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Journal of Virology, January 2009, p. 128-139, Vol. 83, No. 1
0022-538X/09/$08.00+0     doi:10.1128/JVI.01954-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identification of Sequences in Herpes Simplex Virus Type 1 ICP22 That Influence RNA Polymerase II Modification and Viral Late Gene Expression{triangledown}

Thomas W. Bastian and Stephen A. Rice*

Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota 55455

Received 17 September 2008/ Accepted 17 October 2008


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ABSTRACT
 
Previous studies have shown that the herpes simplex virus type 1 (HSV-1) immediate-early protein ICP22 alters the phosphorylation of the host cell RNA polymerase II (Pol II) during viral infection. In this study, we have engineered several ICP22 plasmid and virus mutants in order to map the ICP22 sequences that are involved in this function. We identify a region in the C-terminal half of ICP22 (residues 240 to 340) that is critical for Pol II modification and further show that the N-terminal half of the protein (residues 1 to 239) is not required. However, immunofluorescence analysis indicates that the N-terminal half of ICP22 is needed for its localization to nuclear body structures. These results demonstrate that ICP22's effects on Pol II do not require that it accumulate in nuclear bodies. As ICP22 is known to enhance viral late gene expression during infection of certain cultured cells, including human embryonic lung (HEL) cells, we used our engineered viral mutants to map this function of ICP22. It was found that mutations in both the N- and C-terminal halves of ICP22 result in similar defects in viral late gene expression and growth in HEL cells, despite having distinctly different effects on Pol II. Thus, our results genetically uncouple ICP22's effects on Pol II from its effects on viral late gene expression. This suggests that these two functions of ICP22 may be due to distinct activities of the protein.


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INTRODUCTION
 
Herpes simplex virus type 1 (HSV-1) is a widely studied human alphaherpesvirus that serves as an important model for defining the fundamental pathways used by herpesviruses to replicate in their host cells. During productive infection, the 152-kb double-stranded HSV-1 genome is rapidly translocated to the nucleus where the ~80 viral genes are transcribed by the host cell RNA polymerase II (Pol II) in a temporally orchestrated program that is regulated by viral proteins (reviewed in reference 47). The first genes to be expressed are the immediate-early (IE) genes. Transcription of these genes requires the viral tegument protein VP16 but does not require new viral protein synthesis. Translation of the IE genes results in expression of five proteins, four of which (ICP0, ICP4, ICP22, and ICP27) serve to activate and temporally regulate the ensuing expression of the delayed-early (DE) and late (L) genes.

At the same time that HSV-1 DE and L genes are induced to high levels, host cell gene expression is largely inhibited, a phenomenon known as host shutoff. Host shutoff is a complex process that is mediated at multiple levels of gene expression and is regulated by several viral factors (reviewed in reference 52). A number of studies have investigated whether HSV-1-mediated shutoff involves the inhibition of Pol II transcription on host cell genes (20, 24, 37, 51, 53, 55). Such studies have used either nuclear run-on transcription analysis or metabolic pulse-labeling of RNA to directly measure the transcription rates of specific host genes following infection or, in some cases, of adenoviral or polyomaviral genes that are integrated into host chromosomes. These experiments have indicated that HSV-1 infection strongly inhibits Pol II transcription on many, if not most, host cell genes. On the other hand, several recent microarray analyses, which measure steady-state RNA and not transcription rates, have shown that there is only a modest reduction (less than threefold) in the levels of most host cell mRNAs following HSV-1 infection (19, 33, 44, 56). Such results are not readily consistent with a strong, global shutoff of host cell gene transcription. Thus, further studies are needed to determine whether and to what extent HSV-1 infection inhibits the ability of Pol II to transcribe cellular genes.

The bases for HSV-1-mediated changes to Pol II transcription patterns during productive viral infection are not thoroughly understood but may involve virus-mediated alterations to Pol II itself. Pol II is a large nuclear enzyme consisting of 12 subunits and is responsible for the synthesis of mRNA and small noncoding RNAs (reviewed in references 21 and 59). One important mode of Pol II regulation is via posttranslational modification of the C-terminal domain (CTD) of its large subunit (LS) (10, 26, 32, 36, 61). The CTD consists of multiple repeats (52 in human Pol II) of the heptapeptide consensus sequence YSPTSPS and is the site of extensive phosphorylation by cellular CTD kinases. As a result of this phosphorylation, Pol II exists in two forms in vivo which differ in the extent of CTD phosphorylation: Pol II-A is hypophosphorylated and is the form that enters preinitiation complexes, whereas Pol IIo is hyperphosphorylated and is the form that is actively engaged in transcription. Phosphorylation predominantly occurs on serine-2 and serine-5 (Ser-2 or Ser-5) of the CTD repeat, although recent evidence indicates that serine-7 can also be phosphorylated (11). During transcription, the CTD serves as a scaffold for recruiting mRNA processing and chromatin-modifying factors to the transcribing Pol II complex, and this recruitment is largely regulated by CTD phosphorylation. Phosphorylation on Ser-5 is important for promoter clearance and recruitment of mRNA capping factors, whereas phosphorylation on Ser-2 is important for efficient elongation and recruitment of polyadenylation factors.

Several years ago we discovered that HSV-1 infection induces dramatic changes in CTD phosphorylation (46). Two distinct effects can be defined. First, soon after infection, forms of Pol II that are phosphorylated on Ser-2 (Ser-2P Pol II) are lost in a process that involves ICP22 (12, 45). Later in infection, another pathway that may involve ICP27 also contributes to loss of Ser-2P Pol II (8, 12). Second, and consistent with the loss of Ser-2 phosphorylation, a novel form of Pol II that is predominantly phosphorylated on Ser-5 accumulates (12, 46). This form was named Pol III (for intermediate), since its LS subunit (designated IIi) has an intermediate electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels compared to the normal hyper- and hypophosphorylated forms (known as IIa and IIo, respectively). The ICP22-dependent induction of Pol III can be distinguished from the ICP22-dependent loss of Ser-2P Pol II, as the former but not the latter requires another viral factor, the virion-associated protein kinase UL13 (23). Thus, current evidence suggests that ICP22 may mediate two distinct effects on Pol II. We have previously suggested that these effects may play a role in the shift in transcriptional specificity that occurs in HSV-1-infected cells.

