HCMV encodes a diverse set of proteins that influence the immediate-early environment of the cell (26
). These proteins are either newly expressed upon initiation of viral gene expression or are introduced as components of infectious virions upon virus entry. We have identified here a new HCMV gene product, pUL29/28, that is expressed from the UL29 and UL28 ORFs through the use of multiple splice donor and acceptor sites (Fig. ). Previous reports suggested that the mRNAs within the UL29-UL26 region share a common polyadenylation signal, AUUAAA, within the UL26 3′UTR at nt 32103 (10
). This motif is not present in the UL29/28 mRNAs that we have mapped, and we have identified a second putative polyadenylation signal at nt 31892, CAUAAA, just upstream of the site of poly(A) addition, as seen in the 3′RACE products (Table ).
By using a virus with an epitope tag at the N terminus of the UL29 sequence, BADinUL29F, we demonstrated expression of pUL29/28 protein throughout the course of infection at the predicted size of 79 kDa (Fig. ). In addition, a less abundant, 41-kDa protein specific to infected cells was observed (Fig. ). We also observed these proteins upon transient transfection of expression vectors containing the mapped coding sequences (Fig. ). The 41-kDa protein was not observed during infection using a virus with an epitope tag at the C terminus of the UL28 sequence, BADinUL28F (Fig. ). This is the predicted size of UL29 protein expressed from an unspliced transcript. We observed both spliced and unspliced RNAs associated with polysomes isolated from HCMV-infected cells (Fig. ), which is consistent with the finding that a smaller protein is expressed from this locus. Our results suggest that a 79-kDa protein, pUL29/28, and a 41-kDa protein, pUL29 (Fig. ), are expressed through alternative splicing and share a common amino-terminal domain.
Two mutant viruses, BADsub
UL29 and BADsub
UL28, unable to produce pUL29/28, produced less immediate-early RNA compared to wild-type virus (Fig. ). The UL29 and UL28 genes are adjacent to and expressed in the same orientation as the UL30, UL27, and UL26 genes (Fig. ), and earlier work has demonstrated that disruption of UL26 results in a delay in immediate-early gene expression and reduced virus yield (28
). Given the similarity in phenotypes, we tested whether the disruption of the UL29/28 genes influenced pUL26 expression. Western blot analysis with an antibody to pUL26 demonstrated that the mutations in BADsub
UL29 and BADsub
UL28 still allow for expression of pUL26 during infection (Fig. ). We have monitored the expression of UL27 and UL30 by qRT-PCR and observed RNAs containing both ORFs in BADsub
UL28-infected cells (Fig. ). Similar levels of expression occurred between viruses for RNAs containing the UL30 ORF. However, we observed slightly reduced levels of UL27 RNA expression. Because pUL29/28-deficient viruses result in reduced gene expression, we cannot distinguish between reduced levels of UL27 RNAs mediated from sequence disruptions in UL29/28 versus an overall reduction in pUL29/28-mediated gene expression. However, other reports have demonstrated that disruption of the UL27 ORF results in only a slight growth attenuation in fibroblasts, and no growth phenotype was observed during virus replication in human tissue implanted in SCID mice (10
). In addition, we have recently identified the start site of an RNA containing the UL27 ORF that sits downstream of the UL28 ORF (unpublished observations). Thus, the impaired immediate-early gene expression and growth defect we observed for BADsub
UL29 and BADsub
UL28 (Fig. ) almost certainly result from disruption of pUL29/28 and pUL29 function and not from ancillary effects on neighboring ORFs. Further evidence for this conclusion comes from the fact that both of these proteins can induce activity of the MIEP within transfected cells (Fig. ).
RNAs from the UL26-29 region have been reported to be expressed with early or late kinetics (6
). Given the delay in its expression, how might pUL29/28 and pUL29 be influencing immediate-early gene expression? Although previous characterization of HCMV virion and dense body components by using mass spectrometry did not detect pUL29/28 or pUL29 within particles (48
), we found an association by using a virus with an epitope tag at the N terminus of the UL29 sequence. pUL29/28 and pUL29 both copurify with virions and are protected from protease digestion by the virion envelope (Fig. ). Consistent with their presence in virions, a portion of the tagged proteins is present in the assembly zone late after infection and, as expected for a role in activation of immediate-early gene expression, pUL29/28 is predominantly nuclear at 6 h after infection (Fig. ). Newly synthesized pUL29/28 might also influence immediate-early gene expression. We have demonstrated the presence of pUL29/28 RNA (Fig. ) and protein (Fig. and ) within infected cells very early after infection and, importantly, the amount of RNA containing UL28 and UL29 sequences increases from 2 to 6 hpi (Fig. ). Thus, even though drug sensitivity experiments have categorized this RNA as early-late, it is clearly accumulating during the immediate-early phase of infection. In sum, our data indicate that pUL29/28 and pUL29 are delivered to cells in virions and also expressed very early in infection, and they are predominantly nuclear. Thus, the proteins are present at the start of infection and correctly positioned to assist in the activation of immediate-early genes.
There is ample precedent for tegument proteins such as pUL29/28 and pUL29 to influence immediate-early gene expression. The pUL26 tegument protein was demonstrated to contain a strong transcriptional activation domain (45
), and a UL26 deletion virus exhibited a delay in the onset of immediate-early gene expression (28
). In this case, it is unclear whether pUL26 is directly activating immediate-early expression, since pUL26 can also influence the stability of viral particles (28
). We observed similar levels of infectivity between wild-type virus and the BADsub
UL28 and BADsub
UL29 mutants (Fig. ), so it is not likely that pUL29/28 or pUL29 influences particle stability. The pp71 protein has also been demonstrated to activate immediate-early gene expression (5
), and this transactivation activity is enhanced by the tegument protein pUL35 (43
). pp71 activation depends upon proteasomal degradation of the cellular Daxx protein, a negative regulator of immediate-early gene expression (18
HCMV promoters are, of course, regulated by histone modifications (12
), and we recently discovered that the pUL38 protein interacts with pUL29/28, as well as with the host cell nucleosome remodeling and histone deacetylase (NuRD) complex (27
). The NuRD complex contains histone deacetylases and chromatin-remodeling ATPases, and it can repress transcription (4
). Although we have not yet shown that pUL29/28 and NuRD reside in the same complex, it is conceivable that pUL29/28 acts to protect the viral chromosome from repressive effects of the NuRD complex.