INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

The alphaherpesvirus equine herpesvirus 1 (EHV-1) is the major cause of virus-induced abortion in horses. Additionally, the agent causes respiratory and neurological symptoms (1, 13, 18). Of the more than 76 proteins encoded by EHV-1, most share extensive homology with the prototype member of the virus subfamily, herpes simplex virus type 1 (HSV-1) (30). Among the open reading frames (ORFs) that are not present in HSV-1, the IR6 gene (gene 67) and gene product have been identified (2, 17, 29). Structural homologs of the EHV-1 IR6 protein have been described on the basis of nucleotide sequence analyses in equine herpesvirus 4 (EHV-4), bovine herpesvirus 1 (BHV-1), and canine herpesvirus (CHV) (11, 14, 26, 31). The EHV-1 IR6 gene is present as a diploid gene in both inverted repeat regions in wild-type EHV-1 strains, and its protein product has been shown to form filamentous rod-like structures that localize primarily to the soluble fraction of the cytoplasm in infected cells. In addition, the IR6 protein forms a meshwork surrounding the nuclei of infected cells starting at 6 h postinfection (p.i.), is found in the nuclei of infected cells, and is incorporated into viral nucleocapsids (3, 17, 19). Analysis of EHV-1 viruses that express a mutated IR6 protein has demonstrated that the structure of the IR6 protein is important for its function (19, 20). A viral mutant that is devoid of both copies of the IR6 gene, EHV-1 strain RacH, is apathogenic for the natural host and for laboratory animals. Upon insertion of the IR6 gene, however, the generated IR6 recombinant RacH virus (HIR6-1) was as virulent as the wild-type RacL11 virus (8, 12, 20). Moreover, the temperature-sensitive phenotype of the IR6-negative RacH and the Rac plaque isolates expressing a mutated IR6 protein (RacM24 and RacM36) was restored by the insertion of one copy of the wild-type IR6 gene into the RacH virus (20).

Despite the intensive phenotypical characterization of individual strains expressing various forms of the IR6 protein, the function of the protein remained enigmatic. The observed aggregation of the IR6 protein to the rod-like structures led to the hypothesis that it could interact with cellular proteins that form the cytoskeleton (17). However, no association of the IR6 protein with the investigated proteins actin, tubulin, vimentin, dynein, kinesin, and desmin could be shown (17, 19, 29). To date, the nuclear lamins which represent members of the intermediate filament family have not been analyzed for a putative aggregation with the IR6 protein, although they are expressed in all eukaryotic cells. In vertebrate somatic cells, two major types of nuclear lamins (type B1-B2 and type A/C) can be distinguished, although they are structurally and functionally homologous and may have arisen from the same ancestral gene (reviewed in reference 6). The lamins are located on the nucleoplasmic side of the inner nuclear membrane, are associated with chromatin, and form a meshwork which determines the nuclear architecture. In addition, nuclear lamins play an important role during mitosis and are phosphorylated in the M phase of the cell cycle by the cdc2 protein kinase (25). During mitosis, B-type lamins remain associated with the nuclear membrane, whereas A-type lamins disintegrate and are present as soluble oligomers (16).

