In this study, experiments were conducted to elucidate the role of ETIF, the EHV-1 homologue of HSV-1 VP16 (α-TIF), in virus replication. An ETIF deletion mutant of EHV-1 was constructed in E. coli using an infectious EHV-1 BAC clone and progeny vL11ΔETIF virus was propagated on an ETIF-expressing cell line. The failure to propagate an ETIF-negative virus on noncomplementing cells and the abortive nature of an infection with complemented virus of an ETIF mutant virus suggests an absolute requirement of ETIF for productive virus replication. It could further be demonstrated that absence of ETIF resulted in a massive defect in virus morphogenesis that may be caused by a defect in tegumentation and/or secondary envelopment.
The part of the EHV-1 genome where gene 12
encoding ETIF is located is transcriptionally very active and surrounded by genes expressing homologues of other tegument proteins, e.g., VP13/14 (UL46), VP17/18 (UL47), and VP22 (UL49) (58
). Therefore, the specificity of the essential phenotype of the vL11ΔETIF virus was addressed in two ways. First, a site-specific revertant based on vL11ΔETIF was generated in which the ETIF-encoding sequence was restored. The revertant virus regained wild-type growth properties, indicating that no secondary mutations occurred during isolation of vL11ΔETIF and that the observed lethal phenotype of vL11ΔETIF mapped within the DNA fragment used to rescue the mutation. Second, specificity of the deletion was shown by trans
-complementing the replication defect of vL11ΔETIF using RK-ETIF cells, which constitutively express ETIF. The RK-ETIF cell line efficiently complemented the growth defect in vL11ΔETIF, although titers and plaque sizes were slightly reduced. The reduction in virus growth properties compared to those of parental or revertant virus could be caused by the significantly lower expression levels of ETIF in this cell line compared to virus infection (Fig. ), which also results in relatively less efficient incorporation of ETIF into virus particles (Fig. ). We view it as unlikely that the cellular distribution of ETIF in RK-ETIF cells was altered, because ETIF compartmentalization appeared to be unaffected and consistent with earlier reports showing ETIF in both the cytoplasm and the nucleus (20
). In addition, distribution of ETIF after infection in both RK13 cells and RK-ETIF cells was indistinguishable (data not shown). Localization of ETIF into vesicular compartments in the cytoplasm as described for HSV-1 VP16 could not be observed (31
) but will be investigated in future studies in greater detail.
Previous studies have shown that ETIF accomplishes at least two functions during EHV-1 infection. ETIF serves as a structural component of the tegument (3
) and can stimulate the transcription of the sole EHV-1 immediate-early gene (33
). It shares these functional properties with α-TIFs of HSV-1, BHV-1, PRV, and VZV (5
). In EHV-1, the sole and essential IE gene, transcribed independently of de novo EHV-1 proteins, functions as a transcriptional transactivator of the early genes and initiates the transcriptional cascade of EHV-1 genes. However, EHV-1 DNA is infectious, and virus progeny can be produced after transfection of virus DNA or of plasmids carrying the EHV-1 genome into permissive cells, suggesting that ETIF delivered by incoming virions is not absolutely required for the onset of viral replication (46
; the present study). This is further supported by results obtained from experiments using HSV-1 expressing mutated VP16 in which the transactivation domain was deleted. Such mutants were viable, although both the level and timing of viral gene expression were severely disrupted (66
). The defect in viral gene expression caused by the absence of the transactivation domain of VP16 resulted in significantly reduced virus titers, which could be increased by a high-MOI infection or induction of cellular stress (2
In the case of EHV-1, foci or single infected cells upon transfection of mutant virus DNA lacking the entire coding sequence for ETIF could be identified by an antibody directed against gM. Expression of gM starts in the late phase of virus replication (43
), indicating that virus genome replication and onset of viral gene expression is not dependent on the presence of ETIF, which is consistent with the situation seen in HSV-1 (2
). Production of late viral proteins in the absence of ETIF suggests that the transactivator function of ETIF is dispensable for initiation of the cascade-like transcription pattern of EHV-1 genes. However, the exact role of the ETIF function as transactivator of the IE gene expression and its requirement in the early stages of infection remains to be determined and will be the object of future studies.