ICP22 consists of 420 residues and is encoded by a spliced mRNA transcribed from the US1 gene (see Fig. 1A). It is necessary for efficient HSV-1 growth in animal models of infection (31, 38, 50) as well as for efficient in vitro growth in some, but not all, cultured cells (30, 39, 50). For example, ICP22 mutants grow well in African green monkey kidney (Vero) cells, but not in human embryonic lung (HEL) cells. ICP22 is extensively phosphorylated during infection, primarily by UL13 and another viral protein kinase, US3 (29, 40, 42). In addition to inducing the modification of Pol II, several other functions have been attributed to ICP22; these functions include the induction of certain viral L genes (27, 39, 50), the alteration of cell cycle-related proteins (1-3), and the determination of virion composition (31).


Figure 1
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  		FIG. 1. Expression of mutant ICP22 polypeptides in transfected cells. (A) Diagram of modified ICP22 polypeptides. The bar at the top represents the 420-residue ICP22 polypeptide. The positions of known nuclear localization signals (NLSs) (residues 16 to 31 and 118 to 131) (54) are shown, as is a region conserved among alphaherpesviral ICP22 homologs (residues 161 to 292). The solid black bars below show the ICP22 protein sequences present in the various mutants, with the white bar representing the N-terminal FLAG epitope (F). Dotted lines indicate deletions. The restriction enzymes used to generate the deletions are indicated (BmgBI [B], AleI [A], PmlI [P], and SacII [S]). (B) Immunoblot analysis of transiently expressed proteins. Vero cells were transfected with the indicated plasmids, and protein extracts were prepared after 24 h. The proteins were analyzed by immunoblotting using antisera specific for ICP22 or FLAG. {alpha}-ICP22, anti-ICP22 antibody. (C) Localization of mutant polypeptides. Vero cells were transfected with the various plasmids as indicated and processed for immunofluorescence 1 day posttransfection using FLAG-specific antisera. Plasmids pcDNA22 (22), pcDNAUS1.5 (US1.5), pcDNA22-BA (BA), pcDNA22-AP (AP), and pcDNA22-PS (PS) were used. Representative cells are shown. (D) ICP22-triggered loss of Ser2-P Pol II. Vero cells were transfected with pCMVβgal-c (a) or the ICP22 expression plasmids as indicated (b to f), and processed for immunofluorescence 24 h later. Cells were doubly stained for β-galactosidase (β-gal) (green signal) and Ser-2P Pol II (red signal) (a), or ICP22 (green signal) and Ser-2P Pol II (red signal) (b to f). The panels show the merged green and red images.

The US1 gene also encodes a second less abundant protein, US1.5, from an unspliced mRNA that is transcribed from a promoter downstream of the ICP22 promoter (5). US1.5 is in frame with ICP22 but lacks an N-terminal portion of it. US1.5 was originally reported to initiate at codon 147 of the ICP22 open reading frame (ORF) (5), but a later study by the same group concluded that it initiates at codon 171 (15). More recent work suggests that it may initiate prior to codon 124 (J. J. Bowman and P. A. Schaffer, presented at the 32nd International Herpesvirus Workshop, Asheville, NC, 7 to 12 July 2007). Thus, at present, the exact extent of the US1.5 polypeptide is unclear. Similar to ICP22, US1.5 is extensively phosphorylated and has been reported to possess the L-gene activation function of ICP22 (29).

In this study, we sought to identify the sequences in ICP22/US1.5 that are required for modification of Pol II. To do this, we engineered and analyzed several ICP22 plasmid and virus mutants. Our results reveal that the C-terminal half of ICP22 (residues 240 to 420) is the portion of the polypeptide that is responsible for modification of Pol II. In contrast, both the N-terminal and C-terminal halves of the protein are important for activating viral L-gene expression during infection of HEL cells. Thus, our results genetically uncouple ICP22's effects on Pol II from its effects on viral L-gene activation. This suggests that these two effects may be due to distinct activities of ICP22.


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MATERIALS AND METHODS
 
Cells, viruses, and infections. Vero (African green monkey kidney) and human embryonic lung cells were obtained from the American Type Culture Collection. Cells were grown as monolayer cultures in Dulbecco modified Eagle medium supplemented with 5% (Vero) or 10% (HEL) heat-inactivated fetal bovine serum (FBS), 50 U of penicillin/ml, and 50 µg of streptomycin/ml. All tissue culture reagents except FBS were purchased from Invitrogen (Carlsbad, CA). FBS was purchased from Atlas Biologicals (Ft. Collins, CO).

KOS1.1 was the wild-type (WT) HSV-1 strain used in this study (17). The HSV-1 ICP22 mutant d22lacZ has been described previously (23). Construction of recombinant viruses TF22, TF1.5, TF22BA, TF22AP, and TF22PS is described below. Cells were infected at a multiplicity of infection (MOI) of 10 PFU per cell in phosphate-buffered saline containing 0.1% glucose and 0.1% heat-inactivated newborn calf serum. After 1 h of viral adsorption at 37°C, the inoculum was replaced with medium 199 containing 2% heat-inactivated newborn calf serum, 50 U penicillin/ml, and 50 µg of streptomycin/ml. Infections were then incubated at 37°C. Cycloheximide (CH) reversal experiments were performed as previously described (12).

To analyze viral growth, Vero or HEL cells were infected at an MOI of 10. At 2 hours postinfection (hpi), the cells were treated with acid-glycine buffer (pH 3.0) to inactivate viruses that had not yet entered cells (4). After 24 h, the infections were terminated by freezing at –80°C. The virus was then released from the cells by freeze-thawing three times. Virus yield was determined by plaque assay of the lysates on Vero cells. The medium used for viral plaque assays was the same as for virus infections except that it included 1% (vol/vol) heat-inactivated pooled human serum (MP Biomedicals Inc., Solon, OH). Following infection, plaque assay cultures were incubated for 3 to 4 days to allow plaques to develop.