This study addresses the functional characterization of the EHV-1 IR6 gene product and its colocalization with members of the intermediate filament family, the nuclear lamins. To this end, transient transfection assays of IR6 genes, confocal laser scanning microscopy, coimmunoprecipitation analyses, and electron microscopy were performed using different EHV-1 strains which express wild-type or mutated forms of the IR6 protein. It is shown that the wild-type IR6 protein is transported from cell to cell independently of viral infection, colocalizes with nuclear lamins late in infection, and encodes a function that facilitates the egress of viral nucleocapsids.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Cells and viruses. The equine dermal cell line Edmin337 (8) as well as Rk₁₃ and EHV-1 gB-expressing TCgBf cells (15) were maintained in Dulbecco's minimal essential medium (DMEM) supplemented with 10% fetal calf serum (FCS). EHV-1 strains RacL11, RacM24, and RacH, as well as the IR6-recombinant RacH virus (HIR6-1), were grown and titrated as previously described (19, 20). The gB-negative RacL11 mutant (L11gB) that expresses Escherichia coli -galactosidase instead of the gB ORF and that is deficient in cell-to-cell spread of infectivity was propagated on complementing TCgBf cells (15) before infection of noncomplementing cells. For detection of -galactosidase activity in L11gB-infected cells, cells were fixed with acetone or methanol and stained with -galactosidase staining solution [final concentrations: 300 µg of Bluo-Gal (Gibco-BRL)/ml, 5 mM K₄Fe(CN)₆, 5 mM K₃Fe(CN)₆, 2 mM MgCl₂, 0.1% Triton X-100 in phosphate-buffered saline (PBS)]. A diagram of the genotypes of the different EHV-1 used in this study is presented in Fig. 1.

View larger version (29K): [in this window] [in a new window]   FIG. 1.   Schematic diagram of the genotypes of the viruses used in this study (15, 19, 20). Shown are the BamHI map of EHV-1 strain RacL11 and the magnifications of the US and inverted repeat regions of strains RacL11, RacM24, RacH, and the HIR6-1 virus. The HIR6 virus is a recombinant RacH virus into which one copy of the wild-type IR6 virus was inserted (20). Also shown is the organization of the gB-negative EHV-1 mutant L11gB (15), which is deficient in direct cell-to-cell-spread (ctcs) but expresses the wild-type IR6 protein. BamHI restriction fragment designations are given, and wild-type (wt) as well as mutated (m) IR6 genes (amino acid 134: LeuPro) are indicated. Also shown is the location of the EUS1 gene (gene 68), the EHV-1 US2 homolog (30). The deletion of both copies of the IR6 gene in strain RacH is marked (). The scale is given in kilobase pairs (kb). The US region in the magnification is not drawn to scale.

Plasmids. The recombinant plasmids pDIR6L and pDIR6M24, which contain the RacL11 or the RacM24 IR6 ORF, respectively, under the control of the cytomegalovirus immediate-early promoter, have been described previously (19). The plasmid pC⁺ was constructed by inserting the E. coli lacZ gene released from vector pSV--Galactosidase (Gibco-BRL) into the vector pcDNAI/Amp (Invitrogen) and was used as the reporter plasmid in transient transfection experiments.

Electron microscopy. Rk₁₃ or Edmin337 cells were seeded in 75-cm² cell culture flasks and infected with the indicated viruses at a multiplicity of infection (MOI) of 5. At 4 or 16 h after infection, cells were fixed with 2% glutaraldehyde in PBS for 2 h and washed in PBS. Cells were postfixed with 2% OsO₄ in PBS, washed thoroughly with PBS, and embedded in 1% agarose. Cells were dehydrated stepwise in solutions containing 50 to 100% ethanol. After equilibration in propylene oxide, cells were embedded in Epon (Fluka) and polymerized for 2 to 4 days at 45 to 60°C. Sections of 50 to 80 nm were cut with a microtome, and the sections were counterstained with 2% phosphotungstic acid in PBS and analyzed with a Zeiss EM 10C/CR electron microscope (21).

Transient transfection assays. Transfections of Rk₁₃ cells were done by lipotransfection or calcium phosphate precipitation exactly as previously described (15), and 1 µg of the respective plasmids was transfected into 5 × 10⁵ cells seeded on coverslips. To prevent cell division in transiently transfected cells, cells were incubated after transfection in DMEM without addition of FCS. IR6 import assays were done exactly as described by Elliott and O'Hare in the case of HSV-1 VP22 by extraction with 0.4 M NaCl (4). Briefly, 1 × 10⁶ Rk₁₃ cells were transfected with 10 µg of pDIR6L or pDIR6M24, and cell-free lysates were prepared at 72 h after transfection and added to the medium of Rk₁₃ cells that had been seeded 24 h before. The lysate of approximately 3 × 10³ transfected cells was used to overlay 1 × 10⁴ freshly seeded Rk₁₃ cells. At different times after addition of the lysate, uptake of the IR6 protein into cells was determined by indirect immunofluorescence (IF) using the anti-IR6 antiserum (see below). Before inspection in a fluorescence microscope, nuclei were stained with propidium iodide (10⁶ M in PBS).

