INTRODUCTION

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One of the earliest regulatory events after a productive infection by herpes simplex virus (HSV) is the induction of virus immediate-early (IE) gene expression by VP16, a structural component of the viral particle (33). Transcriptional activation is initiated by the recruitment of VP16 (3), together with two cellular proteins, Oct-1 (31, 34, 41) and HCF (15, 50, 52), into a multicomponent complex formed on the regulatory sites, TAATGARAT motifs, present within each of the IE promoters (for reviews, see references 30 and 48). VP16 also plays an essential role later in infection, in virion morphogenesis (45). Although its precise function(s) at this stage remains to be elucidated, VP16 has been shown to interact with at least two virus proteins, VP22 (7) and the virion host shutoff protein (20, 39), both of which are also assembled into the tegument.

With regard to its role in IE gene expression, previous results from several laboratories indicate that the first step in assembly of the multicomponent complex (called TRF.C in this work) is the formation of a binary complex between VP16 and HCF, which subsequently associates with Oct-1 already bound to the TAATGARAT motifs (8, 15, 46). While a precise mechanistic understanding of the role of HCF remains to be determined, several recent reports have helped elucidate its function in more detail. HCF is synthesized as a large, 2,035-residue precursor protein which is subsequently cleaved at specific reiterated sites located toward the middle of the protein, to yield a family of polypeptides (16, 50). After cleavage, the amino- and carboxyl-terminal portions of HCF remain stably bound together. We and others have shown recently that the region of HCF comprising the first 380 residues binds directly to VP16 (12, 19, 38, 49). This domain of HCF contains six reiterations of about 50 residues (the kelch repeats) and is thought to form a -propeller structure of linked -sheets (2, 6, 53). Residues predicted to be present in repeats 5 and 6 of the kelch domain have been shown to be important for VP16 binding and complex assembly (14).

A BHK-21-derived temperature-sensitive hamster cell line (tsBN67), which undergoes a G₀/G₁ cell cycle arrest at the nonpermissive temperature (29), was rescued by HCF (9). The endogenous HCF in the cell line contained a single amino acid substitution within the N-terminal kelch repeats, and this mutation was shown to be the basis of the block in cell cycle progression. However, the kelch repeat domain itself was not sufficient for rescue of the cell cycle phenotype, which required residues 1 to approximately 900 (49). In vitro analysis of tsBN67 cell extracts has shown that at the nonpermissive temperature, while the stability and processing of HCF were not affected by the mutation, TRF.C formation and VP16 transcriptional activity were abrogated (9). These results indicate that VP16 may interact with HCF determinants which are involved in its cell cycle function.

Although the importance of VP16 in stimulating IE gene expression during HSV type 1 (HSV-1) infection has been previously established from the analysis of viruses encoding mutant VP16 proteins (1, 32, 40), parallel studies of virus replication in cells deficient in cellular components of the VP16 pathway have not been performed. We therefore wished to examine the role and the fate of HCF during HSV infection. In this report, we show that the subcellular distribution of HCF is profoundly altered upon HSV infection and that the protein colocalizes with a proportion of newly synthesized VP16. We also examined the role of HCF in VP16 transcriptional activity by infecting tsBN67 cells at the nonpermissive temperature and showed that VP16-HCF binding was abrogated and that viral gene expression and viral replication were significantly reduced compared to infection of parental BHK-21 cells. In addition, we rescued the tsBN67 proliferation defect by establishing a stable cell line expressing simian virus 5 (SV5)-tagged wild-type (wt) HCF (tsBN67r). Infection of tsBN67r cells at the nonpermissive temperature resulted in the binding of newly synthesized VP16 to exogenous HCF but only partly rescued virus protein synthesis and replication.