Plasmids and transfections. The pcDNA22 and pcDNAUS1.5 plasmids (12) are expression constructs encoding N-terminally FLAG-tagged ICP22 and US1.5 (assuming that US1.5 corresponds to residues 147 to 420 of ICP22), respectively. To construct pcDNA22BA, pcDNA22 was digested with BmgBI and AleI, and the resulting large DNA fragment was self-ligated and transformed into Escherichia coli. Colonies were screened by DNA restriction analysis, and a positive clone was designated pcDNA22BA. To create pcDNA22AP, the same procedure was repeated using the restriction enzymes AleI and PmlI. To engineer pcDNA22PS, pcDNA22 was digested with PmlI and SacII. Blunt ends were generated on the resulting linear fragment using T4 DNA polymerase (Invitrogen). A 10-bp ClaI linker (catalog no. S1089S; New England Biolabs, Ipswich, MA) was then ligated onto the ends. The DNA was digested with ClaI and self-ligated. A clone resulting from these manipulations was designated pcDNA22PS. All mutations were confirmed by DNA sequencing.

To facilitate the introduction of the altered ICP22 genes into recombinant viruses, the mutations were moved into another ICP22 plasmid, pUCNS-NotI, which has a larger extent of flanking HSV-1 sequences. pUCNS-NotI was derived from pUCNS (23) in the following manner. pUCNS was digested at its unique BglII site, located at codons 5 to 7 of the ICP22 ORF. Blunt ends were generated on the resulting linear fragment using T4 DNA polymerase. A NotI restriction site was then introduced by inserting a 12-bp NotI linker (catalog no. 1127; New England Biolabs) at this position. The resulting plasmid was sequenced in the region altered and designated pUCNS-NotI. To introduce mutant ICP22 genes, pUCNS-NotI was doubly digested with NotI and EagI (which cuts at the 3' end of the ICP22 ORF), and the large fragment was purified. pcDNA22 and the pcDNA22 mutant plasmids were also cut with NotI and EagI, and the small fragments containing the ICP22 ORF were purified. Large and small fragments were ligated together to create pUCNS22, pUCNS1.5, pUCNS22BA, pUCNS22AP, and pUCNS22PS. These manipulations resulted in modified ICP22 genes that are N terminally tagged with the sequence MADISPDLAAATMDYKDDDDKSPGGS (the underlined sequence corresponds to the FLAG epitope). These plasmids were used in the marker transfer protocol described below to make recombinant viruses.

Transfections were carried out as previously described (12). Plasmid pCMVβ-c (34) was used to express β-galactosidase in transfection experiments.

Isolation of ICP22 mutant viruses. The isolation of the TF22, TF1.5, TF22BA, TF22AP, and TF22PS viruses was carried out using a marker transfer strategy (23). Briefly, pUCNS22 and mutant plasmids pUCNS1.5, pUCNS22BA, pUCNS22AP, and pUCNS22PS were digested with AgeI, releasing the recombinant ICP22 gene and flanking viral sequences. The DNAs were individually cotransfected into Vero cells with infectious d22lacZ viral DNA. The transfected cells were harvested after 3 to 4 days when most cells exhibited cytopathic effects. Recombinant viruses that did not express β-galactosidase were identified by plaque assay on Vero cells in the presence of 300 µg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside. For each mutant, two independent isolates were obtained from different transfections. All isolates were plaque purified three additional times on Vero cells and designated TF22, TF1.5, TF22BA, TF22AP, and TF22PS. The second isolate of each mutant was given the additional suffix "b." To confirm the structure of the mutant genomes, Southern blotting was performed as described previously, using plasmid pBamN as the radioactive probe (23).

Immunoblotting. Analysis of protein expression by immunoblotting was carried out as previously described (34). To assess ICP22 protein expression, membranes were probed with rabbit antiserum specific for ICP22 (a gift from John Blaho) at a dilution of 1:1,000 or rabbit anti-FLAG serum (Immunology Consultants, Newberg, OR) at a dilution of 1:2,000. To analyze Pol II LS, the mouse monoclonal antibodies ARNA3 (Research Diagnostics, Flanders, NJ), 8WG16 (a gift from Nancy Thompson), and H5 (Covance, Denver, PA) were used at dilutions of 1:500, 1:5,000, and 1:500, respectively. The primary antibodies used for detection of HSV DE and L proteins were a glycoprotein C (gC)-specific mouse monoclonal antibody (Abcam Inc., Cambridge, MA), rabbit polyclonal anti-US11 sera (a gift from Jean-Jacques Diaz), anti-ICP8 mouse monoclonal antibody H1115 (Rumbaugh-Goodwin Institute for Cancer Research, Plantation, FL), a monoclonal antibody specific for VP5 (Abcam Inc.), rabbit polyclonal anti-VP16 sera (58), and rabbit polyclonal anti-VP22 sera (a gift from Gill Elliott); these antibodies were diluted 1:5,000, 1:1,000, 1:300, 1:1,000, 1:10,000, and 1:50,000, respectively. In most experiments, cellular endosomal antigen 1 (EEA1) was also analyzed using a 1:300 dilution of an EEA1-specific mouse monoclonal antibody (BD Biosciences, San Jose, CA). The secondary antibodies used for immunoblot detection were horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse immunoglobulin G (IgG) (GE Healthcare, Piscataway, NJ), diluted 1:7,500 and 1:5,000, respectively. An enhanced chemiluminescence (ECL) detection system (GE Healthcare) was used to detect immunoreactive proteins.

Immunofluorescence. To carry out immunofluorescence, subconfluent cells growing on glass coverslips were fixed in 3.7% formaldehyde and permeabilized in cold acetone (43). For analysis of ICP22 in infected cells, coverslips were incubated at 37°C for 1 h with anti-FLAG M2 mouse monoclonal antibody (Sigma), diluted 1:5,000. Secondary staining was done at 37°C for 1 h with Cy3-conjugated goat anti-mouse IgG, diluted 1:400. For analysis of ICP22 in transfected cells, rabbit anti-FLAG polyclonal antiserum was used at a dilution of 1:300, and secondary staining was done with Cy2-conjugated goat anti-rabbit IgG. In some cases, cells were additionally treated with monoclonal antibody H5 (Covance, Denver PA), specific for Ser-2P Pol II, diluted 1:100. When cells were costained with H5, the secondary staining mixture included a 1:400 dilution of Cy3-conjugated goat anti-mouse IgG. All secondary antibodies were purchased from Jackson Immunoresearch Laboratories (West Grove, PA). Cells were examined using fluorescence microscopy (Olympus BX60). Images were adjusted for brightness and contrast and cropped using Adobe Photoshop (Adobe Systems, Inc., Mountain View, CA).