Antibodies. The anti-IR6 antibody (17) was used at a 1:1,000 dilution. Anti--galactosidase monoclonal antibodies (MAbs) (Boehringer) were purchased and used according to the manufacturer's instructions. Anti-lamin MAbs were kindly provided by Georg Krohne, University of Würzburg, Germany. The antibodies X67, X167, and R27 react with different epitopes of type A lamins, whereas MAb X223 is specific for B-type lamins (7). Each of the lamin antibodies was used at a dilution of 1:200 in Western blot and immunofluorescence analyses. Anti-EHV-1 gB MAb 4B6 and anti-gD polyclonal rabbit antibodies have been previously described (5, 15).

Immunoprecipitation and Western blotting. Rk₁₃ cells were infected at an MOI of 2 with RacL11, RacM24, RacH, or the IR6-recombinant virus HIR6-1. At 16 h after infection, cell lysates were prepared exactly as previously described (15). The cell lysates were adjusted to equal protein concentrations of 5 mg per ml as determined by the BCA kit (Pierce). A mixture of preimmune rabbit serum (10 µl) and the supernatant of an irrelevant hybridoma (500 µl) was added to 500 µl of cell lysate. The mixture was incubated for 1 h on ice, and 80 µl of a protein A-agarose slurry (Bio-Rad) was added for 1 h on ice. After centrifugation, the supernatant was diluted with NET buffer (20 mM Tris-Cl [pH 7.6]; 150 mM NaCl; 5 mM EDTA; 0.1% Nonidet P-40; 0.25% gelatin; 0.02% NaN₃) to 2.5 ml (27). Five hundred microliters of the suspension was mixed with 5 µl of the anti-IR6 antiserum or 5 µl of the anti-lamin A antibodies. The mixture was incubated on ice for 1 h, and protein-antibody complexes were precipitated with protein A-agarose as described above. After four washing steps in NET buffer, beads were suspended in protein sample buffer (27), and the samples were heated to 56°C for 2 min. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10). For Western blot analysis, proteins were transferred to nitrocellulose membranes (Biometra) by the semidry method (9). Free binding sites on the sheets were blocked by the addition of 10% nonfat dried milk in PBS containing 0.05% Tween (PBST) before the antibodies (suspended in PBST) were added. Bound antibodies were detected with anti-rabbit (or anti-mouse) immunoglobulin G (IgG) alkaline phosphatase conjugates (Sigma) and visualized with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Sigma).

Immunofluorescence and confocal laser scanning microscopy. For indirect IF analyses, transfected or infected cells were fixed with ice-cold acetone or methanol for 5 min at different times after transfection or infection. The cells were rehydrated with PBS for 10 min and incubated with anti-IR6 monospecific rabbit antiserum (17) for 30 min after a 45-min blocking period using 10% FCS diluted in PBS. In some experiments, MAbs against -galactosidase or EHV-1 gB were mixed with the anti-IR6 antibody. After thorough washing (twice for 10 min in PBS), an anti-rabbit IgG fluoroisothiocyanate (FITC) conjugate alone or together with an anti-mouse IgG TRITC conjugate (Sigma) was added for 30 min. For detection of nuclear lamins, coverslips were incubated with anti-IR6 and anti-lamin antibodies. After two washes, an anti-mouse IgG biotin conjugate or anti-mouse Cy3 conjugate (Jackson Laboratories) was added for 45 min. In the case of biotinylated antibodies, another two washes with PBS followed, and bound biotin was detected by streptavidin-phycoerythrin (Sigma). After two final washing steps, the samples were analyzed with a fluorescence microscopy (Zeiss Axiovert 25) or by confocal laser scanning microscopy (Zeiss LSM 510).

