The MIEP is a central control unit that processes cellular and viral signals to make a decision between latent or lytic infection of HCMV (26
). Among the viral signals, the tegument proteins ppUL82 and ppUL35 have been shown to interact physically and to transactivate the MIEP alone and cooperatively (24
). We wanted to analyze the biological role of UL35 proteins by generating a virus mutant with deletion of the UL35 gene. One-step growth curves showed that virus production was decreased 1,000-fold when cells were infected with the mutant virus at an MOI of 0.1. This defect could be reversed when the UL35 gene was reinserted. This observation makes the deletion of UL35 the sole reason for the growth defect.
Our observation of reduced virus production by the UL35 deletion virus is supported by a recent large-scale analysis in which a similar deletion of UL35 was generated (13
). In addition, our deletion of UL35 in the genetic background of a clinical isolate showed a comparably reduced virus yield (M. Winkler and K. Schierling, unpublished data). However, in a second large-scale analysis, UL35 was classified as nonessential, with yields reduced less than 10-fold (49
). This discrepancy is likely explained by the different targeting strategies, since in the second approach a transposon insertion into the amino-terminal region was created. Since the UL35 gene encodes two coterminal protein species (24
), the transposon insertion targeted only the full-length ppUL35, leaving the short-form ppUL35A intact. Therefore, either ppUL35A was able to compensate for ppUL35 or the essential part resides in the C termini of the UL35 proteins. The latter possibility is supported by the observation that the addition of a hemagglutinin tag at the end of the UL35 gene in the context of the viral genome leads to a reduction in viral yield similar to that resulting from the deletion of UL35 (Schierling and Winkler, unpublished data).
It was reported previously that ppUL35 has a role in the transactivation of the MIEP and efficient initiation of IE phase (24
). The MOI dependence of the RVdel
UL35 virus yield is compatible with this view, since mutations in other genes implicated in IE regulation and gene expression, such as the ppUL82, ppUL69, ppUL123 (IE1) genes, and enhancer deletions showed similar behaviors (6
). In addition, when infection was done at an MOI determined by plaque titration with an anti-IE antibody, we observed that the DNA amount in that inoculum of the RVdel
UL35 mutant was 10-fold higher than that in the wild-type virus. As the particles appeared to be normal when analyzed by electron microscopy, this observation would mean that a 10-fold-higher number of particles of the mutant virus was required to reach the same infectivity as that of the wild-type virus. Thus, the lack of ppUL35 would be compensated by a higher amount of input viral DNA, by higher levels of other tegument proteins, or by a combination of both. When we standardized our inoculum to equal amounts of input DNA, we observed a delayed IE gene expression and consequently a strongly reduced expression of early and late proteins. This resulted in a significant reduction of viral DNA accumulation and a strongly reduced virus yield. In agreement with an effect on IE gene expression, an MOI-dependent virus yield was observed, rendering UL35 an essential gene when cells were infected at the low MOI of 0.01.
Defects in IE gene expression can be compensated by a high input of viral particles, which has been observed previously in conjunction with an HCMV mutant carrying a deletion in the distal enhancer (25
). A possible explanation for the decreased IE gene expression with the mutant RVdel
UL35 could be that no ppUL82 was packaged into viral particles, since during late phase there was no translocation of ppUL82 towards the cytoplasm. Due to the low yields of the RVdel
UL35 mutant, we were not able to address this point experimentally. When we analyzed the uptake of tegument proteins into cells infected with wild-type or mutant virus, we observed similar numbers of cells staining positive for ppUL82 or ppUL69. Therefore, the defect in IE gene expression should be due mainly to a lack of ppUL35 and a subsequent failure of sufficient cooperative activation of the MIEP.
When infections were carried out using the same MOI for both wild-type and mutant viruses, we observed no difference in viral protein expression. Additionally, the accumulations of viral DNA showed comparable levels and kinetics after infection with wild-type or mutant viruses. As ppUL35 apparently had no influence on early or late gene expression or viral DNA replication, we could use infections standardized to the same MOI to investigate an effect of UL35 during the late phase.
Hereby, we observed an altered virus maturation in cells infected with mutant virus. This alteration was most clearly indicated by the absence of dense bodies. In addition, quantitation of cytoplasmic virus particles showed 10-fold fewer mature enveloped particles when cells were infected with mutant virus. The lack of dense bodies could be explained by the retention of ppUL83 (pp65) in the nucleus, as a virus mutant lacking the UL83 gene also failed to produce dense bodies (39
). It is believed that dense bodies accumulate during high-titer passage of HCMV. Therefore, the mutant virus, which produces much lower titers, should be less prone to accumulate dense bodies. However, our reconstituted wild-type virus had the same passage history, and it was the mutant virus for which the higher titers had to be employed for virus stock production. In addition, immunofluorescence staining of cells demonstrated that several viral proteins (ppUL99 and gpUL55) showed normal accumulations and distributions in the cytoplasm of infected cells. It is therefore likely that the failure to produce dense bodies reflects a specific defect in the envelopement of viral particles.
In the nucleus, large accumulations of electron-dense material were observed. At present, the nature of these structures is unclear. Electron density in the nucleus usually corresponds to DNA, indicating that these structures could represent domains of condensed viral genomes. As viral particles can be seen at the margin of this material, a possible reason for this accumulation could be a packaging defect. However, we observed similar ratios of viral capsid types in the nuclei of cells infected with wild-type and mutant viruses, making a packaging problem unlikely. Therefore, these structures require further investigation.
At present, it is unclear how ppUL35 could be responsible for the translocation of ppUL82 and ppUL83 (pp65) from the nucleus to the cytoplasm. A similar phenotype has been described for a mutant of HCMV with a lack of TRS1 (5
), and a recently published analysis of this mutant argued that pTRS1 has a role in the encapsidation of viral DNA since the number of C capsids was reduced (1
). In our electron microscopic analysis, we did not detect such a difference. In contrast, the lack of dense bodies and the reduced amount of enveloped particles argue for a role of ppUL35 in the cytoplasmic particle assembly. As UL35 proteins interact with ppUL82 but not with ppUL83 (38
), their effect on tegument protein localization might be independent of physical interaction. We therefore favor the hypothesis that ppUL35 of ppUL35A might directly or indirectly influence nucleocytoplasmic transport pathways, either enhancing export of tegument proteins from the nucleus or blocking import of tegument proteins into the nucleus during the late phase.
In summary, we could show that ppUL35 plays an important role in both IE gene expression and virus assembly. Further studies will be required to elucidate the underlying mechanisms and to address the question of whether the effects on IE gene expression and virus maturation are direct or indirect.