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RESULTS
 
A region in the C-terminal half of ICP22 is critical for inducing loss of Ser-2P Pol II. We previously used an immunofluorescence assay to show that transient expression of HSV-1 ICP22 in a variety of mammalian cells is sufficient to trigger the loss of the Ser-2P Pol II (12). We also showed that a C-terminal fragment of ICP22, which is as long as the US1.5 protein is (residues 147 to 420, as it was originally defined [5]), can elicit this same effect. To further map the ICP22 sequences that mediate Pol II alterations, we constructed three new expression plasmids having in-frame deletions in the ICP22 ORF. A diagram of our previous (pcDNA22 and pcDNAUS1.5) and new (pcDNA22-BA, pcDNA22-AP, and pcDNA22-PS) constructs is shown in Fig. 1A. To facilitate analysis, all plasmids were engineered with an N-terminal FLAG epitope tag.

To confirm that the constructs express ICP22 polypeptides of the expected size, we carried out immunoblotting analysis. Vero cells were transfected with the plasmids, and total cell proteins were prepared 24 h later. The extracts were analyzed for ICP22 using rabbit polyclonal serum specific for ICP22 (Fig. 1B, top blot) or FLAG (bottom blot). The results showed that all of the engineered plasmids express FLAG-tagged ICP22 polypeptides of the expected sizes. Additionally, all of the proteins are expressed at roughly comparable levels in Vero cells.

ICP22 is reported to localize to the nucleus in both infected and transfected cells (7, 22, 48, 54). We used immunofluorescence to see whether the mutant proteins were similarly localized. Vero cells were transfected with the plasmids, fixed 1 day later, and processed for immunofluorescence using a rabbit anti-FLAG serum (Fig. 1C). Both WT ICP22 and all mutant forms were highly localized in the nuclei and tended to be excluded from nucleoli. However, the various forms differed significantly in their intranuclear staining. The WT protein (Fig. 1C, panel a) localized diffusely in the nucleus and to discrete nuclear bodies. This pattern is similar to that which has been reported by others for transiently expressed ICP22 (7, 48, 54). In contrast, the US1.5 form of the protein (Fig. 1C, panel b) showed diffuse nuclear localization but lacked nuclear body staining. This is also consistent with published work (48). The BA mutant polypeptide (Fig. 1C, panel c) was similar to US1.5 in generally lacking nuclear body staining, although some cells did exhibit localization in globular regions. The mutant AP protein (Fig. 1C, panel d) showed a pattern that was more similar to the WT protein in that it exhibited a combination of nuclear body and diffuse staining. This mutant also exhibited some larger globular areas of protein accumulation, similar to the BA mutant. These areas tended to be localized to one end of the nucleus. Last, the PS mutant (Fig. 1C, panel e) showed a very distinctive pattern that was similar to the WT pattern with the exception that the ICP22-containing nuclear bodies were significantly more numerous and well-defined.

We next tested the mutants to determine which are able to trigger the loss of Ser-2P Pol II. Transfected Vero cells were fixed 24 h after transfection and analyzed by immunofluorescence for the presence of ICP22 and Ser-2P Pol II (Fig. 1D). For a control, cells were also transfected with an expression plasmid encoding E. coli β-galactosidase. As expected from our previous results (12), cells expressing β-galactosidase (Fig. 1D, panel a, green signal) were similar to the majority of untransfected cells in that they showed readily detectable Ser-2P Pol II in their nuclei (red signal), leading to an orange/yellow nuclear signal in the merged image. Also as reported previously, both ICP22 and US1.5 triggered the loss of Ser-2P Pol II, resulting in a pronounced green nuclear signal in the merged images (Fig. 1D, panels b and c, respectively). The BA and AP mutants (Fig. 1D, panels d and e, respectively) behaved similarly. In contrast, the PS mutant was unable to trigger the loss of Ser-2P Pol II, as demonstrated by the yellow nuclear signals in the merged image (Fig. 1D, panel f). We conclude that amino acids 240 to 340 are required for ICP22's ability to trigger loss of the Ser-2P Pol II in transfected Vero cells. Furthermore, it can be concluded that the N-terminal 240 residues of ICP22 are completely dispensable for this activity.

Isolation of HSV-1 mutants encoding altered forms of ICP22. The above results indicate that a sequence in the C-terminal half of ICP22 is involved in modifying Pol II in transfected cells. To see whether this region is also critical for Pol II modification during infection, we used a marker transfer approach to introduce the various mutant alleles into the HSV-1 genome in place of the WT gene. The resulting viral recombinants are shown in Fig. 2A. The mutant encoding the WT FLAG-tagged ICP22 was designated TF22, while the mutants encoding the altered forms were designated TF1.5, TF22BA, TF22AP, and TF22PS. For each mutant, two genetically independent isolates were obtained, with the second isolate being given the suffix "b." Two independent isolates were obtained so that we could confirm that any mutant phenotypes were due to the engineered alterations as opposed to unrelated adventitious mutations that could occur in a single isolate.


Figure 2
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  		FIG. 2. Characterization of viral ICP22 mutants. (A) Expected genome structures of viral mutants. At the top is shown a diagram of the HSV-1 genome as well as a blow-up of the ICP22 gene contained on the BamHI N restriction fragment. The spliced US1 transcript is indicated. The white and black bars denote the HSV-1 genome repeats RL and RS, respectively. The sizes (in kilobases) of expected BamHI fragments in the region of the ICP22 gene are shown; the parentheses denote deletions. B, BamHI restriction sites. (B) Southern blot analysis. Total DNA was purified from infected Vero cells, digested with BamHI, and analyzed by Southern blotting using 32P-labeled pBamN as a probe. The positions of DNA size standards are shown to the left of the blot. The asterisk denotes the 1.9-kb BamHI fragment arising from the opposite US/RS junction. (C) Immunoblotting analysis. Vero cells were mock infected or infected with the indicated viruses at an MOI of 10. Protein extracts were prepared at 6 hpi, and immunoblotting was carried using ICP22- or FLAG-specific antisera. The migration positions of protein molecular mass markers (in kilodaltons) are shown to the left of each image. {alpha}-ICP22, anti-ICP22 antibody.

