescent cells in an aliquot of each of the transfected-infected cell suspensions was determined. To compare data between experiments, ICP27 was included as a standard in every experiment, and all other complementation was normalized to it. Complementation of viral growth was measured by plaque assay on 2-2 cells.

Viral growth assays.

Vero cells were infected at a high MOI (5 PFU/cell) or a low MOI (0.1 PFU/cell) with vBSΔ27, vSM/TK-Δ27, or v27/TK-Δ27. Infections were stopped by freezing the cells at −80°C at the indicated times postinfection. Virus was released from infected cells by four freeze-thaw cycles, and yields were determined by titration on 2-2 cells. To determine the relative ability of each virus to grow under nonselective conditions, 2-2 cells were infected with each virus at an MOI of 0.1, and virus yields at the indicated times postinfection were determined by titration on 2-2 cells. Plaquing efficiency was determined by growing each of the indicated viruses on both 2-2 and Vero cells and calculating the ratio of titers.

Sequence analysis of vSM/TK-Δ27 isolates.

Six individual plaques from the 96-h time point of the kinetic growth analysis were isolated on Vero cells, and virus from these plaques was amplified on 2-2 cells. Cytoplasmic viral DNA was prepared as previously described (46) for each isolate and used as a template in PCRs to amplify the SM ORF. Each PCR was done in duplicate, and each replicate was subcloned into a plasmid for sequencing. A panel of SM-specific primers was used to sequence all isolates and, as controls, the wild-type SM ORF amplified from two independent isolates of the wild-type virus.

Viral DNA replication.

Equivalent amounts of infected cell lysates (1.3 × 10⁵ cell equivalents) taken directly from cell suspensions prepared for protein analysis were digested with 200 μg of proteinase K per ml in a solution containing 50 mM Tris (pH 7.5), 5 mM EDTA, 100 mM NaCl, 0.5% SDS, and 100 μg of RNase A per ml for 3 h at 50°C. Digested samples were incubated in denaturing solution (0.25 M NaOH, 0.5 M NaCl) for 10 min at room temperature, and then samples were blotted to GeneScreen Plus membranes (Schleicher & Schuell) with a Minifold II slot blot system (Schleicher & Schuell). DNAs were cross-linked by UV irradiation with a Stratalinker 2400 (Stratagene). A ³²P-labeled ICP4 probe was synthesized by random priming from a 4.5-kb gel-purified HindIII restriction fragment isolated from pCPC-CMV4 and hybridized to the blots at 68°C according to the manufacturer's instructions. The blots were washed according to the manufacturer's instructions and visualized by autoradiography.

Protein synthesis assays.

Vero cells (6.5 × 10⁵) were infected with vBSΔ27, vSM/TK-Δ27, v27/TK-Δ27, or wild-type HSV-1 strain KOS at an MOI of 10 PFU/cell. At the indicated times postinfection, the cells were washed three times with Met-free and Cys-free DMEM. Cells were pulse-labeled for 30 min in 300 μl of Met-free and Cys-free DMEM supplemented with 1% dialyzed fetal calf serum and 50 μCi of Tran³⁵S label (1,214 Ci/mmol; ICN Pharmaceuticals Inc., Costa Mesa, Calif.). Following the labeling period, cells were washed with PBS and resuspended in 300 μl of 1.5× SDS-PAGE sample buffer. Proteins (50 μl of each sample) were electrophoresed through 7.5% polyacrylamide gels, and bands were visualized by fluorography and autoradiography.

RESULTS

Phylogenetic relationships among ICP27 homologs parallel subfamily classifications.

The ICP27 gene from HSV-1 has homologs in all of the herpesvirus genomes that have been sequenced. At the amino acid level, these proteins exhibit the highest degree of sequence conservation at the carboxy termini, while the amino-terminal sequences are more divergent. A phylogenetic analysis generated from an amino acid alignment of the carboxy-terminal conserved portions of the proteins reveals that evolutionary relatedness correlates with subfamily classification (Fig. 1). While only a few of these proteins have been studied in any detail, there are sufficient data demonstrating that these proteins have retained some functional homology. Although the alphaherpesvirus proteins from HSV-1 (ICP27) and VZV (ORF4) are not functionally interchangeable (53, 61), several members of the gammaherpesvirus subfamily share many biological properties with ICP27 from HSV-1 (1, 4, 8, 26, 29, 38-40, 42, 72, 78, 92). In the following series of experiments, we examined the ability of the EBV SM protein from the gammaherpesvirus subfamily to complement some of the known functions of ICP27.

FIG. 1.

Evolutionary relationships among HSV-1 ICP27 homologs. An unrooted phylogenetic tree was generated from an amino acid alignment of the conserved carboxy-terminal portion of sequences corresponding to ICP27 and its homologs (HSV-1 ICP27 amino acid residues 264 to 508). The sequences represent viruses from the α (HSV-1, HSV-2, BHV, EHV, PRV, MDV, and VZV), β (CMV and HHV-6), and γ (EBV, HVS, and KSHV) herpesvirus subfamilies. Criteria for inclusion in the analysis were BlastP homology to the HSV-1 strain 17 ICP27 protein (HSV-2, CMV, MDV, PRV, and VZV) or to the CMV UL69 protein (EBV, KSHV, and HVS). Bootstrap values for each branch are shown at the node as the number of occurrences in 100 bootstrap test trees.

Homologs of HSV-1 ICP27 complement growth of an ICP27 deletion mutant.

The functional similarities between HSV-1 ICP27 and the homologous proteins from the gammaherpesvirus subfamily suggested the possibility of overlapping functions. To determine if ICP27 homologs were capable of providing any ICP27 function, the ability of the homologs to complement the growth of an ICP27 deletion mutant, vBSΔ27, was examined. Vero cells were transiently transfected with expression plasmids for HSV ICP27, EBV SM, EBV SMLRRΔ, VZV ORF4, or HCMV UL69 and subsequently infected with vBSΔ27. vBSΔ27 yield was measured by plaque assay on the ICP27-complementing cell line 2-2 (Fig. 2A). For all transfections, expression of the homologs was confirmed by immunoblotting a portion of the cell lysate with antibodies reactive with the individual proteins (Fig. 2B).

