We generated a library of mutants in pRhCMV/BAC-Cre by using random transposon insertion and linear recombination (24
). In total, the library covers 75 RhCMV ORFs that correspond to annotated HCMV genes and five large, nonoverlapped genes that are unique to RhCMV (Table ). With our current understanding of the coding capacity of the RhCMV genome, this represents ca. 50% of the known HCMV orthologues in RhCMV (17
Twenty-seven mutants carry lesions within RhCMV ORFs that are essential for replication in fibroblasts, and, with the exception of UL74 and UL97, the HCMV orthologues are essential for HCMV replication in at least one strain (11
). As noted above, UL74 and UL97 have been defined as strongly augmenting ORFs, i.e., viruses lacking these functions exhibit severe growth defects (18
). Consequently, it is not surprising that they have scored as essential in our RhCMV screen. Four mutations reside in ORFs that augment RhCMV replication, and 49 identify ORFs that are nonessential for replication in cultured telo-RFs. The augmenting and nonessential RhCMV ORFs exhibit growth phenotypes analogous to those described for the corresponding HCMV ORFs (11
), further validating RhCMV as a model for HCMV.
We used primary RRPE cells (5
) to screen for RhCMV genes that are nonessential for replication in fibroblasts but required for replication in epithelial cells. RhCMV infects these cells inefficiently, exhibiting a block prior to immediate-early gene expression (Fig. ). This phenotype might result from the absence of a UL128 orthologue in pRhCMV/BAC-Cre and its parental strain 68-1 (17
). pUL128 is essential for the efficient infection of epithelial cells by HCMV (37
). An orthologue of this ORF is present in the 180.92 strain of RhCMV (31
). In spite of the inefficient infection, the BAC-derived virus used in the present study produces substantial yields of infectious virus over an extended period of time in RRPE cells. Indeed, we have harvested virus weekly from infected RRPEs for a period of 20 weeks, with titers in the medium >105
/ml at each time point (data not shown).
Four of the 45 RhCMV ORFs that are nonessential for replication in cultured telo-RFs proved to be required for efficient replication in RRPE cells. Mutations in Rh01 (an HCMV TRL1 orthologue), Rh159 (UL148), Rh160 (UL132), and Rh203 (US22) caused moderate to severe growth defects in RRPE cells (Fig. ). To the best of our knowledge, this is the first demonstration that these four genes influence the tissue tropism of a CMV. It is worth noting that mutations in the orthologues of UL130 and UL131A did not affect the replication of RhCMV in RRPE cells (data not shown). UL128, UL130, and UL131A in HCMV are believed to form a virion protein complex that is required for entry into epithelial and endothelial cells (1
). The fact that the corresponding RhCMV mutants showed no phenotype probably results from the fact that the complex is already disrupted in RhCMV BAC-derived virus due to the missing UL128 ORF (17
The defect in Rhsub01 was characterized. At an input multiplicity of 3 TCID50/cell with centrifugal enhancement, the mutant virus entered RRPE cells, expressed representative viral proteins and accumulated viral DNA as efficiently as wild-type virus (Fig. to ). Further, the pRh01-deficient mutant produced an amount of infectious extracellular virus similar to that of the wild-type virus during the first week after infection of RRPE cultures (Fig. , left panel). Importantly, however, the mutant virus failed to produce increasing amounts of progeny during weeks 2 to 6 after infection as was the case for the wild-type virus (Fig. , right panel). Further, although mutant and wild-type virus particles produced during the first week after infection were equally infectious (Fig. , left panel), mutant particles produced during weeks 2 to 6 were less infectious (Fig. , right panel). This might reflect the production of defective particles in cells that have been infected by the mutant for an extended period of time.
The failure of the mutant to produce increasing amounts of infectious progeny following the first week after infection of RRPE cultures (Fig. ) is consistent with the observation that the number of pRh156-expressing cells increases to a very limited extent over time after infection with Rhsub01 (Fig. ), and it leads to the conclusion that the mutant fails to spread after producing an initial burst of virus from the first set of cells infected in RRPE cultures.