To confirm that the mutant viruses had the expected genomic changes, Southern blotting was carried out. Infected-cell DNA preparations were digested with BamHI, transferred to a nylon membrane, and probed with plasmid pBamN, which has an insert of the 4.8-kb, ICP22 gene-containing BamHI N fragment from the HSV-1 genome (Fig. 2A). As expected, the WT KOS1.1 sample showed a ~4.8-kb hybridizing band (Fig. 2B), while mutant d22lacZ showed a ~6.8-kb band due to the insertion of the E. coli lacZ gene (23). Since the pBamN probe contains sequences from the HSV-1 RS repeat (Fig. 2A), all viral DNAs also showed a ~1.9-kb hybridizing band (asterisk in Fig. 2B) that is derived from the opposite US/RS junction. In addition to this common fragment, all of the FLAG-tagged strains displayed two other fragments. This is consistent with expectations, since the inserted FLAG sequence possesses a BamHI site. One fragment of ~1.3 kb was shared by all mutants, while the larger ones varied as expected according to the size of the engineered deletions.

We also used immunoblotting to analyze the ICP22 polypeptides expressed by the viral mutants in Vero cells (Fig. 2C). Again, the results were consistent with expectations in that all of the recombinants expressed FLAG-tagged ICP22 proteins that migrated on sodium dodecyl sulfate-polyacrylamide gels with mobilities that were consistent with their engineered mutations. Together, the results of the Southern and immunoblotting analyses confirm that the engineered viral mutants have the expected genomic structures.

Growth of viral ICP22 mutants. Previous studies have shown that HSV-1 ICP22 mutants exhibit a cell type-dependent growth defect, i.e., they replicate near normally in some cultured cell lines, including Vero cells, but are defective for growth in others, including HEL cells (23, 30, 39, 50). To characterize the growth phenotypes of the new mutants, we carried out single-cycle viral yield assays in both Vero and HEL cells infected at an MOI of 10. Infections were terminated at 24 hpi, and virus yields were determined by plaque assay of the infected-cell lysates on Vero cells (Fig. 3). As expected from our previous work (23), the ICP22 null mutant d22lacZ exhibited a modest growth defect in Vero cells compared to KOS1.1 (~60-fold) but was significantly more attenuated in HEL cells (~800-fold defect). The FLAG-tagged viruses varied in their growth phenotypes, although in all cases both isolates of each recombinant were very similar. Importantly, TF22 was indistinguishable from KOS1.1 in its efficient replication in both cell lines. This establishes that the N-terminal FLAG epitope does not affect the ability of ICP22 to promote viral growth. In contrast, all of the other FLAG-tagged viruses showed some deficiency in growth compared to KOS1.1. The two that were the most impaired were TF1.5 and TF22PS. TF1.5 grew only slightly better than d22lacZ, with replication defects of ~35-fold and ~135-fold in Vero and HEL cells, respectively. TF22PS grew similarly to TF1.5 in Vero cells but exhibited a slightly more severe defect in HEL cells (a ~300-fold defect relative to KOS1.1). In contrast, the TF22BA and TF22AP mutants grew more efficiently in both Vero and HEL cells than TF1.5 and TF22PS did, although they did not grow as well as KOS1.1 or TF22. Overall, the results indicate that both TF1.5 and TF22PS are similar to the null mutant d22lacZ in their cell type-dependent growth phenotype. Thus, sequences in both the N- and C-terminal halves of ICP22 are required for efficient viral replication in HEL cells.


Figure 3
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  		FIG. 3. Growth of ICP22 mutants in Vero and HEL cells. Confluent monolayers of Vero (A) or HEL (B) cells were infected in triplicate with the viruses indicated at an MOI of 10 and incubated for 24 h. Virus yield in the infected cells was determined by plaque assay of the cell lysates on Vero cells. Bars denote the mean virus yield; error bars represent the standard error of three triplicate infections.

TF22PS is unable to induce Pol III. We next examined the viral mutants to see whether they were able to induce Pol III. Vero cells were mock infected or infected with KOS1.1, d22lacZ, or the various FLAG-tagged derivatives at an MOI of 10. At 6 hpi, total protein extracts were prepared and subjected to immunoblotting using two monoclonal antibodies directed against the Pol II LS. ARNA3 binds to the body of the LS and thus reacts with all phosphorylation variants, while 8WG16 binds to the CTD in a phosphospecific manner (32, 46). It recognizes the hypophosphorylated IIa form and the HSV-1-specific IIi form but does not efficiently recognize the normal hyperphosphorylated IIo form (32, 46). Consistent with previous findings, mock-infected Vero cells showed approximately equal amounts of IIo and IIa subunits and did not exhibit IIi subunit (Fig. 4, lanes 1 and 8), whereas KOS1.1-infected cells exhibited a loss of IIo coupled with an induction of IIi (lanes 2 and 9). Also as reported previously, the ICP22 null mutant d22lacZ was unable to efficiently induce IIi but did cause a significant loss of IIo (Fig. 4, lanes 3 and 10). The FLAG-tagged recombinants showed two distinct phenotypes. Consistent with its WT growth, TF22 was similar to KOS1.1 in being able to efficiently induce Pol III (Fig. 4, lanes 4, 5, 11, and 12). Likewise, mutants TF1.5 (Fig. 4, lanes 6 and 7), TF22BA (lanes 13 and 14), and TF22AP (lanes 15 and 16) all induced Pol III. In contrast, TF22PS (Fig. 4, lanes 17 and 18) failed to induce Pol III. For a control, separate regions of the blots were probed for EEA1, a cellular antigen that does not change in abundance during HSV-1 infection (13). The levels were comparable in all lanes, indicating approximately equal loads of proteins. Additional immunoblotting confirmed that all viruses expressed comparable levels of ICP27, indicating that cells were successfully infected. Together, these results demonstrate that residues 240 to 340 of ICP22 are required for induction of Pol III during viral infection.


Figure 4
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  		FIG. 4. Induction of Pol III in ICP22 mutant-infected cells. Vero cells were mock infected or infected with the indicated viruses at an MOI of 10. Protein extracts were prepared at 6 hpi and analyzed by immunoblotting using the Pol II LS-specific antibodies ARNA3 or 8WG16. The same samples were also probed for ICP27 and EEA1. The Pol II LS form IIa migrates at approximately 200 kDa; II0 migrates at approximately 240 kDa. The two blots show the results of separate experiments.