FIG. 2.

Complementation of vBSΔ27 growth by HSV-1 ICP27 homologs. Vero cells were transiently transfected with CMV promoter-based expression plasmids encoding HSV-1 ICP27, EBV SM, EBV SMLRRΔ, HCMV UL69, VZV ORF4, or, as a control, the empty parental plasmid. At 24 h posttransfection, the cells were infected with vBSΔ27, and the transfected-infected cells were collected at 24 h postinfection. To normalize for transfection efficiency in individual experiments, a green fluorescent protein expression plasmid was included in all transfection reactions. To make comparisons between experiments, all experiments included a wild-type ICP27 expression plasmid, and data were normalized to complementation by wild-type ICP27. (A) Complementation of viral growth was measured by plaque assay on 2-2 cells. The y axis denotes virus growth on a logarithmic scale, and the x axis denotes the transiently expressed protein (HSV-1 ICP27, EBV SM, EBV SMLRRΔ, HCMV UL69, or VZV ORF4). If any viral growth was stimulated in cells transfected with the empty control vector, this value was subtracted from growth stimulated in the presence of the viral proteins. The dashed line indicates the limit of detection of the assay. The data represent the averages of at least three independent experiments. (B) The expression of HSV ICP27 or EBV SM protein synthesized in complementation assays was measured by immunoblotting with antiserum specific for ICP27 (Clu38), SM (α-SM), UL69 (α-HA), or ORF4 (α-VZV ORF4). At the top of each lane, the transfected plasmids are labeled as C (control), 27 (pCMV27), SM (pCMVSM), EBV SMLRRΔ (pVR114), UL69 (pCGNUL69), or ORF4 (pVZV4).

As described previously (48), ICP27 complemented the vBSΔ27 defect. The other alphaherpesvirus protein tested, VZV ORF4, did not complement any detectable growth of vBSΔ27 despite the parsing of these two proteins to the same branch of the phylogenetic tree (Fig. 1). These results agree with previous unsuccessful attempts to complement ICP27 mutants with the VZV ORF4 protein (53). While it may appear from this graph that the HCMV UL69 protein complements vBSΔ27, in two experiments viral yield barely exceeded the limit of detection of the assay (50 PFU/ml), and in a third experiment there was no measurable yield. Therefore, UL69 does not complement the ICP27-null mutant above the limit of detection of the assay (note that the error bars overlap the lower boundary limit of 50 PFU/ml). By contrast, the SM protein encoded by the gammaherpesvirus EBV reproducibly resulted in a 100- to 1,000-fold higher yield of vBSΔ27. However, this yield was considerably less than that induced by ICP27. Additionally, an SM mutant, SMLRRΔ, that was previously characterized as defective for transactivation activity and nucleocytoplasmic shuttling (4) also failed to complement the ICP27-null mutant (again, note that the error bars overlap the lower boundary limit of 50 PFU/ml). These experiments demonstrate that the EBV SM protein compensates, at least in part, for ICP27 defects and also suggest that shuttling between the nucleus and cytoplasm is an important component of complementation.

Construction of a recombinant SM-expressing herpesvirus.

To examine the ability of SM to substitute for ICP27 in the context of a viral infection and to have a means of measuring SM-complemented functions, a recombinant herpes simplex virus expressing the EBV SM protein in an ICP27 null background was constructed (Fig. 3). The recombinant virus (vSM/TK-Δ27) was derived from vBSΔ27 and expresses SM from the thymidine kinase (tk) locus of vBSΔ27. The replacement was targeted to the tk locus rather that the ICP27 locus for several reasons: (i) tk is not required for viral growth in cell culture, (ii) tk replacement viruses have been successfully constructed and characterized previously, and (iii) expression of SM from the tk locus would allow the construction of a panel of recombinant viruses with different ICP27 backgrounds to test for complementation of various existing ICP27 mutants in this system.

FIG. 3.

Construction of EBV SM-expressing recombinant herpes simplex viruses. To analyze growth complementation of an ICP27 deletion mutant by the EBV SM protein in the context of a viral infection, a recombinant vBSΔ27-based virus containing the EBV SM ORF in the tk locus was constructed. The EBV SM ORF was placed under the control of the ICP27 promoter and cloned into a plasmid containing the complete tk gene to provide flanking homology with tk (p27PSM/TK fragment, panel C; expanded tk locus, panel B). In the resulting targeting plasmid, the SM insertion interrupts the tk ORF. Recombinant viruses were constructed in a vBSΔ27 background (A) by cotransfection of a linear p27PSM/TK fragment with infectious vBSΔ27 nucleocapsids into the ICP27-complementing cell line 2-2. Recombinants from two independent cotransfections were selected by plaquing on 2-2 cells in the presence of 5-bromodeoxycytidine. vSM/TK-Δ27 recombinants were screened by PCR (D) for the retention of lacZ coding sequence at the ICP27 locus (top panel). Individual isolates of plaques PCR positive for SM are labeled across the top of the lanes. The lane labeled Δ27 is a control reaction from a vBSΔ27 plaque. Potential vSM/TK-Δ27 recombinants were screened by PCR for the presence of SM coding sequence and simultaneously for the absence of tk coding sequence with a three-primer reaction described in detail in Materials and Methods (bottom panel). Individual isolates of PCR-positive viruses are labeled across the top of the lanes. The lane labeled MP is a control PCR from a reaction containing two plasmids, pTK-29/-19 (tk containing) and p27PSM/TK (SM containing), and the lane labeled Δ27 is a control reaction from a vBSΔ27 plaque. For Southern hybridization of recombinant virus genomic DNA, cytoplasmic DNA from vBSΔ27- or vSM/TK-Δ27-infected cells was digested with SalI and hybridized with a random-primed probe complementary to tk as described in Materials and Methods (E). The locations of the probe and the hybridization signals (B and C) diagnostic for wild-type tk (5,948 bp) and SM-replaced tk (5,374 and 2,474 bp) are indicated. Homology between the SM-replaced tk locus and the wild-type tk locus is indicated by shading.