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

The EHV-1 IR6 protein colocalizes with nuclear lamins. The appearance of the EHV-1 IR6 protein-specific rod-like structures in infected cells suggested a colocalization with proteins that influence the cell's architecture (17, 19, 20, 29). To test the hypothesis that the IR6 protein colocalizes with lamin proteins which are associated with the inner nuclear membrane and are involved in cell division (6), two different approaches were taken.

View larger version (23K): [in this window] [in a new window]   View larger version (29K): [in this window] [in a new window]   FIG. 2.   Laser confocal scanning images of RacL11-infected Rk13 cells fixed at 12 h p.i. (A) or 2 to 10 h p.i. (B). Cells were fixed with acetone, and slides were probed with anti-IR6 antibodies and a mixture of anti-lamin antibodies as described in Materials and Methods. Green FITC fluorescence (IR6) and red phycoerythrin or Cy3 fluorescence signals (lamins) were recorded separately by using appropriate filters. Overlay of the IR6 and lamin fluorescent signals is shown in yellow. At the 12-h time point (A), serial 750-nm sections through an infected cell are shown.

View larger version (35K): [in this window] [in a new window]   FIG. 3.   Laser confocal scanning images of RacM24- (A) or RacH-infected cells (B) at 18 h p.i. Cells were reacted with anti-lamin antibodies which were detected with anti-mouse IgG Cy3 conjugate (red). In RacM24-infected cells, simultaneous staining with the anti-IR6 antibody detected with an anti-rabbit IgG FITC (green) was performed. In the case of the IR6-negative RacH virus, infection of cells was analyzed with anti-gD antibody detected with anti-rabbit IgG Cy5 (blue). No disintegration of nuclear lamins or their colocalization with RacM24 IR6 could be observed.

View larger version (19K): [in this window] [in a new window]   FIG. 4.   (A) Western blot analysis of immunocomplexes obtained with anti-IR6 antibodies (-IR6) or a mixture of anti-lamin A antibodies (-lamin A). Lysates of cells that were infected with RacL11, RacM24, or RacH or were mock infected were prepared at 16 h p.i. and were subjected to immunoprecipitation with the indicated antibodies (-IR6 or -lamin A) as described in Materials and Methods. The immunocomplexes were separated by SDS-10% PAGE and transferred to nitrocellulose. Both IR6 and lamin immunoprecipitates were probed with the anti-IR6 antiserum. Lysates of RacL11-infected (+) and mock-infected cells () that were not subjected to immunoprecipitation were included as controls. The IR6-specific band is indicated by an arrow, and the putative IR6 dimer (19) in the positive control lysate (+) is marked with an arrowhead. The circle indicates rabbit IgG (Mr of 70,000 to 80,000). (B) Control Western blot of Rk13 cell lysates probed with anti-lamin A antibodies. Rk13 cell lysates were prepared and separated by SDS-10% PAGE, transferred to nitrocellulose, and probed with the mixture of anti-lamin A antibodies. Molecular weights of a prestained molecular weight marker (Gibco-BRL) are indicated in thousands.