	MATERIALS AND METHODS

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Virus and cells. The BHK-21-derived temperature-sensitive cell line tsBN67 (29) was obtained from RIKEN Gene Bank (Tsukuba, Japan). Vero, wt BHK-21, and tsBN67 cells were maintained in Dulbecco's modified minimal essential medium supplemented with 10% newborn calf serum. Vero and BHK-21 cells were propagated at 37°C, whereas tsBN67 cells were propagated at 33.5°C (permissive temperature). Previous results have demonstrated that tsBN67 cells undergo a reversible G₀/G₁ block after incubation at 39.5°C (nonpermissive temperature). The rescued cell line (tsBN67r) was maintained at 39.5°C in the medium described below, supplemented with G418 (0.8 mg per ml). Virus infections were carried out with HSV-1 strain 17. Vero cells were infected at a multiplicity of infection (MOI) of 10 PFU/cell. BHK-21 and its derivatives tsBN67 and tsBN67r were seeded 2 days prior to infection (4 × 10⁵ cells/dish) at the assay temperature (33.5 or 39.5°C). Separate cultures of each cell types were counted immediately prior to infection to ensure identical MOIs. Infected cells were harvested at the indicated times postinfection (p.i.), and samples were processed for either immunoprecipitation, Western blotting, or immunofluorescence. Total viral progeny was assayed by plaque assay on Vero cells.

Immunoprecipitations. Mock-infected or infected cells were washed in cold phosphate-buffered saline, and high-salt extracts were prepared as previously described (11, 51). VP16 was immunoprecipitated with the anti-VP16 monoclonal antibody (MAb) LP1 (1:100) in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1% NP-40. Immune complexes were recovered by adding protein A-Sepharose. Beads were washed five times in the incubation buffer and then lysed in 2× sodium dodecyl sulfate (SDS) loading buffer.

SDS-PAGE and Western blotting. Mock-infected or infected cells were washed in cold phosphate-buffered saline, and total lysates were prepared by adding 250 µl of 1× SDS loading buffer. Samples were briefly sonicated prior to electrophoresis. Equal amounts of total proteins were fractionated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto Hybond-C membranes. Primary antibodies for immunodetection were diluted as follow: anti-IE110k (MAb 11060, 1:10,000), anti-IE175k (MAb 10176, 1:5,000), anti-HSV thymidine kinase (1:2,000), anti-VP16 (MAb LP1 [1:4,000] or polyclonal rabbit antibody POS1 [1:2,000]), anti-VP22 (polyclonal rabbit antibody AGV30, 1:50,000), antiactin (MAb AC-40, 1:5,000; Sigma), and anti-HCF (rabbit polyclonal antibody, 1:10,000). Reactive proteins were visualized by enhanced chemiluminescence (Pierce).

Immunofluorescence. Cells grown on coverslips were mock infected or infected with HSV at an MOI of 10 PFU/cell and were fixed in 100% methanol for 15 min at the indicated times p.i. Immunofluorescence reactions were carried out as described previously (7). Primary antibodies were diluted as follows: LP1, 1:400; anti-HCF, 1:200; anti-RNA polymerase II (Pol II) reacting against the carboxy-terminal tail, 1:200; and anti-SV5, a mouse MAb against a short peptide region of SV5 M protein used as an epitope tag in HCF constructs, 1:2,000. Secondary antibodies were fluorescein-conjugated anti-mouse immunoglobulin G (1:100) and tetramethyl rhodamine isocyanate-conjugated anti-rabbit immunoglobulin G (1:200). Cells were examined in a Zeiss LSM410 or Bio-Rad MRC600 confocal microscope with an ×40 or ×63 objective lens. Images were collected by scanning each channel separately, and control assays demonstrated no significant cross-channel leak-over. After collection, the images were processed and annotated with Adobe Photoshop software.

Rescue of the tsBN67 temperature-sensitive defect. A stable cell line expressing SV5-tagged HCF was constructed as follow. tsBN67 cells grown for 2 days at 39.5°C were transfected with 2 µg of pSL25, a plasmid expressing SV5-tagged full-length HCF (18), together with 2 µg of pSV2Neo, a plasmid expressing the selectable marker for neomycin resistance. Cells were transfected by the calcium phosphate method modified by the inclusion of N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid-buffered saline as previously described (10). Two days after transfection, cells were washed and incubated in the presence of G418 (0.8 mg/ml) for 2 weeks. Individual colonies were selected and examined for expression of exogenous HCF by Western immunoblotting and immunofluorescence using the anti-SV5 and anti-HCF antibodies. A suitable clone (tsBN67r) was selected and expanded for analysis.