The SM ORF was cloned into a plasmid downstream of the ICP27 promoter so that SM would be expressed with kinetics similar to those of ICP27 during infection. This expression cassette was then subcloned into a plasmid containing the entire tk ORF and 5′ control region to provide flanking homology with tk (p27PSM/TK). The SM expression cassette replaced the 5′ end of the tk ORF and extended upstream beyond the tk transcriptional start site (Fig. 3C). To construct the virus, a linear fragment of p27PSM/TK was cotransfected into 2-2 cells with infectious vBSΔ27 nucleocapsids, and recombinants were selected by plaque purification in the presence of BdC (Fig. 3A to C). Potential recombinants were screened by PCR for the presence of the SM coding sequence and simultaneously for the presence or absence of the tk coding sequence in the three-primer reaction described in Materials and Methods (Fig. 3D). Following three rounds of plaque purification in the presence of BdC, the sequence arrangement at the tk locus of the resulting recombinant virus was confirmed by Southern hybridization with a tk-specific probe (Fig. 3E). All virus stocks were prepared on 2-2 cells to eliminate any selection for adaptive mutations, and they were screened by PCR for the presence of SM and lacZ and for the absence of ICP27 (data not shown). As a control, a virus expressing ICP27 under the control of its own promoter from the tk locus of vBSΔ27 was constructed in parallel by the same method (v27/TK-Δ27).

Growth of vSM/TK-Δ27 at a high MOI.

As an initial assessment of the ability of vSM/TK-Δ27 to grow, Vero cells were infected with vBSΔ27, vSM/TK-Δ27, or v27/TK-Δ27 at an MOI of 5 PFU/cell. Infected cells were collected at 6, 12, 24, and 48 h postinfection, and virus yields were measured by plaque assay on 2-2 cells (Fig. 4A). The ICP27-expressing control virus, v27/TK-Δ27, exhibited a significant increase in titer by 24 h postinfection Throughout the time course, plaque formation was measurable for vSM/TK-Δ27, but there was no measurable increase in the titer of this virus at any time point relative to vBSΔ27. The titer observed for vBSΔ27 in this experiment represents unadsorbed viral particles that remain attached to the cell surface. Based on the observation that a low-pH wash reduces the titer to zero, the residual virus does not represent actual viral growth. Experiments prior to the kinetic growth analysis demonstrated that vSM/TK-Δ27 formed plaques on Vero cells, and therefore, it was clear that the virus had some capacity for growth. Therefore, these results suggested that the growth kinetics of the recombinant virus were delayed so that replication was not detectable during the time period of analysis. Alternatively, if the amount of virus produced in a given infected cell is very low, an increase in yield may not occur in high-MOI infections, where amplification through a culture would be restricted. In other experiments, no growth was detectable at any time point in cells infected with vBSΔ27 (data not shown). When 2-2 cells were infected with vSM/TK-Δ27 or v27/TK-Δ27 at the same MOI, viral yields were nearly equivalent at all time points for both viruses, indicating that there were no defects in vSM/TK-Δ27 growth when ICP27 was provided in trans (Table 1).

FIG. 4.

Growth of vSM/TKΔ27in low- and high-MOI infections. (A) Vero cells were infected with vSM/TK-Δ27 (SM/TK) or v27/TK-Δ27 (27/TK) at an MOI of 5 PFU/cell, and the infected cells were collected at the indicated times postinfection. The resulting viral yields were determined on 2-2 cells. Data points represent the averages of three independent infections, each performed in duplicate. (B) Vero cells were infected with two independent isolates of vSM/TK-Δ27 (SM/TK.B1 or SM/TK.A16), v27/TK-Δ27 (27/TK), or vBSΔ27 (Δ27) at an MOI of 0.1 PFU/cell, and the infected cells were collected at the indicated times postinfection. The resulting viral yields were determined on 2-2 cells. Data points represent the averages of two independent infections, each performed in duplicate.

TABLE 1.

Virus yields from high- and low-MOI infections of 2-2 cells^a

Time postinfection (h)	Virus yield (PFU/ml) ± SD
	Low-MOI infection		High-MOI infection
	vSM/TK-Δ27	v27/TK-Δ27	vSM/TK-Δ27	v27/TK-Δ27	vBSΔ27
6	2.8 × 105 ± 1.0 × 104	2.5 × 105 ± 2.9 × 104	2.6 × 103 ± 2.9 × 102	6.9 × 103 ± 3.0 × 103	3.4 × 103 ± 3.5 × 102
24	2.5 × 108 ± 3.5 × 107	1.8 × 108 ± 1.7 × 107	2.1 × 107 ± 1.0 × 106	2.2 × 107 ± 3.5 × 106	NDb
48	2.5 × 108 ± 4.7 × 107	1.5 × 108 ± 0	4.0 × 108 ± 2.6 × 107	2.6 × 108 ± 5.5 × 107	5.5 × 108 ± 1.6 × 108

^aValues represent the averages of three independent infections (two for VBSΔ27 and for v27/TK-Δ27 at 48 h), each performed in duplicate.

^bND, not determined.

Kinetics of vSM/TK-Δ27 growth are delayed.

The failure to detect an increase in viral yield in high-multiplicity infections suggested that the replication kinetics of vSM/TK-Δ27 were delayed. However, it was not possible to examine vSM/TK-Δ27 growth over a prolonged time course in a high-multiplicity infection because the high particle input on the cells had generalized toxic effects; therefore, the growth kinetics of vSM/TK-Δ27 were determined in low-multiplicity infections. Vero cells were infected with v27/TK-Δ27, vBSΔ27, or two independent isolates of vSM/TK-Δ27 at an MOI of 0.1 PFU/cell, and viral yields were measured over a 96-h time course (Fig. 4B).

