Although a great deal is known about the envelope of alphaherpesviruses, our understanding of the composition of the HCMV envelope is still incomplete. The recent analysis by Varnum et al. (47
) identified 19 virally encoded glycoproteins as components of the viral particle. In that study, mass spectrometry was used to characterize the HCMV proteome and gpUL132 was found to be a component of the virus. The abundance was calculated to be around 0.4%, which classifies gpUL132 as a low-abundance structural component of HCMV. While mass spectrometry provides valuable information on the overall protein composition of viral particles, it also has limitations, e.g., for the identification of protein complexes or posttranslational modification of proteins.
In this report we have shown that gpUL132 exists in two forms, 45 to 60 kDa and 22 to 28 kDa, in infected cells as well as in extracellular virions from low-passage and high-passage HCMV strains. The sensitivity of the protein to PNGase F indicated that the 45- to 60-kDa form of gpUL132 in infected cells and virions carries complex N-linked sugars. The sensitivity of the 45- to 60-kDa form of gpUL132 to digestion with PNGase F also provided proof that gpUL132 is a type I glycoprotein with its amino-terminal domain being on the outside of infected cells or virions. The two potential consensus sequences for the addition of N-linked carbohydrates (NMT34 and NMT63) are both found between the predicted signal sequence (amino acids 1 to 24) and the transmembrane anchor (amino acids 84 to 106).
In addition to complex N-linked carbohydrates, gpUL132 most probably carries O-linked sugars. This is indicated by the diffuse migration of the protein after removal of the N-linked carbohydrates. According to computer-based predictions, gpUL132 carries 17 sites for addition of O-linked glycosylation (NetOGlyc 3.1 server; http://www.cbs.dtu.dk
). Additional modifications such as phosphorylation, which has also been detected on other HCMV structural glycoproteins such as gB, could further add to the diffuse migration pattern seen in PAGE (34
The 22- to 28-kDa form of gpUL132 did not appear to carry N-linked sugars based on our finding that digestion with endoglycosidase H or PNGase F failed to yield a more rapidly migrating protein. The origin(s) of this smaller form of gpUL132 form is unclear at present. Splicing of the mRNA and thus the generation of a smaller ORF is unlikely since we found only a single 3.6-kb RNA in infected cells; however, we cannot eliminate the possibility that the smaller form of gpUL132 arose from a minor population of spliced RNA that was not detected in this analysis. From data derived with RV-UL132-M2 it is clear that the carboxy-terminal domain of the protein is intact, indicating that the difference in mass between the two forms of gpUL132 must reside in the amino-terminal part of the protein.
Several explanations for the origin of the smaller forms gpUL132 are possible. The theoretical molecular mass of the unmodified UL132 polypeptide of strain AD169 is 29.9 kDa. Thus, it is conceivable that the small protein could represent a minimally modified form of gpUL132 showing a slightly aberrant migration in PAGE. However, the presence of some modification on this form of the molecule is indicated by its diffuse migration in PAGE as well as the multiple forms recognized by the anti-UL132 serum in a number of HCMV strains. Alternatively, the protein might represent a proteolytic cleavage product or originate from an internal in-frame start codon, resulting in a truncated form of the protein. Internal initiation has also been observed for other HCMV structural proteins (43
). Interestingly, the smaller gpUL132 form was seen only in infected cells and extracellular virions but was not produced in transfected cells, indicating that the context of virus infection is prerequisite for the generation of the small forms of the gpUL132. In addition, a pulse-chase experiment of AD169-infected cells failed to reveal a precursor-product relationship between the larger and smaller forms of gpUL132, suggesting that the smaller form was not a proteolytic cleavage product of the larger form of gpUL132 (data not shown). Although the nature of the protein and the underlying mechanism(s) responsible for the generation of the small form of gpUL132 have yet to be defined, it is important to note that both forms are incorporated into virus particles in nearly equivalent amounts. This observation raises the possibility that both forms of gpUL132 could have roles in the replication and infectivity of HCMV.
Deletion of ORF UL132 from the genome of the laboratory strain AD169 as well as the low-passage isolate PAN resulted in a pronounced replication deficit. This effect could not be attributed to second-site mutations within our recombinant viruses because different and independent strategies were used to generate the two mutant viruses RV-delUL132/AD and RV-delUL132/PAN, making it unlikely that in both cases an additional mutation could result in the observed replication deficit, and a revertant virus that was constructed for strain AD169 showed replication kinetics identical to the wild type. Finally, strains AD169 and PAN contain ORF UL132 in a different genomic context, making it highly unlikely that disturbance in transcription or translation of adjacent genes accounts for the altered replication.