TF22PS is defective for triggering loss of Ser-2P Pol II. We previously showed that ICP22 triggers loss of Ser-2P Pol II during the IE stage of HSV-1 infection (13). This effect is distinct from induction of Pol III because Pol III induction requires UL13 but Ser2P Pol II loss does not (23). Our transient-expression analysis (Fig. 1D) showed that the PS mutant is the only one that is incapable of triggering loss of Ser-2P Pol II in transfected cells. To look at this activity in infected cells, we employed a "cycloheximide reversal" protocol, a treatment that extends and exaggerates the IE phase of infection (13). Specifically, cells are infected in the presence of CH for 5 h, leading to the enhanced accumulation of IE mRNAs (16). After removal of the CH, the cells are incubated for another 2 h to allow a period of high-level IE protein expression before harvesting the proteins and subjecting them to immunoblotting analysis to assess Ser-2P LS levels. We have previously shown that under these conditions, loss of Ser-2P Pol II is highly dependent on ICP22 (13). The results of this analysis are shown in Fig. 5. For comparison, we also carried out a parallel 7-h infection in the absence of CH. As seen previously, KOS1.1 infection (Fig. 5, lanes 3, 4, 17, 18, 31, and 32) triggered a striking reduction in Ser-2P Pol II compared to mock-infected cells (lanes 1, 2, 15, 16, 29, and 30). In contrast, and also as reported previously, d22lacZ-infected cells exhibited relatively high levels of Ser-2P Pol II compared to KOS1.1, particularly under CH reversal conditions (Fig. 5, lanes 6, 20, and 34). The FLAG-tagged recombinants displayed two phenotypes. TF22, TF1.5, TF22BA, and TF22AP all resembled KOS1.1, triggering a near complete loss of Ser-2P Pol II under CH reversal conditions. In contrast, TF22PS resembled d22lacZ in its inability to efficiently cause loss of Ser-2P Pol II under CH reversal conditions (Fig. 5, lanes 26 and 28), although it appeared to be slightly less deficient in this activity. This suggests that the PS mutant may have some residual activity. These results implicate residues 240 to 340 of ICP22 in the depletion of Ser-2P Pol II during the IE stage of HSV-1 infection.


Figure 5
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  		FIG. 5. Loss of Ser-2P Pol II in ICP22 mutant-infected cells. Vero cells were mock infected or infected with the indicated viruses. In one set of infections (odd-number lanes), the cells were left untreated, and protein extracts were prepared at 7 hpi. In the other set of infections (labeled CHR; even-number lanes), cells were infected in the presence of 50 µg/ml CH for 5 h. The CH was then removed, and the cells were incubated for two more hours prior to harvesting. Protein extracts were analyzed by immunoblotting using the H5 antibody or an antibody specific for EEA1. The blots on the left and in the middle show the results of one experiment, whereas the blot on the right shows the results of a separate experiment.

Localization of ICP22 mutant proteins during HSV-1 infection. To examine the relationship between ICP22's effects on Pol II and its cellular localization, we used immunofluorescence to determine the localization of the FLAG-tagged ICP22 polypeptides in infected Vero cells at various times after infection (Fig. 6). Mock infection did not result in detectable staining of ICP22 at any time point, nor was there significant staining for any virus at 1.5 or 2 hpi (data not shown). At 3 hpi, a small percentage of infected cells expressed the corresponding ICP22 recombinant protein. At this time point, the TF22-encoded WT protein (Fig. 6a) and the US1.5 (Fig. 6d), ICP22BA (Fig. 6g), and ICP22AP (Fig. 6j) mutant proteins all had similar localization patterns. The proteins were found diffusely throughout the nucleus in a fine granular pattern but were excluded from nucleoli. The ICP22PS protein also localized to the nucleus in a diffuse granular pattern at 3 hpi. However, approximately half of the cells expressing ICP22 protein showed further localization to small nuclear bodies (Fig. 6m). At 6 hpi, the localization of ICP22 to large nuclear structures that are likely replication compartments became clearly visible for all the infections (Fig. 6b, e, h, k, and n). In addition, nearly all TF22-infected cells showed ICP22 in small nuclear bodies, although the number of structures in these cells was less than in TF22PS-infected cells at this time point. Nuclear bodies could also be seen in TF22AP-infected cells (Fig. 6k) but were absent in TFUS1.5- and TF22BA-infected cells. Late in infection (10 hpi), the localization of ICP22 changed significantly for TF22, TF22AP, and TF22PS. TF22-infected cells exhibited an increase in the number of nuclear bodies, but what was most notable was that the bodies were much smaller in size than at previous time points (Fig. 6c). The nuclear bodies also were reduced in size in TF22PS-infected cells (Fig. 6o). The most dramatic change at 10 hpi was for the AP protein, which became localized almost exclusively to nuclear bodies that were larger than those previously seen (Fig. 6l). In addition, there was a significant reduction in the intensity of AP staining in the rest of the nucleus. At 10 hpi, the localization of the US1.5 and BA proteins remained mostly diffuse throughout the nucleus.


Figure 6
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  		FIG. 6. Localization of ICP22 polypeptides during infection. Subconfluent monolayers of Vero cells were mock infected or infected with the mutants shown. At the times indicated, cells were fixed and processed for immunofluorescence using an anti-FLAG antibody.

Two conclusions can be made from the infected-cell localization data, combined with the results from transfected cells (Fig. 1C). First, since the US1.5 and ICP22BA proteins are unable to localize to these structures, it can be concluded that the N-terminal 213 residues of ICP22 are important for localization to nuclear bodies. Second, we can state that the localization of ICP22 to nuclear bodies is not required for ICP22's ability to modify Pol II, as both the US1.5 and BA proteins do not localize to these structures yet are fully capable of altering Pol II.