In this experiment, the v27/TK-Δ27 yield peaked by 48 h postinfection, and no growth of vBSΔ27 was detectable. The growth of both vSM/TK-Δ27 isolates proceeded with identical kinetics; the yield for both viruses increased 10-fold between 12 and 24 h postinfection, but no further increases in yield were measured through 96 h of infection. The final yield of vSM/TK-Δ27 was 10,000-fold less than the final yield of v27/TK-Δ27. These results demonstrate that SM can partially compensate for ICP27 function, although the inability of vSM/TK-Δ27 to reach titers similar to those of v27/TK-Δ27 suggests that the two proteins are not completely interchangeable. The failure to detect further amplification of the vSM/TK-Δ27 isolates may in part reflect the state of the cells at these times postinfection or a steady state between viral death and replication. Nevertheless, the results clearly demonstrate that expression of SM supports growth of an ICP27-null virus. When 2-2 cells were infected with vSM/TK-Δ27 or v27/TK-Δ27 at an MOI of 0.1 PFU/cell, viral yield measured on 2-2 cells was nearly equivalent at all time points for both viruses, indicating that there were no defects in vSM/TK-Δ27 growth when ICP27 was provided in trans (Table 1).

Adaptive mutations do not account for vSM/TK-Δ27 growth.

The prolonged time course of vSM/TK-Δ27 growth suggested the possibility that the increase in viral yield may not have resulted from SM stimulation of HSV growth, but rather the acquisition of mutations in the SM ORF that allowed replication. If SM mutants were responsible for the observed growth, then by 96 h postinfection these mutants should represent a majority of all viruses present at this time point. To identify potential SM mutants, the pool of virus collected at 96 h postinfection from the low-MOI kinetic analysis (Fig. 4B) was plaque purified on Vero cells, and six individual isolates were selected for sequencing of the SM ORF. Following plaque purification, individual isolates of virus were amplified on 2-2 cells, and the nucleotide sequence of the SM ORF in each isolate was determined. Each PCR was prepared in duplicate, and all PCR products were cloned into pZero for sequencing of the entire SM ORF. No SM mutations were detected in any of the isolates (data not shown). Additionally, after extended passage of the 96-h viral yield on Vero cells, there were no discernible increases in growth rate or plaque size.

Although no SM mutations were detected, the presence of extragenic mutations was a concern, as there is a precedent for extragenic mutations mitigating the phenotype of temperature-sensitive ICP27 mutants (9). To address this issue, the pool of virus collected at 24 h postinfection from the low-MOI kinetic analysis (Fig. 4B) was plaque purified. This time point was selected for analysis because it represented the first and most significant increase in viral growth over the time course. Ten individual isolates were selected and amplified on 2-2 cells. The plaquing efficiency of all 10 isolates on 2-2 cells relative to Vero cells was identical to the plaquing efficiency of the original vSM/TK-Δ27 stock (data not shown). Therefore, HSV growth is stimulated by the expression of SM in the absence of ICP27.

Alterations in vSM/TK-Δ27 plaquing efficiency.

The ability of vSM/TK-Δ27 to initiate productive infections was examined directly in a virus spread assay. For these experiments, Vero cells seeded onto glass coverslips were infected with vSM/TK-Δ27, vBSΔ27, v27/TK-Δ27, or wild-type HSV-1 KOS 1.1 at an MOI of 0.01 PFU/cell. At the indicated times postinfection, the coverslips were processed for indirect immunofluorescence with an antibody reactive with the immediate-early protein ICP4 (Fig. 5). At 5 h postinfection, only single-cell infections could be detected for all viruses. vBSΔ27 infections never spread to neighboring cells during 72 h of infection, consistent with its requirement for a complementing cell line to grow. Cells infected with either wild-type HSV or v27/TK-Δ27 displayed similar growth patterns through 48 h postinfection.

FIG. 5.

Analysis of viral spread by indirect immunofluorescence. Vero cells were seeded onto glass coverslips and infected with vSM/TK-Δ27 (SM/TK-Δ27), vBSΔ27 (Δ27), v27/TK-Δ27 (27/TK-Δ27), or HSV-1 KOS (wild type) at an MOI of 0.01 PFU/cell. At the indicated times postinfection, cells on the coverslips were fixed and processed for indirect immunofluorescence with a monoclonal antibody reactive with the immediate-early ICP4 protein H1114. Representative fields of cells are shown for each time point. Two representative fields are presented for vSM/TK-Δ27 at 24, 48, and 72 h postinfection to illustrate the heterogeneity of these infections. U, upper panels; L, lower panels.

Although many singly infected cells were detected in these cultures at 11 h postinfection, several infections had spread to adjacent cells. By 24 h postinfection, large foci of very brightly stained infected cells predominated. These spread throughout the entire culture by 48 h postinfection (data not shown). The staining pattern of cells infected with vSM/TK-Δ27 was more heterogeneous than that in wild-type or vBSΔ27 infections. Therefore, two representative fields from each of the later time points are shown. Many foci of infections with vSM/TK-Δ27 were detectable by 24 h postinfection (Fig. 5, SM/TK-Δ27, U panels), although in several cases there was no detectable spread (Fig. 5, SM/TK-Δ27, L panels). Some of the infected foci spread very slowly by 72 h postinfection (Fig. 5, SM/TK-Δ27, U), but small foci that never progressed beyond a few rounds of replication were detected at all time points (Fig. 5, SM/TK-Δ27, L). These results parallel the kinetic analysis of vSM/TK-Δ27 in low-MOI infections and demonstrate that although vSM/TK-Δ27 grows on Vero cells, productive infections are initiated less frequently and progress more slowly than with viruses expressing ICP27.

The growth defect of vSM/TK-Δ27 on Vero cells is reflected in the altered PFU to FFU ratio for this virus. FFU was measured for each virus by indirect immunofluorescence. Vero cells infected with serial dilutions of vSM/TK-Δ27, v27/TK-Δ27, vBSΔ27, or wild-type HSV-1 KOS 1.1 were stained with an antibody reactive with ICP4 at 7 h postinfection. In parallel, PFU was determined for each virus by titration on Vero cells. The ratio of PFU to FFU measures the ability of each virus to establish productive infections. The results in Table 2 demonstrate that KOS and v27/TK-Δ27 have very similar PFU to FFU ratios. Consistent with the reduction in yield for vSM/TK-Δ27 in the kinetic growth analysis, the PFU-to-FFU ratio is reduced by 1,000-fold. These results suggest that many of the infections initiated with vSM/TK-Δ27 are not productive.