	RESULTS

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The intracellular distribution of HCF is altered in HSV-infected cells. To determine the effect of HSV infection on HCF localization, a time course of infection was carried out with Vero cells. Cells were mock infected or infected at an MOI of 10, fixed at various times p.i., and stained for HCF and VP16 (Fig. 1). In mock-infected cells (Fig. 1, Mock), HCF exhibited a diffuse nuclear pattern with the exclusion of nucleoli, in agreement with previous results for other cell types (16, 18). At 1 h p.i. we observed subtle but distinct changes in HCF localization, with the appearance of speckled foci, which became more prominent by 4 h p.i. (Fig. 1, 4 h, arrows). By 6 h p.i. significant reorganization of HCF distribution was readily apparent (Fig. 1, 6 h, arrow, left panel), with a substantial amount of the protein accumulating in large intranuclear domains reminiscent of HSV replication compartments. Finally, at later times of infection (8 to 12 h), HCF localization progressed to nuclear honeycomb-like structures, with a minor but reproducible component in the cytoplasm (Fig. 1, 8 to 12 h, arrows). The very early alterations in HCF localization after infection are illustrated in a separate experiment with higher-magnification images which show more clearly the appearance of the protein in speckled foci between 1 and 2 h p.i., small bright foci by 4 h p.i., and the start of the coalescence into larger globular domains at 6 h p.i. (Fig. 2a).

View larger version (46K): [in this window] [in a new window]   FIG. 1.   Intracellular compartmentalization of HCF and VP16 in HSV-1-infected Vero cells. Vero cells were mock infected (Mock) or infected with HSV-1 (strain 17) at an MOI of 10 and fixed at the various times indicated. Double immunofluorescence was carried out with anti-HCF and anti-VP16 (LP1) antibodies. The panel for each time point shows the pattern of detection of HCF or VP16 in the same field of cells. VP16 was first detected between 4 and 6 h p.i., while concentration of HCF into speckled foci could be detected by 2 h p.i. The later nuclear colocalization of HCF and VP16 is readily apparent.

View larger version (38K): [in this window] [in a new window]   FIG. 2.   HCF reorganization and colocalization with RNA Pol II. (a) Vero cells were mock infected (Mock) or infected with HSV-1 (strain 17) at an MOI of 10 and fixed at the various times indicated. Cells were stained with anti-HCF antibody alone and analyzed by confocal microscopy using an ×63 objective combined with an ×2.5 digital zoom. (b) Mock-infected or HSV-infected cells (6 h p.i.) were double labeled for RNA Pol II and HCF. For the mock-infected cells, only RNA Pol II is shown; for the infected cells, the panels show the same field of cells stained for RNA Pol II or HCF, with clear colocalization of the two proteins in globular or coalesced foci (arrows).