Expression of L proteins in HEL cells requires sequences from both the N- and C-terminal halves of ICP22. Previous studies have shown that ICP22 is critical for the efficient expression of a subset of L genes in cells that are restrictive for the growth of ICP22 mutants (27, 39, 41, 50). These ICP22-dependent genes include US11, UL38, and UL41. To see which regions of ICP22 are involved in L-gene expression, we mock infected HEL cells or infected them with KOS1.1 or the various ICP22 mutants. At 8 hpi, total protein extracts were prepared and analyzed by immunoblotting using antibodies specific for various HSV-1 DE and L proteins (Fig. 7). For all strains analyzed, the DE protein ICP8 and the L proteins VP5, VP16, and VP22 were expressed at approximately equivalent levels. However, significant differences were seen in the expression of two L genes, gC and US11. KOS1.1, TF22, TF22BA, and TF22AP all expressed these proteins efficiently, although TF22, TF22BA, and TF22AP were consistently slightly less robust in gC/US11 expression compared to KOS1.1 (Fig. 7, compare lanes 4, 6, and 7 to lane 2; also data not shown.) This suggests that the FLAG tag may have a slight inhibitory effect on ICP22 function. However, in contrast to KOS1.1, TF22, TF22BA, and TF22AP, the mutants d22lacZ, TF1.5, and TF22PS all showed quite pronounced defects in gC and US11 expression. This was consistently observed in multiple repeat experiments. Thus, we conclude that both the N- and C-terminal segments of ICP22 are important for efficient viral L-gene expression in HEL cells.


Figure 7
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  		FIG. 7. Expression of DE/L proteins in ICP22 mutant-infected cells. HEL cells were mock infected or infected with the indicated viruses. At 8 hpi, protein extracts were prepared, and immunoblotting was carried out to detect the indicated viral DE/L proteins. Cellular protein EEA1 was analyzed as a loading control.


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DISCUSSION
 
Identification of sequences in ICP22 that are critical for Pol II modification. In this study, we used a genetic approach to determine which sequences in ICP22 are involved in modifying host cell Pol II. We find that residues 240 to 340 of the 420-residue ICP22 polypeptide are critical for Pol II alterations. In contrast, sequences N terminal to residue 240 are not required. The finding that the C-terminal half of ICP22 is critical for Pol II modifications is consistent with prior data obtained using the ICP22 mutant 22/n199, which encodes only the N-terminal 199 residues of ICP22 (31). This mutant is unable to induce Pol III (45), attesting to the importance of the C-terminal half of ICP22 in this function.

At this time, it is unclear whether residues C terminal to amino acid 340 play a role in the ability of ICP22 to modify Pol II. We attempted to address this question by engineering a plasmid that expresses an ICP22 polypeptide that is truncated at residue 340 due to the introduction of a stop codon. However, for unknown reasons, this construct and a similar truncated construct were expressed very poorly in transfected cells (data not shown). As a result, we did not investigate these mutants further. Additional work is now required to understand how the C-terminal sequences of ICP22 affect its expression in transfected and possibly infected cells and to determine whether they play a role in altering Pol II.

Our previous work indicated that ICP22 mediates two distinct changes to Pol II (12, 23). One change, which is UL13 independent, is the rapid loss of Pol II forms bearing Ser-2 phosphorylation. A second change, which is UL13 dependent, is the appearance of Pol III. If these two events are due to separable activities of ICP22, then it might be possible to isolate an ICP22 virus mutant that can mediate one Pol II alteration but not the other. Thus, it is notable that in our study we did not separate these two effects by mutation. That is, all mutants were either capable of inducing both effects on Pol II (TF1.5, TF22BA, and TF22BS) or incapable of inducing either (TF22PS). Although it is certainly feasible that further mutagenesis will succeed in separating these functions, another possibility is that the two effects of ICP22 on Pol II are due to a single activity that is modulated by UL13-dependent phosphorylation. Such a model has been proposed by Durand et al. and Purves et al. to explain how UL13 regulates ICP22-dependent viral L-gene expression (9, 41).

The biochemical mechanism by which ICP22 affects Pol II is currently unknown. However, it is certainly intriguing that ICP22 is reported to physically interact with cdk9 (9), the principal CTD kinase phosphorylating Ser-2 (35, 60). It is possible that ICP22's interaction with cdk9 inhibits or alters its CTD kinase activity, resulting in a loss of Ser-2P Pol II and/or induction of Pol III. If so, our results predict that the cdk9-binding region of ICP22 resides in its C-terminal half.

Schwyzer et al. (49) have pointed out a core sequence in ICP22, corresponding to residues 161 to 292, that is conserved in all known members of the subfamily Alphaherpesvirinae. The PS deletion, which abrogates ICP22's Pol II-modifying function, impinges on this sequence (Fig. 8A). This suggests that core sequence could be involved in the effects of Pol II. However, the AP deletion also impinges on this sequence but does not affect ICP22's ability to induce Pol II modification. Recently, we have noted that there is an additional conserved sequence in ICP22, corresponding to residues 295 to 420 (Fig. 8A and B). This sequence is conserved among all known members of the genus Simplexvirus (one of the of the four genera of the Alphaherpesvirinae), but it is not conserved across genera. We hypothesize that this sequence is important for the Pol II-modifying function of ICP22. If so, then this regulatory function may be expressed only by the members of the Simplexvirus genus, which so far consists mostly of primate viruses associated with urogenital and neuronal infections (57). This hypothesis is consistent with our previous finding that the ICP22 homolog of varicella-zoster virus (a member of the genus Varicellovirus) does not trigger Ser-2P Pol II loss in a transient-transfection assay (12). Further mutagenesis of both conserved sequences will be needed to define their relative contributions to ICP22's effects on Pol II.


Figure 8
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  		FIG. 8. Conserved sequences in HSV-1 ICP22. (A) The bar represents the 420-residue ICP22 polypeptide. The region conserved among members of the subfamily Alphaherpesvirinae is indicated, as is as a C-terminal sequence conserved only in members of the genus Simplexvirus. The sequence deleted in the PS mutant is shown at the top. (B) Alignment of C-terminal sequence conserved in simplexviruses. The C-terminal sequences of ICP22 and four homologs from other simplexviruses (cercopithecine virus 2 [CeHV2] [simian agent 8], cercopithecine virus 16 [CeHV-16] [herpesvirus papio 2], cercopithecine virus 1 [CeHV-1] [monkey B virus], and herpes simplex virus type 2 [HSV-2]) were aligned using Clustalw (http://www.ebi.ac.uk/). Single and double dots indicate weakly and strongly conserved residues, respectively; asterisks indicate identical residues. Gaps introduced to maximize alignment are indicated by dashes.