TABLE 2.

Ratio of PFU to FFU^a

Virus	PFU/ml	FFU/ml	PFU/FFU ratio
vBSΔ27	<50	1 × 1010	NAb
vSM/TK-Δ27	7.8 × 105	7.8 × 109	0.0001
v27/TK-Δ27	1.2 × 109	8.1 × 109	0.14
HSV-1 KOS	2.8 × 109	2.0 × 1010	0.14

^aFFU were calculated at 7 h postinfection by immunostaining with an antibody reactive with ICP4.

^bNA, not applicable.

The plaquing efficiency of vSM/TK-Δ27 was determined by titration on 2-2 and Vero cells (Table 3). This analysis reveals that the titers of two independent isolates of vSM/TK-Δ27 (A16 and B1) were reduced by nearly 1,000-fold on Vero cells compared to the titers obtained on 2-2 cells. On 2-2 cells, the sizes of vSM/TK-Δ27 and v27/TK-Δ27 plaques were comparable, but vSM/TK-Δ27 took longer than v27/TK-Δ27 to form plaques on Vero cells. All vSM/TK-Δ27 plaques were comparable in size on Vero cells. These results support the data from the complementation assay, the kinetic growth analysis, and the measurements of PFU-to-FFU ratios.

TABLE 3.

vSM/TK-Δ27 viruses exhibit reduced plaquing efficiency on Vero cells^a

Virus	PFU/ml		Ratio, 2-2/Vero
	2-2	Vero
Wild-type HSV-1 KOS	3.7 × 109	3.1 × 109	1.1
v27/TK-Δ27	1.7 × 109	1.5 × 109	1.1
vSM/TK-Δ27 A16b	2.3 × 108	3.2 × 105	718
vSM/TK-Δ27 B1b	8.2 × 108	1.1 × 106	745
vBSΔ27	3.4 × 109	<50	NAc

^a2-2 cells are an ICP27-complementing cell line.

^bTwo independent isolates of vSM/TK-Δ27 were titrated on both 2-2 and Vero cells.

^cNA, not applicable.

Nucleocytoplasmic shuttling is a determinant of complementation by SM.

The ability to transactivate genes and the ability to shuttle between the nucleus and cytoplasm are two well-characterized functions of both SM and ICP27. An SM mutant, SMLRRΔ, has a significantly reduced ability to transactivate reporter genes and is also defective for nucleocytoplasmic shuttling (4). This mutant also failed to complement growth of vBSΔ27 (Fig. 2), suggesting that these activities are relevant to the ability of SM to substitute for ICP27. An HSV recombinant expressing this allele of SM was constructed, and the yield of this virus was measured in a low-MOI infection of Vero cells (Fig. 6). The kinetics of vSM/TK-Δ27 growth were comparable to what was observed in Fig. 4B. However, there was no measurable increase in titer for vSMLRRΔ/TK-Δ27 at any time point. This result agrees with the complementation assay data and demonstrates that in the context of a viral infection, there is a correlation between the ability of these regulatory proteins to shuttle and to support HSV replication.

FIG. 6.

Growth of vSMLRRΔ/TK in low-MOI infections. Vero cells were infected with vSMLRRΔ/TK (SMLRRΔ/TK), vSM/TK-Δ27 (SM/TK), v27/TK-Δ27 (27/TK), or vBSΔ27 (Δ27) at an MOI of 0.1 PFU/cell, and the infected cells were collected at the indicated times postinfection. The resulting viral yields were determined on 2-2 cells. Data points represent the averages of three independent infections, each performed in duplicate.

HSV late proteins are synthesized in vSM/TK-Δ27-infected cells.

The previous experiments demonstrate that vSM/TK-Δ27 grew on Vero cells. Therefore, the general profile of viral protein synthesis in vSM/TK-Δ27-infected cells was examined to determine the effect of SM expression on accumulation of HSV proteins. For this experiment, cells were infected with vSM/TK-Δ27, v27/TK-Δ27, or vBSΔ27 at an MOI of 0.1 PFU/cell, and infected cells were collected at 10, 24, and 48 h postinfection. Accumulation of HSV proteins representative of the α, β, and γ kinetic classes was measured by immunoblotting (Fig. 7).

FIG. 7.

Accumulation of viral proteins in vSM/TK-Δ27-infected cells. Vero cells were mock infected (uninfected) or infected with vSM/TK-Δ27 (SM/TK-Δ27), vBSΔ27 (Δ27), or v27/TK-Δ27 (27/TK-Δ27) at an MOI of 0.1 PFU/cell, and lysates of infected cells were prepared at the indicated times (hours) postinfection. The relative abundance of the indicated HSV proteins was determined by immunoblotting with antibodies reactive with UL38, gC, VP16, ICP8, ICP4, EBV SM, and ICP27. These proteins are representative of all HSV kinetic classes (α, β, γ₁, and γ₂). The asterisks on the SM immunoblot of SM/TK-Δ27-infected cells denote a cross-reacting protein induced by viral infection that is also present in cells infected with vBSΔ27 and v27/TK-Δ27.