HCF interacts with VP16 in HSV-infected cells. Biochemical assays have indicated that VP16 interacts with HCF in the absence of the other components of TRF.C, but to date there are no reports which specifically address HCF interaction with VP16 during HSV infection. Since we observed that HCF colocalizes with VP16, we next wished to determine whether the two proteins directly interact with one another during infection. Vero cells were mock infected or infected with HSV-1 at an MOI of 10 PFU/cell, and soluble extracts were prepared at 3, 6, 9, 12, and 16 h p.i. (Fig. 3). Samples were first analyzed directly by Western blotting to evaluate the relative amounts of proteins present in each extract (Fig. 3a). Reaction with an antiactin antibody demonstrated that approximately equivalent protein levels were loaded for each sample (Fig. 3a, actin). VP16 was first detected at 6 h p.i., increasing in abundance as infection progressed (Fig. 3a, VP16). Analysis of HCF with a polyclonal antibody raised against the C terminus of the protein showed that its steady-state level remained relatively constant throughout the course of infection. Note that the slight change in ratio of the different C-terminal species seen at 16 h p.i. was not significant and was not reproducibly observed (see, e.g., Fig. 4). The samples were next immunoprecipitated with a MAb against VP16, and VP16 was then visualized with a rabbit polyclonal antibody (Fig. 3b, VP16). Coprecipitated HCF was detected by Western blotting of the immunoprecipitates with the anti-HCF antibody (Fig. 3b, HCF). While no HCF was observed from the mock- or early-infected samples, as infection progressed HCF was coprecipitated in increasing amounts, corresponding to the amounts of precipitated VP16. While it is reasonable to expect from previous biochemical analyses that HCF interacts with VP16 immediately after infection, the amounts of VP16 present at that time were below our levels of detection in either total or immunoprecipitated extracts (Fig. 3). More sensitive assays will have to be developed to examine the HCF-VP16 interaction immediately after infection. Nevertheless, these results demonstrate that HCF associates with newly synthesized VP16 in HSV-1-infected cells, and it is likely that the interaction occurs in replication compartments where the two proteins accumulate throughout infection.

View larger version (53K): [in this window] [in a new window]   FIG. 3.   Direct interaction between HCF and VP16 in HSV-1-infected Vero cells. Vero cells were mock infected (lane M) or infected with HSV-1 at an MOI of 10, and soluble cell extracts were prepared at 3, 6, 9, 12, and 16 h p.i. VP16 was immunoprecipitated with LP1 (1:100), immune complexes were resolved in an SDS-10% polyacrylamide gel, and proteins were transferred onto a Hybond-C membrane. HCF and VP16 products were detected with anti-HCF and POS1 antibodies, respectively, before (a) and after (b) immunoprecipitation (I.P.). An antiactin antibody was used to ensure that similar amounts of proteins were present in each extract prior to immunoprecipitation.

Reduced association of VP16 with HCF in infected tsBN67 cells. In transfection assays, VP16 fails to activate IE transcription in tsBN67 cells at the nonpermissive temperature, with the defect being attributed to reduced TRF.C formation (9, 49). We wished to examine the consequences of the defect in HCF on VP16 function in virus-infected cells and specifically to determine if VP16 binds to HCF in infected tsBN67 cells at the nonpermissive temperature. Parental BHK-21 and tsBN67 cells grown for 2 days at 33.5 and 39.5°C were infected at an MOI of 10 PFU/cell or were mock infected, and soluble cell extracts were prepared at 16 h p.i. (Fig. 4). As before, controls with the antiactin antibody confirmed that similar levels of proteins were present in all cell extracts (data not shown). Levels of HCF and VP16 were first assessed prior to immunoprecipitation (Fig. 4a). HCF was present in similar amounts in mock-infected and infected cells, both BHK-21 and tsBN67, with little difference in relative abundance of individual species (Fig. 4a, HCF). In infected cells, VP16 was expressed at similar levels in each of the cell types; in particular for tsBN67, there was little reduction in expression at the elevated temperature (Fig. 4a, VP16).

View larger version (52K): [in this window] [in a new window]   FIG. 4.   Interaction between HCF and VP16 in HSV-1-infected BHK-21 and tsBN67 cells. BHK-21 and tsBN67 cells grown for 2 days at 33.5 or 39.5°C were mock infected (lanes M) or infected (lanes I) at an MOI of 10. Soluble extracts were prepared 16 h p.i., and VP16 was immunoprecipitated with LP1. Detection of the input and immunoprecipitated proteins was as for Fig. 3.

