In this study, we have shown that ORF57 is essential for KSHV virion production and lytic gene expression after induction of the lytic cycle by constructing a KSHV recombinant in which the ORF57 gene was insertionally inactivated. ORF57 is the KSHV gene belonging to the herpesvirus gene family of multifunctional gene regulatory proteins that also includes HSV ICP27, EBV SM (Mta, EB2, and BMLF1), VZV ORF4, CMV UL69, and herpesvirus saimiri ORF57 (for review, see reference 37
). The general importance of these genes in lytic herpesvirus replication is suggested by the presence of an ORF57 homolog in a large number of mammalian herpesviruses whose genomes have been sequenced, including ovine, equine, bovine, alcelaphine, murid, and various primate species. In addition, the requirement of these genes for virus replication has been directly tested in VZV, HSV, and EBV (11
). In these three viruses and in KSHV, as we have shown here, ORF57 or its homolog is essential. The ability of ORF57 transfection to reconstitute virus production to only 20 to 30% of wt levels is likely due at least in part to the fact that, whereas all wt KSHV-infected cells were transduced by ORF50, only a fraction of cells was transfected by ORF57.
As we have shown here, early and late lytic KSHV gene expression is highly dependent, at the mRNA level, on KSHV ORF57. The essential character of the ORF57 family of genes is likely to derive from their role in facilitating accumulation of lytic mRNA transcripts. ICP27 and EBV SM have been shown to interact with various components of the cellular export machinery, facilitating export of unspliced mRNAs (4
). The majority of early and late lytic herpesvirus genes are intronless and may therefore lack the ability to independently recruit components of the cellular exon junction complex, a multiprotein complex deposited near the exon junction of processed mRNAs (for review, see reference 12
). Components of the exon junction complex, particularly REF/Aly, interact with TAP, the primary mediator of nuclear mRNA export, which interacts with the nuclear pore complex and facilitates cytoplasmic mRNA transfer. Current models for the mechanism of action of ORF57 include an interaction of ORF57 with viral mRNA and recruitment of REF/Aly by direct protein-protein interaction, and published evidence indicates that one region of ORF57 directly binds to REF/Aly (23
We have also demonstrated that ORF57 and EBV SM are each unable to substitute for the other in rescuing productive replication of SM-null and ORF57-null recombinant viruses, respectively. Although the ORF57 family proteins are homologous, there are significant differences in sequence and function, particularly among the alpha-, beta-, and gammaherpesviruses. In no case has one member of this family been able to substitute for another in rescuing virus replication. EBV SM has been inserted into an HSV genome with ICP deleted, and the recombinant, although it does replicate, is almost as defective in replication as the parent Δ27 mutant (3
). Similarly, CMV UL69 and ICP27 were not capable of trans
-complementing a recombinant EBV with SM deleted to efficiently rescue virion production (15
). This exclusive requirement for the specific regulatory gene of each virus also applies within herpesvirus families, since VZV ORF4 mutants cannot be complemented by HSV ICP27 (11
). Although these viral proteins behave similarly in reporter assays, transactivating reporter genes, they are clearly not equivalent in biologic function. Somewhat surprisingly, EBV SM was as efficient as ORF57 in enhancing expression of ORF57 and mCP genes from ORF57-null virus and even more active than ORF57 on the ORF9 gene. The inability of SM to rescue infectious KSHV production despite apparent activity on KSHV transcripts has also been observed in other systems, where ICP27 cannot rescue EBV SM or VZV ORF4 mutants despite activity on heterologous viral mRNAs.
These functional differences are likely to be due to one or more aspects of the mechanism of action of ORF57 homologs. Most importantly, there is some degree of specificity in terms of the responsiveness of various target genes to the ORF57 family of proteins. Such differences have been observed both in reporter assays and with viral genes (30
). For example, some EBV lytic genes are more highly SM dependent than others (15
). It is likely that these differences in target gene responsiveness are at least partly due to the regulatory proteins having different affinities for different mRNA species, as suggested by yeast three-hybrid experiments with ICP27, which demonstrated that ICP27 has a preferential affinity for a subset of HSV transcripts (40
). Thus, although SM does act on several essential KSHV lytic genes, it may be that its mRNA specificity is distinctive enough that it does not permit appropriate accumulation of all heterologous KSHV transcripts. This hypothesis can now be directly tested by using the ORF57-null and EBV SM-null recombinants in combination with viral gene arrays to examine the differences in viral transcript accumulation when trans
-complemented with the heterologous regulatory gene. It should be noted that the dependence of late lytic genes on ORF57 and its homologs is complicated by the likely involvement of these proteins in regulation of genes involved in viral DNA replication. Thus, ORF57 and its homologs are likely to affect late gene expression via both direct effects (on mRNA levels) and indirect effects (on DNA template numbers).
A second important difference between the various herpesvirus homologs that does not allow them to be functionally interchangeable may lie in their effects on host cell gene expression. For example, EBV SM has significant growth-inhibitory effects, whereas we have not observed such an effect with ORF57 (unpublished observations). In addition, SM induces a specific subset of host cell genes, and some of these induced gene products may be important in lytic EBV gene expression (27
). Thus, the functional specificity of the various ORF57 homologous proteins may also derive from unique effects on host cell gene expression that are required for efficient replication.
Finally, the utility of producing this specific ORF57-null mutant is demonstrated by the ability to directly determine whether ORF57 is involved in regulation of a specific pathway or expression of a particular gene. It has been hypothesized that ORF57 may be involved in regulation of PAN expression (20
), and using the ORF57-null mutant, we have demonstrated that maximal PAN expression during lytic replication does in fact require ORF57. While this finding is compatible with the interpretation that ORF57 synergizes with ORF50 to stimulate PAN transcription, it is also possible that ORF57 enhances nuclear stability of PAN posttranscriptionally. The actual role of a physical interaction between ORF57 and ORF50 proteins in PAN gene transcription in vivo is also called into question by the finding that EBV SM is as effective in enhancing PAN accumulation from the ORF57-null KSHV as ORF57 itself. The ORF57-null recombinant will allow us to address these and other mechanistic questions regarding the regulation of gene expression during lytic KSHV replication in the context of the entire viral genome.