The results of this experiment can be summarized as follows. (i) ICP27 is synthesized only in v27/TK-Δ27-infected cells, and SM is synthesized only in vSM/TK-Δ27-infected cells; neither protein is detectable in cells infected with vBSΔ27. The bands that appear in the SM blots of cells infected with vBSΔ27 and v27/TK-Δ27 are from cross-reactive host proteins induced by virus infection. They are also detected in cells infected with vSM/TK-Δ27 and are identified in this panel by the asterisks adjacent to the lanes. (ii) Levels of the α protein ICP4 and the β protein ICP8 are consistent among all infections. (iii) Accumulation of VP16, a member of the γ₁ class of late proteins, is very similar in cells infected with each of these viruses. However, this protein is not as dependent upon ICP27 for expression as proteins expressed from the γ₂ class of HSV late genes, as evidenced by the synthesis of VP16 in cells infected with vBSΔ27 (48). (iv) Levels of gC and UL38, two γ₂ proteins, increase over time in cells infected with either vSM/TK-Δ27 or v27/TK-Δ27. In contrast, these proteins are not detected in cells infected with vBSΔ27. The accumulation of γ₂ proteins in cells infected with vSM/TK-Δ27 demonstrates that expression of SM supports HSV late gene expression. However, this experiment does not differentiate a role for SM in late gene expression that is independent of its role in the initiation of DNA replication (see below) and the concomitant increase in template availability. The involvement of SM in HSV γ₂ gene expression parallels the activity of ICP27 in HSV late gene expression and further demonstrates the functional similarity of these proteins.

SM complements HSV DNA replication.

Western analysis of viral protein accumulation indicated that two proteins from the γ₂ class of HSV genes were synthesized in cells infected with vSM/TK-Δ27 but accumulated to lesser amounts. In combination with the observation that vSM/TK-Δ27 grew, the dependence of γ₂ genes on viral DNA replication for expression suggested that SM was complementing HSV DNA replication. To measure viral DNA replication, aliquots of infected cells collected for measurement of protein synthesis were slot blotted onto a membrane and then hybridized with a radioactive probe specific for the gene encoding ICP4. Figure 8 shows the accumulation of viral DNA at 10, 24, and 48 h postinfection in Vero cells. In cells infected with vSM/TK-Δ27 and v27/TK-Δ27, viral DNA accumulated over the 48-h time period. However, the amounts of vSM/TK-Δ27 DNA synthesized in these infected cells were reduced by comparison to the amounts of viral DNA that accumulated in cells infected with v27/TK-Δ27. Previous studies in our laboratory have demonstrated that vBSΔ27 does not replicate DNA in high- or low-MOI infections (82), and therefore, these results indicate that expression of SM allows HSV DNA replication.

FIG. 8.

Viral DNA replication. Vero cells were either mock infected or infected with vSM/TK-Δ27 (SM/TK-Δ27), vBSΔ27 (Δ27), or v27/TK-Δ27 (27/TK-Δ27) at an MOI of 0.1 PFU/cell. Aliquots of infected cells were collected at the indicated times (hours) postinfection, and 10-fold dilutions (0.1× and 0.01×) were slot blotted onto nylon membranes. The DNAs were hybridized with a ³²P-labeled probe complementary to the ICP4 loci.

Effects of SM on cellular protein synthesis.

HSV infection induces profound alterations in the pattern of host gene expression. The majority of cellular gene expression is inhibited in infected cells largely because of the activity of the virion-associated protein vhs and ICP27 (21, 30, 31, 55, 56, 85). To determine if SM has similar effects on the pattern of host cell gene expression, a kinetic analysis of the profile of proteins synthesized in cells infected with vSM/TK-Δ27, v27/TK-Δ27, vBSΔ27, or wild-type HSV-1 KOS 1.1 was compared to the profile of host proteins expressed in mock-infected cells (Fig. 9). A reduction in the synthesis of cellular proteins was evident in cells infected with wild-type virus or v27/TK-Δ27 at 11 h postinfection, and viral proteins were synthesized at significant rates by this time (arrows). In contrast, cellular proteins were detectable throughout the 11-h time period that was examined in cells infected with vBSΔ27, and there was an obvious deficiency in viral protein synthesis.

FIG. 9.

Synthesis of cellular proteins in vSM/TK-Δ27-infected cells. Vero cells were either mock infected (M) or infected with vSM/TK-Δ27 (SM/TK-Δ27), vBSΔ27 (Δ27), v27/TK-Δ27 (27/TK-Δ27), or wild-type HSV-1 KOS at an MOI of 10 PFU/cell. The cells were pulse-labeled with Tran³⁵S Label at 3, 7, and 11 h postinfection (hpi). Total-cell extracts were analyzed by SDS-PAGE, and labeled proteins were visualized by autoradiography. Arrows on the right identify viral proteins; solid circles on the left identify cellular proteins that exhibit a reduction in synthesis as vSM/TK-Δ27, v27/TK-Δ27, or wild-type HSV-1 infections progress, and asterisks indicate cellular proteins that exhibit reduced synthesis in infections where ICP27 is expressed but continue to be synthesized in vSM/TK-Δ27 infections.

Viral protein synthesis was detectable in cells infected with vSM/TK-Δ27, but the cellular protein profile in cells infected with vSM/TK-Δ27 was variable. Some cellular proteins were synthesized throughout 11 h of infection, suggesting that expression of SM during the course of an HSV infection does not result in shutoff of host cell protein synthesis (Fig. 9, asterisks). However, synthesis of other cellular proteins was inhibited as effectively as in infections where ICP27 was expressed (Fig. 9, solid circles). Therefore, SM is not as effective as ICP27 at repressing cellular protein synthesis.

DISCUSSION

The experiments presented here demonstrate that the EBV SM protein is an ortholog of HSV-1 ICP27. Molecular phylogenetic analysis shows that evolutionarily, SM and ICP27 are distantly related proteins. However, SM is able to provide ICP27 functions that allow growth of an HSV-1 ICP27-null mutant both in a transient-expression-based complementation assay and in the context of a viral infection.

ICP27 has a well-defined role in the posttranscriptional regulation of viral late gene expression that is genetically separable from its regulation of early gene expression (74, 76, 89). At late times postinfection, ICP27 shuttles between the nucleus and cytoplasm and increases the cytoplasmic accumulation of a subset of viral late mRNAs (51, 62, 63, 75, 81). These data suggest that ICP27 regulates viral late gene expression through a direct involvement in viral mRNA export. However, the mechanism by which ICP27 mediates the nucleocytoplasmic export of viral mRNA remains unclear.