HSV protein synthesis is severely impaired in tsBN67-infected cells at the nonpermissive temperature. Previous analyses have shown that the effects of mutations in certain viral proteins, including VP16 (1), may be more distinct at low MOIs. Therefore, it was possible that any consequences of reduced binding of HCF to VP16 in the tsBN67 cells would be manifest at low MOI. To address this question, BHK-21 and tsBN67 cells were grown for 2 days at 33.5 or 39.5°C and then infected at high (10 PFU/cell) and low (0.1 PFU/cell) MOIs. Total cell lysates prepared at various times p.i. were analyzed by Western blotting using a variety of antibodies to different virus-encoded proteins (Fig. 5). We first analyzed the appearance of an IE protein, IE110k (ICP0), at the high MOI (Fig. 5a). At both temperatures, IE110k appeared slightly earlier in BHK-21 cells than in tsBN67-infected cells. (Note that despite its optimal rates of synthesis at IE times, accumulation of IE110k to maximal amounts occurs late in infection.) There was, however, no significant difference in the expression of IE110k in tsBN67 cells infected at the permissive compared to the nonpermissive temperature. Similar results were obtained for the other virus proteins (data not shown). This result suggests that at a MOI, viral gene expression proceeds normally despite the defect in HCF.

View larger version (61K): [in this window] [in a new window]   FIG. 5.   HSV-1 protein synthesis in infected BHK-21 and tsBN67 cells. (a) BHK-21 and tsBN67 cells grown for 2 days at 33.5 or 39.5°C were infected with HSV-1 at an MOI of 10. Total cell lysates were prepared at 0 (lanes M), 2, 4, 6, 10, 14, 18, and 24 h p.i. Proteins were fractionated in an SDS-10% polyacrylamide gel and transferred onto a Hybond-C membrane, and IE110k was detected with a MAb. (b) BHK-21 and tsBN67 cells were infected with 0.1 PFU/cell, and total cell lysates were prepared at 0 (lanes M), 4, 8, 12, 18, 24, 32, and 48 h p.i. Proteins were fractionated in an SDS-10% polyacrylamide gel, transferred onto a Hybond-C membrane, and reacted with antibodies directed against IE (IE110k and IE175k), early (thymidine kinase [tk]), and late (VP16 and VP22) viral proteins, HCF, and actin, which served as a control for the relative amounts of proteins present in each sample. Note that threefold more lysates were used for the detection of IE175k.

Intracellular localization of HCF and VP16 in tsBN67-infected cells. The biochemical analysis by coprecipitation indicated that at both MOIs, VP16 binding to HCF was defective in tsBN67 cells at the nonpermissive temperature. We next wished to investigate whether this defect would affect the intracellular localization of HCF and VP16 during HSV infection. BHK-21 and tsBN67 cells were mock infected or infected at 39.5°C with an MOI of 10 PFU/cell. Cells were fixed at 6 h p.i. and stained with the anti-HCF antibody (Fig. 6a). In mock-infected BHK-21 cells, HCF exhibited a diffuse nuclear localization pattern (Fig. 6a1) identical to that observed in Vero cells (see above). In tsBN67 cells however, while the distribution pattern of the protein was predominantly nuclear, somewhat more cytoplasmic HCF was also detected (Fig. 6a2). A similar pattern was observed at the permissive temperature (data not shown). In infected cells, however, HCF was relocalized in both BHK-21 and tsBN67 cells (Fig. 6a3 and -4), with patterns again very similar to those observed in infected Vero cells. Therefore, despite the defective association between HCF and VP16 observed in coprecipitation assays, the protein was still relocalized into replication compartments during HSV infection.

View larger version (45K): [in this window] [in a new window]   FIG. 6.   Intracellular distribution of HCF and VP16 in BHK-21 and tsBN67 cells infected at 39.5°C. BHK-21 and tsBN67 cells grown for 2 days at 39.5°C were mock infected or infected with HSV-1 at an MOI of 10 and fixed 6 h later. Indirect immunofluorescence was carried out with the anti-HCF polyclonal antibody (a) or LP1, the anti-VP16 MAb (b).