It is clearly possible to infect RRPE cells with wild-type and mutant viruses at the same efficiency by using virus stocks produced in telo-RFs at a multiplicity of 3 TCID50
/cell with centrifugal enhancement (Fig. to ). It also is evident that extracellular, infectious mutant virus is produced after the initial infection (Fig. , left panel), but this virus fails to initiate secondary infections (Fig. and , right panel). Perhaps Rhsub
01 is inherently deficient for entry into RRPE cells, and a combination of high input multiplicity and centrifugal enhancement compensates for the deficiency. Low-speed centrifugation at the time of infection has been shown to enhance infection by a variety of different viruses, including CMV (19
), but the mechanism remains obscure. Centrifugal enhancement might alter the physiology of RRPE cells to bypass the need for pRh01 during the entry process. Alternatively, the composition of Rhsub
01 virions might be different when produced in telo-RFs compared to RRPE cells. A change in the mutant virion might then preclude subsequent infection of RRPE cells, even though these virions can infect telo-RFs in assays for the production of infectious progeny. It is noteworthy that pRh01 accumulates in the cytoplasm (Fig. ), where it could potentially influence tegumentation and the final assembly of virions, but it does not appear to be a constituent of virus particles. We were unable to detect pRh01-FLAG in virions by immunoblot assay (data not shown), and its HCMV orthologue, pTRL1, has not been found in HCMV virions (35
If it is not a constituent of virions, how could pRh01 influence the infectivity of virus particles produced in RRPE cells? In polarized epithelial cells, there are distinct sorting pathways for cargo destined for the apical and basolateral membranes (12
). Although the RRPE cells used in the present study were not polarized, it is tempting to speculate that their sorting pathways present are nevertheless distinct from those in fibroblasts. pRh01 may facilitate the transport of viral proteins and possibly virions to the proper compartments in RRPE cells. It is possible that pRh01 serves a similar role in telo-RFs. RRPE cells may simply be very stringent filters that, since they are infected at relatively low effective multiplicities of infection, magnify the effect of minute, possibly multiplicity-dependent replication defects that are inconsequential in the fully permissive telo-RFs. Finally, it is conceivable that pRh01 plays no role in virion assembly but regulates the expression or secretion of viral or cellular proteins that normally modulate the innate immune response of neighboring RRPE cells to facilitate spread of the virus.
Thus far, it has not been possible to investigate the composition of Rhsub
01 virions produced in RRPE cultures due to the very low yields of particles. We have, however, noted a difference in wild-type versus Rhsub
01 virus stocks produced in telo-RFs. When cells are infected with the mutant, they contain much more pRh112 at 1.5 h after infection than when they are infected with wild-type virus (Fig. ). pRh112 accumulates with late kinetics (Fig. ), and its HCMV orthologue, UL83-coded pp65, is an abundant virion protein. Thus, the pRh112 detected at 1.5 h after infection is almost certainly delivered to the cells by infecting particles. Both virus stocks were used at a multiplicity of 3 TCID50
/cell, and the TCID50
/genome ratios of wild-type and mutant virions are similar, so the same number of genome-containing virions were delivered in the mutant and wild-type virus infections. Consequently, the greater amount of pRh112 suggests that mutant genome-containing virions contain more pRh112 than wild-type virions or that the mutant stock contains more noninfectious particles lacking genomes, i.e., so-called noninfectious enveloped particles or dense bodies (26
), than does the wild-type virus stock. Alternatively, pRh112 in mutant virions could be modified so that it is more stable after infection, or mutant virions could disassemble more slowly (although there is no apparent delay in pRh156 expression), and this could stabilize the virion protein. No matter what the cause of the differential level of pRh112 at 1.5 h after infection of telo-RFs, it does not lead to an observable difference in the kinetics of mutant compared to wild-type virus replication in this cell type (Fig. ).
In conclusion, we have generated a library of RhCMV mutants and screened it to identify four viral genes that influence replication in epithelial cells but not fibroblasts. These four genes have the potential to markedly influence the spread and pathogenesis of the virus within its primate host.