Proteins with homology to ICP27 have been identified in all three herpesvirus subfamilies (α, β, and γ). These families are catalogued based on different biological properties of the viruses, such as the length of the replication cycle and the host range of the virus. Molecular phylogenetic analysis of ICP27 homologs parallels the subfamily characterization (Fig. 1). At the amino acid level, the VZV ORF4 protein and ICP27, both members of the alphaherpesvirus subfamily, are more closely related than ICP27 and its homologs in CMV and EBV. However, the functional data that have emerged for many of these proteins suggest different relationships. In contrast to the posttranscriptional regulatory activity of ICP27, the VZV ORF4 protein is a transcriptional regulator. When tethered to a GAL4 DNA-binding domain, ORF4 activates transcription, and ORF4-responsive cis-acting elements have been identified in the promoters of genes regulated by ORF4 (61). Although posttranscriptional regulatory activity cannot be ruled out for ORF4, its activity remains distinct from ICP27 because ORF4 does not repress expression of genes with introns (53).

By contrast, the more distantly related SM protein from EBV, a gammaherpesvirus, has significant functional similarity to ICP27. In transient-expression assays, SM activates expression of reporter genes with particular 3′ processing signals and represses expression of genes with introns (38, 39, 45, 72). SM also binds RNA in vitro and in vivo and shuttles between the nucleus and cytoplasm, consistent with a role in mRNA export (7, 71, 72, 78). These similarities indicate that certain regulatory functions of ICP27 are conserved and suggest that these proteins may have a common mechanism for regulating gene expression.

The ability of ICP27 homologs to compensate for the defects of an ICP27-null virus was examined in a transient-expression-based complementation assay. In these experiments, the ability of the homologs to complement the growth of an ICP27 deletion mutant paralleled the functional data available for the proteins. No complementation was observed for the VZV ORF4, which confirms the results of a previous study (53). HCMV UL69 does not complement the null virus to a statistically significant level (Fig. 2). This result also confirms previous analyses (95) and is not surprising because several properties of UL69 are distinct from those of ICP27 and the other homologs. In HCMV-infected cells, UL69 mRNA does not accumulate in the presence of protein synthesis inhibitors and therefore is not expressed with immediate-early kinetics (95). Additionally, UL69 is a component of the HCMV virion and regulates the release of infected cells from the G₁ phase of the cell cycle (32).

The EBV SM protein reproducibly complemented replication of vBSΔ27 growth. By contrast, a mutant allele of SM (SMLRRΔ) that neither shuttles nor transactivates failed to complement (4). This suggests that despite the sequence differences between SM and ICP27, a relevant shared function may be nucleocytoplasmic shuttling. Although complementation by wild-type SM was incomplete, the results of the complementation analysis with both the wild-type and mutant SM proteins are intriguing given that the replication characteristics of HSV and EBV are markedly different and that inhibition of one feature shared by ICP27 and SM resulted in a failure to complement.

To examine the ability of SM to compensate for ICP27 defects in the context of a viral infection, a recombinant virus expressing SM in an ICP27-null background was constructed. This virus grows on Vero cells, confirming the results of the transient-transfection-based assay. In high-MOI infections, no increase in viral titer was observed through 48 h of infection, suggesting that viral growth is significantly delayed relative to that of a virus expressing ICP27. Viral growth was also measured over a longer time period in low-MOI infections. In a virus spread assay (Fig. 5), newly initiated vSM/TK-Δ27 infections were evident at 24 h postinfection, and virus continued to spread gradually through 72 h postinfection However, this spread was considerably restricted by comparison to v27/TK-Δ27 infections, which had spread through the entire culture by 48 h postinfection Analysis of vSM/TK-Δ27 yield over a 96-h time course in a low-MOI infection of Vero cells demonstrated a 10-fold increase in growth, although the final yield of vSM/TK-Δ27 infections was significantly reduced by comparison to v27/TK-Δ27 yields. Yield measurements from vSM/TK-Δ27 infections of the human foreskin fibroblast T18 cell line were similar (data not shown).

Compared to that for the wild-type viruses, the PFU/FFU ratio for vSM/TK-Δ27 was also significantly reduced, indicating that not every infection initiated by vSM/TK-Δ27 results in viral replication. Additionally, the plaquing efficiency of vSM/TK-Δ27 was reduced by nearly 1,000-fold on Vero cells compared to a cell line that provides ICP27 in trans. These results suggest that SM provides a function that allows HSV growth but that SM and ICP27 are not completely interchangeable.

A biochemical analysis of macromolecular synthesis in cells infected with vSM/TK-Δ27 indicated that the basis for growth was a result of SM-induced stimulation of HSV DNA replication and late protein synthesis. In these experiments, DNA replication and late protein synthesis were reduced compared to these events in an ICP27-expressing control virus, again indicating that the two proteins are not completely interchangeable. When these experiments were repeated in T18 cells, similar results were obtained (data not shown). However, the ability of vSM/TK-Δ27 to grow on a noncomplementing cell line and the ability of SM to stimulate HSV DNA replication and late gene expression suggest conservation of some functions between these two proteins.

It is possible that the multiple defects associated with ICP27-null mutants may limit the degree of SM complementation. For example, SM may function interchangeably with ICP27 in late gene expression but not complement effects on early gene expression and DNA replication completely. Similarly, less effective shutoff of cellular protein synthesis by SM may limit its ability to substitute for ICP27 and consequently result in decreased viral yields. Complementation of ICP27 mutants with defects in specific functions will resolve this issue.

The functional overlap between SM and ICP27 may be reflected in shared functional domains. Several of these domains are potentially relevant to the activity of both proteins in mRNA export. ICP27 and SM each contain a leucine-rich region (LRR) that fits the consensus sequence for nuclear export signals identified in other nucleocytoplasmic shuttling proteins (2, 41, 91). The cellular exportin CRM1 binds to nuclear export signal-containing proteins and facilitates cytoplasmic translocation in a process that is sensitive to the drug leptomycin B (23, 24, 58, 84). Several ICP27-dependent activities are sensitive to leptomycin B, including nuclear export of ICP27 and accumulation of particular late HSV mRNAs in the cytoplasm of infected cells (83).