Rescue of the tsBN67 cell defect by HCF restores VP16 interaction. To confirm that the decrease of viral protein synthesis and replication in tsBN67 cells at the nonpermissive temperature was caused by the defect in HCF, we rescued the temperature-sensitive phenotype by establishing a stable cell line expressing full-length wild-type HCF. The tsBN67 cells were transfected with a vector expressing SV5-tagged HCF (18), individual colonies were selected for growth at the nonpermissive temperature, and a suitable clone (tsBN67r) was chosen for further experiments. Growth at 39.5°C was restored in these cells, with a doubling time similar to that of wild-type BHK cells (results not shown). Expression of exogenous HCF was confirmed by Western blotting (data not shown) and by immunofluorescence analyses using the anti-SV5 and anti-HCF antibodies (Fig. 7a). As expected, staining of tsBN67 and tsBN67r cells with the anti-HCF antibody revealed that HCF was present in higher amount in the nuclei of tsBN67r cells (compare Fig. 8a2 and -4). Using the anti-SV5 antibody to detect the N terminus of the introduced wt HCF, we observed that while HCF was also largely nuclear, there was also some cytoplasmic staining (Fig. 8a3). In virus-infected cells, the SV5-tagged HCF was also relocalized into nuclear replication compartments (data not shown). Although this result was not unexpected, it confirms the relocalization that was observed in the Vero and tsBN67 cells with an independent antibody and removes the possibility that this may have been due to the anti-HCF antibody cross-reacting with virus components in the replication compartments.

View larger version (41K): [in this window] [in a new window]   FIG. 7.   Characterization of a stable tsBN67 cell line expressing SV5-tagged HCF. (a) Parental tsBN67 cells (top) and cells rescued with the SV5-tagged wt HCF (tsBN67r cells; bottom) were grown on coverslips at 39.5°C for 2 days before fixation. Indirect immunofluorescence was performed with either the anti-SV5 MAb (panels 1 and 3) or the anti-HCF polyclonal antibody (panels 2 and 4). (b) tsBN67 or tsBN67r cells grown for 2 days at 33.5°C or 39.5°C were mock infected (lanes M) or infected (lanes I) with HSV at an MOI of 10. Soluble extracts were prepared at 16 h p.i., and VP16 was immunoprecipitated (I.P.) with LP1 and detected by Western blotting of the immunoprecipitates with POS1. Coprecipitated HCF was detected with the anti-HCF antibody. The increased amount of HCF in the rescued line is demonstrated in the top panel, showing a Western blot of the total amount of HCF in each sample prior to the immunoprecipitation (input). The difference in efficiency of HCF coprecipitation at 39.5°C between the two lines can be seen by comparing the longer (for tsBN67 cells; see also Fig. 4) and shorter (tsBN67r cells) exposures (exp.). An antiactin antibody was used as a control to assess the relative amounts of proteins present in each extract.

HSV protein synthesis and replication in tsBN67r cells. We next wished to determine whether expression of exogenous wt HCF would reduce the delay in virus protein synthesis and replication observed in tsBN67 cells at the nonpermissive temperature. In parallel, tsBN67 and tsBN67r cells were infected (MOI of 0.1 PFU/cell) at both temperatures, and total cell lysates were prepared at 10, 24, and 48 h p.i. Samples were analyzed for levels of IE110k, VP22, VP16, and actin, which again served as a control for the total protein load in each sample (Fig. 8). At 33.5°C, similar amounts of viral proteins were synthesized in both cell lines (compare lanes 3 and 4 with lanes 7 and 8), which could be expected since HCF is functional in the tsBN67 cells at this temperature. But it is of note that the higher levels of HCF in the rescued tsBN67r cells did not accelerate the progression of the HSV replicative cycle. At 39.5°C, IE110k, VP22, and VP16 were only marginally more abundant in tsBN67r cells than in tsBN67 cells (compare lanes 11 and 12 with lanes 15 and 16). Although this effect was modest, viral protein synthesis was detected earlier in tsBN67r cells. Nevertheless, perhaps the most salient feature of the results is that in both cell lines, and in contrast to what was found for the parental BHK-21 cells (Fig. 5b), lower amounts of viral proteins were detected at 39.5°C than at 33.5°C (compare lanes 3 and 4 with 11 and 12 and lanes 7 and 8 with 15 and 16). Even at later times of infection at 39.5°C, virus protein levels never accumulated to those observed at 33.5°C. Therefore, the delay in viral protein synthesis at 39.5°C is only partly rescued in tsBN67r cells, and generally levels of protein synthesis remained lower than those observed at 33.5°C or in BHK-21 cells infected 39.5°C.