Both N- and C-terminal sequences of ICP22 are required for efficient L-gene expression and growth in HEL cells. ICP22 mutants exhibit a cell type-dependent replication defect that correlates with an inability to efficiently express several L proteins (27, 39, 50). We used our mutants to map the L-gene expression function of ICP22. The results indicate that both the N- and C-terminal regions of ICP22 are required for efficient L-gene expression in HEL cells, as both TF1.5 and TF22PS show very similar defects in growth and expression of L proteins US11 and gC (Fig. 7). Although it was previously reported that gC expression is independent of ICP22 (42), we have reproducibly found that this L protein is ICP22 dependent in HEL cells (data not shown). This is consistent with a prior study that showed that gC gene transcription is reduced in ICP22 mutant-infected HEL cells (45), as well as with work demonstrating that ICP22 mutant virions contain reduced levels of gC (31).

Our conclusion that sequences from both the N- and C-terminal halves of ICP22 are required for late viral gene expression is consistent with the work of Sears et al. (50), who found a defect in viral L-gene expression in cells infected with ICP22 mutant R325-βTK+, which encodes only the N-terminal 199 residues of ICP22. On the other hand, our work is in apparent conflict with the results of another study. We found that TF1.5, which expresses residues 147 to 420 of ICP22, is defective for viral L-gene expression in HEL cells. In contrast, Ogle and Roizman generated and analyzed a similar mutant, R7805, and concluded that residues 1 to 146 of ICP22 are not required for L-gene expression in rabbit skin cells, which like HEL cells are restrictive for ICP22 mutants (29). At present, we do not understand the basis for the differing results, although it is possible that it reflects the different cells used in the two studies. We also found that TF1.5 is growth defective in HEL cells, a result that is consistent with its defect in viral L-gene expression. Thus, our studies indicate that both N- and C-terminal sequences of ICP22 play important roles in allowing HSV-1 to replicate efficiently in HEL cells.

The mechanism by which ICP22 enhances viral gene expression is not yet known. At least two models have been put forth. First, it has been proposed that ICP22 enhances L-gene expression by activating the cell cycle regulator cdc2, which helps to recruit topoisomerase II{alpha} to viral genomes to enable optimal expression of L genes (2). Second, we have proposed that ICP22-dependent Pol II changes could enhance expression of HSV-1 DE/L genes if such genes have different requirements for CTD phosphorylation relative to cellular genes (45). Our present study is relevant to the second model. The key finding is that sequences from both the N- and C-terminal halves of ICP22 are required for L-gene expression in HEL cells, whereas only the C-terminal region is required for modification of Pol II. Thus, we can conclude that Pol II modification alone is insufficient for optimal L-gene expression in HEL cells. Furthermore, it is conceivable given our results that Pol II modification does not play a role in L-gene expression but rather has evolved to play another role in HSV-1 biology (see below). However, at present, our results do not rule out an effect of Pol II modification on viral L-gene expression. Further work will be required to resolve this important question.

Localization of ICP22 to nuclear bodies. An interesting but poorly understood aspect of ICP22 is its ability to localize to small nuclear bodies. Work by Jahedi et al. and Leopardi et al. (18, 22) in Roizman's laboratory originally showed that ICP22 localizes to these bodies early in infection, then moves to a more diffuse distribution in viral replication compartments as infection progresses. This movement was shown to be UL13 dependent. Our results are broadly consistent with those findings in that we observe that ICP22's localization to nuclear bodies is more pronounced in transfected cells than infected cells, where UL13 is present. The nature of the ICP22-containing nuclear bodies is currently unknown, although their presence in transfected cells indicates that they are either preexisting cellular structures or are induced de novo by ICP22. Recent work indicates that they do not correspond to PML domains, Cajal bodies, or SC35-containing nuclear speckles (7, 48), although they appear to have a paired spatial relationship with SC35 speckles (7, 48). Whatever their physical nature, our results demonstrate two important points concerning their relationship to ICP22. First, the N-terminal 213 residues of ICP22 are critical for its localization to the nuclear bodies. Second, ICP22 does not need to localize to these structures to induce its characteristic changes to Pol II.

Potential biological role of ICP22-induced Pol II modification. As discussed above, our results raise the possibility that modification of Pol II by ICP22 does not play a role in HSV-1 L-gene expression, although more work is required to address this point. If Pol II modifications are indeed not needed for efficient viral L-gene expression, what might be their role in HSV-1 biology? One possibility is that this activity has evolved to inhibit host genome transcription at an early stage of infection where rapid host responses are critical to achieve an effective antiviral defense. Even if the putative transcription inhibitory effect is only transient, it may be sufficient to provide a significant replication benefit to the virus. Inhibition of cellular gene transcription by ICP22 is consistent with its documented ability to bind the cdk9 subunit of P-TEFb (positive transcription elongation factor b), which is needed for transcription of most host genes (6). Transcription of the HSV-1 genome, on the other hand, may proceed efficiently without the need for P-TEFb and/or Ser-2 phosphorylation of Pol II, as is the case for a minority of cellular Pol II-transcribed genes (14, 25).

The function of ICP22-dependent Pol II modifications in HSV-1 biology may ultimately be revealed by studying the phenotype of a viral ICP22 mutant that is competent for L-gene expression but defective for Pol II modification, assuming that it is possible to isolate such a mutant. Our present study provides the groundwork for attempts to do so. If such a mutant can be obtained, it will be intriguing to see whether it is defective for in vitro replication or growth in experimental animals and whether it differs from the WT virus in its effects on host cell genome transcription.


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ACKNOWLEDGMENTS
 
We thank John Blaho, Jean-Jacques Diaz, Gill Elliott, Nancy Thompson, and Steve Weinheimer for generously supplying antibodies used in this study. We also acknowledge Katy Fraser for her helpful comments on the manuscript.

This research was supported by a grant to S.A.R. from the NIH (RO1-AI50127).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, University of Minnesota Medical School, Mayo Mail Code 196, 420 Delaware St. S.E., Minneapolis, MN 55455. Phone: (612) 626-4183. Fax: (612) 626-0623. E-mail: ricex019{at}umn.edu Back

{triangledown} Published ahead of print on 29 October 2008. Back


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Journal of Virology, January 2009, p. 128-139, Vol. 83, No. 1
0022-538X/09/$08.00+0     doi:10.1128/JVI.01954-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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