Recently, a CRM1-independent mechanism of mRNA export has also been documented for ICP27 (43). These studies demonstrate a direct physical interaction between ICP27 and the cellular mRNA export factor REF. In microinjected Xenopus laevis oocytes, no effect of leptomycin B on ICP27 shuttling was observed, and a mutant of ICP27 that fails to bind REF was inactive in viral mRNA export (43). However, consistent with previous observations, the presence of an intronless late viral mRNA stimulated the cytoplasmic accumulation of ICP27 in coinjected oocyte nuclei (43). Collectively, it appears that ICP27 can mediate export of viral mRNAs through CRM1-dependent and -independent pathways.

Leptomycin B also has effects on the activity of the SM protein. In transiently transfected cells, leptomycin B decreases the transactivation activity of the wild-type SM protein and inhibits the nuclear export of SM (4). Mutations or deletions of the LRR sequence in SM restore transactivation activity and nuclear export in the presence of the drug (4). However, the same mutations have effects on the intracellular localization of SM independent of CRM1 association, and CRM1-independent nuclear export of SM has also been reported (4, 19). Although the exact export pathway(s) remains to be confirmed for both proteins, transit between the nucleus and cytoplasm is a relevant activity for both proteins. An HSV recombinant expressing a mutant SM allele that is not capable of nucleocytoplasmic shuttling fails to replicate, demonstrating that this shared function is important for promotion of HSV growth by both ICP27 and SM.

Consistent with potential roles as viral mRNA export proteins, ICP27 and SM also physically associate with RNA both in vitro and in vivo (6, 7, 50, 52, 71, 72, 75, 78, 83). ICP27 contains four domains with homology to known RNA-binding motifs, including an RGG box and three carboxy-terminal KH domains (52, 83). Through mutational analyses, both the RGG box and the third KH domain have been implicated in mediating interactions with RNA (52, 75, 83). SM physically associates with RNA in vitro (7, 72, 78) and in vivo (71). Although no RNA-binding domains have been confirmed in SM by genetic methods, a potential KH domain at the carboxy terminus of SM is identifiable by sequence comparison (data not shown).

Conservation of functional domains in proteins that are otherwise not very similar in overall amino acid sequence allows retention of function. The human immunodeficiency virus type 1 (HIV-1) Rev and the human T-lymphotropic virus type 1 Rex proteins are encoded by two evolutionarily distinct families of human retroviruses. Although Rev and Rex exhibit very little amino acid homology, the Rex protein can partially rescue growth of a replication-defective HIV-1 Rev mutant (69). Both proteins facilitate nuclear export of unspliced viral RNAs through physical interactions with structured RNA response elements present in the target RNAs and contain two conserved domains with essential functions: (i) a nuclear export signal (22, 91) and (ii) an arginine-rich motif required for interactions with viral mRNA. Rex stimulates nuclear export of unspliced HIV-1 mRNAs through a physical interaction with the HIV-1 Rev-responsive element (RRE) (3). However, consistent with their lack of sequence similarity, Rex and Rev recognize the HIV-1 RRE with distinct specificities (3).

Although homologous domains are present in SM and ICP27, conservation of functional domains is not a requirement for retention of functions between proteins. The HSV-1 VP16 protein is a potent transcriptional activator. Recruitment of VP16 to a DNA binding complex and transcriptional activation are separable activities that map to discrete domains: an amino-terminal domain required for assembly into a DNA binding complex, and a carboxy-terminal acidic activation domain (12, 28, 88). The VP16 homolog from equine herpesvirus 1 (EHV-1) has homology with the amino-terminal portion of VP16 but lacks nearly the entire carboxy-terminal acidic region (16). Despite the absence of a similar acidic activation domain, the EHV-1 homolog is a strong activator of both EHV-1 and HSV-1 transcription (16, 27, 44, 65). However, conservation of the amino-terminal domain of the EHV homolog is not sufficient for incorporation into a DNA binding complex (27).

Likewise, a chimeric protein consisting of the carboxy-terminal domain of the EHV homolog fused to the amino terminus of VP16 is a very weak transcriptional activator (27). Therefore, the EHV-1 protein does not follow the two-domain model of transcriptional activation established for VP16. Similarly, the functional overlap between ICP27 and SM may be achieved through the combined features of the individual proteins. Given the limited sequence similarity between these two proteins, it may be that functions conserved between ICP27 and SM require a complete three-dimensional protein structure rather than smaller homologous domains. These proteins may have evolved to regulate gene expression through a common mechanism despite unique overall protein arrangements.

A number of ICP27 mutants have been generated and analyzed in significant detail. With regard to this work, the phenotype of mutant d1-2 (17, 68) is comparable to the phenotype of vSM/TK-Δ27. This mutant has four amino-terminal residues deleted that comprise part of the NES (75, 81). In transient-expression assays, a plasmid carrying the d1-2 mutant allele also complements the growth of an ICP27 null virus, but it is more effective at complementation than SM. Although the d1-2 mutant virus synthesized nearly wild-type levels of gC, reductions in the synthesis of other viral late proteins are comparable between d1-2 and vSM/TK-Δ27. Previous studies have demonstrated that disruptions in the ICP27 NES impair shuttling and viral replication (51, 75, 83). The similar phenotypes observed in d1-2 infections, where the NES is not fully functional (51), and in vSM/TK-Δ27 infections, where the NES may be suboptimal, strongly support a role for shuttling in the regulation of HSV late gene expression.

The ability of SM to compensate for ICP27 defects suggests that certain functions of the two proteins may be retained. It remains to be determined if SM and ICP27 facilitate gene expression through a common pathway or if these proteins have distinct activities that result in a common phenotype. In conjunction with identification of protein domains required for RNA recognition, identification of the mRNAs bound by each protein and the particular mRNA binding site will provide a basis for understanding the relationship between the regulatory functions of these proteins. The conservation of function between these two evolutionarily distant proteins emphasizes the importance of this pathway for the regulation of gene expression in herpesviruses.