View larger version (42K): [in this window] [in a new window]   FIG. 8.   HSV-1 protein synthesis in infected tsBN67 and tsBN67r cells. tsBN67 and tsBN67r cells were grown for 2 days at 33.5 or 39.5°C exactly and then mock infected or infected with HSV at an MOI of 0.1; protein synthesis analyzed as for Fig. 5.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

HCF and VP16 interaction and compartmentalization in infected cells. While biochemical aspects of mechanism of transcriptional activation by VP16 are understood in some detail, there are few reports on the interactions between VP16 and its associated cellular factors in vivo in virus-infected cells. In this study, we analyzed HCF compartmentalization during HSV infection in wt cells and in cells containing a mutant HCF defective in VP16 binding. We show that HCF undergoes a subtle but distinct recruitment into small speckled foci within 1 to 2 h p.i., and a pronounced recruitment into concentrated and merged large globular aggregates was observed beginning at 6 to 8 h p.i. It would be reasonable to predict that the speckled foci of HCF which are seen very early after infection may represent sites of VP16 recruitment and activity. However, using a variety of antibodies and conditions for immunofluorescence, we have been unable to follow the fate of input virion-associated VP16 immediately after infection.

Importance of HCF in stimulating the HSV lytic cycle. At low MOIs in the temperature-sensitive cell line encoding a mutant HCF, HSV-1 protein synthesis and replication were significantly reduced at the nonpermissive temperature. Our results are consistent with previous studies using HSV strains encoding mutant VP16 proteins which are defective in TRF.C formation or transcriptional activation (1, 40). However, previous reports have indicated that HSV replicates more efficiently in cycling than in growth-arrested temperature-sensitive cell lines (43, 54), and this could be an additional contributory factor in the reduced virus expression in the tsBN67 cells at 39.5°C. However, in the cell line rescued for HCF function in cell cycle progression, only partial rescue of HSV replication was observed. At low MOI, virus protein synthesis was only marginally higher in the rescued cells, and the ratio of virus yield at 39.5°C to that at 33.5°C was not substantially different from the parental temperature-sensitive line. While these results indicate that the reduced protein synthesis and replication were not simply due to defective cell cycle progression, they also prompt the question as to why the introduction of HCF did not restore virus replication to that observed in wt BHK cells. HCF may have additional roles in virus replication, but if anything the rescued line expressed greater amounts of HCF, and in any case (unless this was positively detrimental to replication) all relevant functions of HCF would have been expected to be restored. Perhaps one explanation could be that since the tsBN67 cells were generated by broad chemical mutagenesis, there was disruption of additional cellular functions which while not involved in the cell cycle block were nevertheless important for normal levels of virus replication. These considerations indicate that it is formally difficult to attribute the defect in virus expression to a defect in HCF function and illustrate the complexities in attempting to provide supporting genetic evidence for the biochemical data implicating HCF in VP16 activity and virus replication. However, recent biochemical results on the selective assembly of HCF into the VP16 complex, together with studies of HCF compartmentalization indicating that HCF is cytoplasmic in neurons but translocates to the nucleus following stimuli which reactivate latent HSV infections, indicate that the protein plays an important role in virus replication (14, 17). Our results on HCF colocalization in replication compartments, independently of direct VP16 binding, indicate that the protein may have as yet unidentified roles in HSV replication. Studies are now under way to relate the fate of input VP16 to early HCF localization and to examine late HCF compartmentalization with respect to possible subcompartments for replication or assembly.