The second function of ETIF is of a structural nature. ETIF is a virion component (34
), and we could detect at least three different sizes of ETIF-specific bands in both virus-infected cell lysates and purified virions by Western blot analysis using an MAb directed against ETIF. These various forms of ETIF are also found in lysates of RK-ETIF cells and of RK13 cells transfected with pc-ETIF. The nature of the multiple bands of ETIF in Western blot analysis is not completely understood and could reflect different phosphorylation states or be the result of proteolytic cleavage. The most likely explanation, however, is the use of alternative start codons that can be found in the sequence of ETIF and would result in the expression of shorter forms with predicted molecular masses corresponding to the observed smaller bands. In fact, recent data indicate that mutation of the first start methionine leads to absence of the largest form (60 kDa) of ETIF, while the two lower ETIF species were still expressed (59
The presence of ETIF in phenotypically complemented vL11ΔETIF, which carry the risk of “phenotypical leakage,” may explain seemingly contradictory results of the transfection and infection experiments. High-MOI infection of RK13 cells with vL11ΔETIF propagated on RK-ETIF cells resulted in extracellular titers that were significantly reduced compared to those of wild-type or rescuant virus but were still measurable and reached 3 × 105
PFU/ml. This result was unexpected and was in stark contrast to the essential requirement of ETIF for virus egress as observed in transfection experiments (the present study) and previous reports on a VP16-null mutant of HSV-1 showing loss of infectivity after infection in one-step growth kinetics (64
). One explanation for this phenomenon could be that delivery of functional ETIF by incoming complemented virions results in “recycling” for at least another round of virion assembly. However, it cannot be entirely ruled out that the requirement of ETIF for virus growth can be overcome by infections at high MOI. Recycling of ETIF would require that the incoming protein is stable and remains functional so that it can be sequestered and serve as a structural component of newly synthesized virions. In fact, in immunoblot analyses, incoming ETIF was stable beyond 6 h p.i. (Fig. ) and is therefore available long enough to be recycled for virion assembly. In addition, the higher particle/PFU ratio of vL11ΔETIF propagated on RK-ETIF cells compared to wild-type and ETIF-rescuant virus will result in larger amounts of ETIF delivered to RK13 cells by incoming virus particles. The fate, however, of incoming ETIF is not completely understood. Dissociation of HSV-1 VP16 from the incoming particle and localization to the nucleus early in infection was reported (31
). It was also shown that HSV-1 VP16 is relatively stable upon entry into cells, does not undergo any apparent changes, and can be detected up to 4 h after infection (40
). Similar results, i.e., dissociation of the UL48 protein from the incoming virion after entry, were recently obtained by immunoelectron analyses of the early stages of infection of RK13 cells with PRV (19
). Although there are no supporting data available, dissociation of incoming ETIF from the EHV-1 particle and subsequent localization to the nucleus is likely. Considering that ETIF can be recycled, it is conceivable that ETIF is added to progeny virus capsids within the nucleus or relocated to the cytoplasm, where it is added to newly formed nucleocapsids. This latter hypothesis will be addressed in future experiments.
Our hypothesis of recycling of ETIF after infection with phenotypically complemented ETIF-negative EHV-1 is also supported by the results of the low-MOI infections, which minimized the amount of incoming ETIF. Infection of noncomplementing RK13 cells with low MOIs of complemented ETIF-null virus resulted in strictly MOI-dependent titers of virus progeny that were up to 10-fold higher than input titers. In contrast to infection with wild-type virus or infection of complementing cell line RK-ETIF, complete infection of all cells was never observed after infection of RK13 cells with vL11ΔETIF. Upon serial propagation of vL11ΔETIF in RK13 cells, virus titers diminished until no infectious virus could be recovered, usually around the fourth passage. These data indicate that input ETIF protein derived from infecting virions is not sufficient to serve for more than few rounds of replication. Recycling of ETIF for production of finally enveloped particles would likely require incorporation into newly formed particles. In purified virions of ETIF-negative virus propagated once on noncomplementing cells, no ETIF, however, could ever be detected by Western blotting. This likely indicates that the amounts of ETIF that need to be incorporated into virions for successful secondary envelopment are minute. Taken together, our results suggested that infection of RK13 cells with complemented vL11ΔETIF and subsequent passage on RK13 cells is a self-limiting infection, which support data from transfection experiments that ETIF is required for productive virus assembly and egress.
The assembly and the egress properties of ETIF mutant viruses were addressed by electron microscopy. In the late phase of infection of the ETIF-null mutant on RK13 cells, a clear defect in virus morphogenesis was detected, similar to the observations of VP16-negative HSV mutants, which were severely impaired in virus egress, presumably caused by a defect in an assembly step after primary envelopment (41
). Characteristic for this mutant was that capsid formation, DNA encapsidation, and nuclear egress seemed to be identical to the situation in cells infected with wild-type virus. However, many naked capsids in the cytoplasm but not enveloped particles in the cytoplasm or the extracellular space could be detected (41
). Similarly, a UL48-negative mutant of PRV exhibiting a severe growth defect but able to grow on noncomplementing cells showed a massive defect in virus morphogenesis that resulted in accumulation of nucleocapsids in the cytoplasm (17
). The molecular and mechanistic details of herpesvirus egress are still not completely understood. In a proposed model comprising the sequential envelopment/de-envelopment/re-envelopment process for herpesvirus maturation (36
), tegumentation of herpesvirus capsids occurs at two different sites (reviewed in reference 37
). One site is the nucleocapsid in the cytoplasm after de-envelopment where UL36 and UL37 are added, and the other is the future budding site involving other tegument proteins and their interaction with cytoplasmic domains of viral glycoproteins located at membranes of cytoplasmic vesicles. Previous studies and the present study indicate that HSV-1 VP16, the PRV UL48 protein, and EHV-1 ETIF are apparently not required for the initiation of both processes (17
). It is therefore plausible that this tegument protein plays an important structural role in linking tegument proteins to each other, tegument proteins with envelope proteins, or may be required for efficient connection of the tegumented capsid with the glycoprotein-containing vesicle during budding. Multiple interactions of HSV-1 VP16 with tegument (e.g., VP22, Vhs [the UL41 product]) and envelope glycoproteins (e.g., gH) have been reported and emphasize its important structural role (13
). The interaction of VP16 with Vhs, which precludes degradation of viral mRNA mediated by Vhs, is an important posttranscriptional regulatory function of VP16 (32
). In EHV-1, a similar interaction between ETIF and Vhs might be possible and could contribute to some extent to the phenotype of vL11ΔETIF. Studies on Vhs in EHV-1 have shown that no host protein shutoff was mediated by the UL41 homologous protein, although the shutoff function is conserved as shown by transient-transfection experiments of vhs
). It is not known, however, whether ETIF interacts with Vhs and thereby modulates its function. Further studies will be conducted to identify possible interaction partners of ETIF and the exact role of EHV-1 ETIF in virus maturation, with particular emphasis on the nature and role of the three different ETIF protein species detected both in infected cells and purified EHV-1 virions.