The IR6 protein is involved in nucleocapsid egress. Previous observations have demonstrated that the inability of EHV-1 strains that either lack the IR6 gene (RacH) or express a mutated form of the protein (RacM24 and RacM36) to replicate efficiently at an elevated temperature of 40°C is caused by a defect in virus maturation and/or egress but not a failure to express individual late genes (20). To identify more precisely the role of the IR6 protein in EHV-1 replication, electron microscopical studies were performed. Edmin337 or Rk₁₃ cells were infected at an MOI of 5 with either RacL11, RacM24, RacH, or the IR6-recombinant RacH virus HIR6-1 (20), and incubated at 37 or 40°C for 4 to 16 h. Infected cells were subsequently processed for electron microscopy. Examination of ultrathin sections of cells infected with the different viruses at 4 h p.i. revealed that infection of the cells occurred at both temperatures irrespective of the virus strain and the incubation temperature: few newly synthesized viral capsids could be observed in the nuclei of infected cells (data not shown). These data confirmed previous observations which indicated that an efficient expression of late gene products was observed in all virus strains at both 37 and 40°C (20). When infected cells were analyzed at 16 h p.i., no differences among the various viruses in nucleocapsid formation and translocation of newly synthesized virions to the cytoplasm or the extracellular space were observed at 37°C (Fig. 5A). Similarly, when cells infected with RacL11 were analyzed after incubation at 40°C for 16 h, an efficient production and translocation of capsids to the cytoplasm and to the extracellular and intercellular space were observed (Fig. 5C). In contrast, when ultrathin sections of cells infected with RacH at 40°C for 16 h were examined by electron microscopy, virtually no virions could be detected in the cytoplasm of infected cells or in the extra- and intercellular space. However, large amounts of immature and mature capsids (23, 24) were present in the nuclei of RacH-infected cells at the elevated temperature (Fig. 5B). As estimated from the inspection of a large number of different sections of RacH- and RacL11-infected cells, there was no marked difference in the total number of capsids produced at either 37 or 40°C. A similar pattern of virion distribution in infected cells was obvious after infection with RacM24 at the elevated temperature (data not shown). When cells infected at 40°C for 16 h with the engineered HIR6-1 virus (a RacH mutant expressing the wild-type IR6 protein [20]) were analyzed, efficient egress of capsids from the nuclei of the cells was observed, and the nucleocapsid egress of the IR6-recombinant RacH virus was indistinguishable from that of the RacL11 virus (Fig. 5D). These observations demonstrated that the temperature-sensitive (ts) phenotype of the RacH virus that lacks a functional IR6 gene is caused by a failure in capsid egress at the elevated temperature, and that the process of capsid egress is completely restored in the presence of one copy of the wild-type IR6 gene present in the HIR6-1 virus. In addition, only the wild-type form of the IR6 protein is able to confer the property of capsid egress at elevated temperatures to EHV-1 because the RacM24 virus expressing a mutated IR6 protein also exhibits a ts phenotype that is indistinguishable from that of RacH. Thus, a functional IR6 protein facilitates efficient EHV-1 capsid egress from nuclei of infected cells.

View larger version (146K): [in this window] [in a new window]   FIG. 5.   Electron micrographs of Edmin337 cells infected with RacH virus at 37°C (A), and with RacH (B), RacL11 (C) or HIR6-1 (D) at 40°C. Cells were fixed at 16 h p.i. Ultrathin sections were prepared and viewed with a Zeiss electron microscope (EM 10C/CR). N, nucleus; Cy, cytoplasm. Whereas nucleocapsids in the nuclei and enveloped viruses in the cytoplasm and extracellular space are visible in RacH-infected cells at 37°C and in cells infected with RacL11 or HIR6-1 at 40°C, no virions in these compartments were observed in RacH-infected cells at 40°C. For size comparisons, capsids are approximately 120 nm in diameter.

The IR6 protein is transported from cell to cell and enters cells in the absence of virus infection. In the last series of experiments, the question of whether the EHV-1 IR6 protein is able to cross cellular boundaries independently of virus infection was addressed. Previous observations had suggested such a possibility because (i) after transfection of the wild-type IR6 gene into COS7 cells, almost every cell appeared to contain the IR6 protein at 96 h after transfection (19), and (ii) after infection of cells with RacL11 at an MOI of 0.1, the IR6 protein was detected in >60% of the cells at 10 h p.i. (22). To test the possibility of IR6 movement between cells and to determine whether the IR6 protein can enter cells from the outside, independently of virus infection, four different approaches were taken.

View this table: [in this window] [in a new window]   TABLE 1.   Total number of IR6-positive and -galactosidase-positive cells after transfectiona

View larger version (8K): [in this window] [in a new window]   FIG. 6.   Confocal laser scanning image of Rk13 cells cotransfected with either wild-type (RacL11) (A) or mutated (RacM24) IR6 gene (B) and reporter plasmid pC+. The IR6 protein and -galactosidase were detected at 72 h after transfection by double immunofluorescent staining as described in Materials and Methods. The cells were analyzed by confocal laser microscopy. IR6 staining (green) and -galactosidase staining (red) are shown. -Galactosidase-expressing cells are marked with an arrowhead.

View larger version (72K): [in this window] [in a new window]   FIG. 7.   Photographs of Rk13 cells infected with the gB-negative RacL11 virus mutant (L11gB) that expresses -galactosidase. Infected cells were fixed at 10, 20, or 36 h p.i. Top panels show Bluo-Gal staining of L11gB-infected cells, and bottom panels show immunofluorescence labeling of the IR6 protein in the same views. Photographs were taken with a Zeiss Axiovert 25 microscope. Magnification, ×240. Infected cells are marked with an arrowhead.

View larger version (7K): [in this window] [in a new window]   FIG. 8.   Confocal laser scanning image of TCgBf cells that had been transfected with pDIR6L and were mixed with Rk13 cells. Cells were mixed at a ratio of approximately 1:30 (1 transfected TCgBf cell per 30 normal Rk13 cells). The IR6 protein was detected with the rabbit antibody and visualized with anti-rabbit IgG FITC, while EHV-1 gB was detected with MAb 4B6 (15) and visualized with anti-mouse IgG TRITC. Overlay of IR6 and gB fluorescences are shown. A TCgBf cell containing IR6 protein is marked with an arrowhead.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

This study describes experiments that were performed to characterize the properties of the EHV-1 IR6 protein. Though nonessential for virus growth (19) and absent in the prototype of the alphaherpesviruses, HSV-1, the IR6 protein is involved in EHV-1 virulence (20), and homologs of the protein have been identified in EHV-4 as well as BHV-1 and CHV (11, 14, 26, 31), suggesting that IR6 homologous proteins might be important for perpetuation of virus infection in vivo. The formation of rod-like structures induced by the wild-type IR6 protein that is reminiscent of structures formed by proteins that make up the cellular architecture led us and other investigators to address the possible interaction of the IR6 protein with members of the filament families. While the cellular protein(s) associated with the IR6 protein was not identified in these previous studies (17, 19, 29), the laser confocal microscopy data and the coimmunoprecipitation experiments presented in this study demonstrate a colocalization of the wild-type IR6 protein and cellular type A lamins. However, we were not able to demonstrate precipitation of lamin proteins from cell lysates by using a glutathione S-transferase-IR6 fusion protein although the fusion protein itself was precipitated as demonstrated by both Coomassie blue staining and Western blot analysis (19, 22). In addition, we view it as unlikely that unspecific precipitation of the IR6 protein by the anti-lamin antibodies occurs because no precipitation of other EHV-1 structural proteins, such as gD or gB, by the anti-lamin antibodies could be demonstrated by immunoblotting (data not shown). Similarly, the wild-type IR6 protein was not coprecipitated by using antibodies directed against other filament proteins such as tubulin, actin, or desmin (19). Nevertheless, the interaction of the IR6 protein with lamins is a very late event in virus infection and remains restricted to approximately 10% of infected cells. Also, the rod-like structures do not contain lamin proteins in all cases. It is therefore possible that the IR6-lamin interaction is restricted to specific cell types or to cells that are in a certain stage of the cell cycle. At present, it is difficult to interpret the relationships between the formation of the IR6 rod-like structures, phosphorylation of the IR6 protein, and the interaction of the IR6 protein with type A lamins with an efficient export of nucleocapsids that is less efficient (i) in the absence of the IR6 protein (EHV-1 strain RacH), and (ii) in the presence of a mutated form of the abundantly produced protein encoded by RacM24 and RacM36 (reference 20 and this study). The ability of viruses that are devoid of a wild-type IR6 protein to replicate efficiently at an incubation temperature of 37°C but not at 40°C suggests that the IR6 function can be compensated by another viral or cellular protein(s) in cultured cells. The fact that RacM24, RacM36, and RacH are apathogenic for the natural host and laboratory animals (8, 12, 20) may also support the interpretation that the IR6 protein exerts a cell type-specific function. Ongoing studies to elucidate the in vivo function of the IR6 protein include the generation of IR6-transgenic animals.

A striking feature of the wild-type IR6 protein is its ability to cross cellular boundaries in the absence of virus infection. The results from a series of independent experiments clearly demonstrated that the wild-type IR6 protein is able to move from cell to cell in the absence of infection because (i) the number of IR6-positive cells exceeded that of the cells expressing a reporter gene by 17- to more than 30-fold after transient transfection, (ii) the wild-type IR6-positive cells appeared to form groups or lineages of cells, (iii) IR6-positive cells surrounded cells infected with a gB-negative RacL11 virus mutant that is defective in cell-to-cell spread, (iv) migration of wild-type IR6 protein between different cells was demonstrated, and (v) wild-type IR6 protein entered cells from the extracellular space. In contrast, the mutated IR6 protein encoded by pDIR6M24 was severely impaired in these properties inasmuch as the numbers of IR6-positive cells only marginally exceeded that of the -galactosidase-positive cells at any time point after transient transfection, and no migration between cell types or uptake of mutated IR6 from the medium could be demonstrated.

The intrinsic property of a herpesviral protein to spread from cell to cell independently of virus infection is not unprecedented as the HSV-1 tegument protein VP22 also exhibits this property. It must be noted, however, that the intercellular transport of the IR6 protein does not appear to be as efficient as that of VP22. In addition, it has been shown that purified VP22 protein can enter cells via an actin-dependent transport mechanism, making it a good candidate for protein delivery to target cells (4). Although the IR6 protein exhibits similar features, the exact mechanism by which the IR6 protein is transported from cell to cell, is taken up from the medium, and moves inside cells is not known. Unlike the HSV-1 VP22 tegument protein, however, this transport appears not to be dependent on the actin cytoskeleton, because IR6 rod formation and transport were not affected by cytochalasin D treatment in Rk₁₃ or COS7 cells (19, 22). The existence of herpesviral proteins that are transported to neighboring cells might point to a similar mechanism by which yet-uninfected cells are prepared for subsequent infection via a virus-encoded messenger to facilitate infection, perhaps by interference with proteins involved in the cell's architecture and division machinery. An alternative explanation for IR6 movement to neighboring cells may be, however, that certain cell types or cells in a specific phase of the cell cycle are rendered resistant to infection after uptake of the IR6 protein to allow virus replication in fully permissive cells only. These hypotheses are currently being tested and are consistent with the fact that both the HSV-1 VP22 and EHV-1 IR6 proteins are (very) early gene products which are produced starting at 2 to 4 h p.i. (4, 17, 19).

In sum, this report describes the characterization of the EHV-1 IR6 protein on a cellular and molecular level. These data and our previously published observations indicate that the aggregation of the IR6 to filamentous structures (i) is an important factor that determines EHV-1 virulence, (ii) is responsible for its colocalization with nuclear type A lamins, (iii) facilitates efficient egress of viral capsids from nuclei of infected cells, and (iv) enables the viral protein to be transported from cell to cell independent of virus infection.