In addition, the replication deficit that was observed in our study using RV-delUL132/AD is in agreement with data generated during genomewide transposon-mediated insertional mutagenesis in strain AD169 (14
). In contrast, a recent study using the low-passage clinical isolate FIX came to the conclusion that deletion of ORF UL132 had no influence on the replication of the mutant virus in fibroblasts, although the virus replicated more slowly in endothelial cells (16
). We also noted that deletion of the UL132 ORF in a clinical virus isolate, PAN, had a lesser effect on virus replication in fibroblasts. This finding is somewhat difficult to interpret directly because none of these three viruses is isogenic and additional viral genes could influence the growth phenotype of these viruses in fibroblasts. However, a possible explanation for these discrepant findings could be a compensation in strain FIX of the defect introduced by the deletion of UL132 by additional gene products.
HCMV FIX represents a genetically complete virus since it encodes a number of functions, including endothelial cell tropism and transmission to leukocytes, that have been lost by AD169 and PAN. The genetic basis of the loss of function for these two strains has not been completely determined, but genes from the Ulb′ region, in particular UL128 to UL132 and UL146/147, are most probably key to these functions (16
). The genome of strain AD169 does not contain the Ulb′ region and consequently this virus has lost endothelial cell tropism as well as the capacity to be transmitted to leukocytes. The PAN virus represents an intermediate between FIX and AD169 in that it contains a Ulb′ region but has lost the endothelial cell tropism and leukocyte transfer potential. It is conceivable that the intermediate phenotype of PAN compared to FIX and AD169 with respect to replication of the mutant viruses might be the result of a partial compensation of the UL132 defect by a function provided by the Ulb′ region.
We can only speculate on the function of gpUL132 in the viral replication cycle, but at this point, it appears to have an important role in production of infectious progeny. This assumption is based on several observations.
(i) Quantitative real-time PCR showed that the number of HCMV genome copies in the supernatants of RV-UL132del/AD-infected cells was reduced in parallel to the reduction in infectious titers, indicating that smaller numbers of DNA-containing virions were produced by the deletion mutant.
(ii) The multistep growth curves showed that in the case of the RV-HB5 and RV-delUL132/AD viruses, titers in the tissue culture supernatant reached a plateau within the observation period of 11 days, indicating that the defect in the UL132 mutant virus is not simply a delay in replication. For strain PAN and the respective UL132 deletion mutant, peak virus titers were reached at the same day postinfection, again pointing to the fact that production of infectious virions was not delayed in the UL132 mutant virus.
(iii) The entry of UL132 mutant virus was not impaired compared to wild-type RV-HB5, indicating that gpUL132 has no major role in attachment and/or penetration of HCMV to fibroblasts.
The current model of herpesvirus morphogenesis postulates that capsids obtain a primary envelope as they pass through the inner nuclear membrane. On exit from the outer nuclear membrane, they undergo deenvelopment. Capsids that are released in the cytoplasm are subsequently coated with tegument proteins and finally wrapped by membranes of a post-Golgi compartment that has been termed the assembly compartment (36
). This compartment has been shown to contain proteins found in the trans-Golgi network and late endosomes (18
). Thus, the structural glycoproteins must be targeted to this compartment and, late in infection, become concentrated within this organelle. This proposed assembly pathway appears to be a default assembly pathway for all herpesviruses (29
). However, it should be emphasized that the number of proteins required for assembly of an infectious particle and their specific function(s) in virion morphogenesis likely vary significantly between individual families of the herpesviruses (29
). Thus, it could be difficult to extrapolate findings from studies of the assembly pathway of a specific herpesvirus to other members of this diverse family.
The finding that recombinant gpUL132 can be localized to the trans-Golgi network in the absence of other viral functions demonstrates that, similar to other structural glycoproteins of HCMV such as gB, gpUL132 contains all of the cis
-acting elements necessary for trans-Golgi network localization. This is in contrast to a number of other HCMV structural glycoproteins, including gH, gM, gN, and gpTRL10, which require complex formation with other viral proteins in order to reach the more distal parts of the secretory system (22
). Inspection of the gpUL132 cytoplasmic domain for the presence of trafficking motifs that are known to function in these processes reveals a number of motifs that may be involved in this intracellular trafficking. Three tyrosine-based motifs, YXXΦ (where Y is tyrosine, X is any amino acid, and Φ is any bulky hydrophobic amino acid), are found at positions YQRL162
. YXXΦ motifs mediate the incorporation of membrane proteins into transport vesicles due to interaction with cellular adaptor proteins (reviewed in reference 4). In addition, gpUL132 contains acidic cluster motifs which potentially interact with the connector protein PACS-1 (phosphofurin acidic cluster sorting protein 1), which redirects proteins from the endosomes to the trans-Golgi network, a proposed site of tegument assembly and virion envelopment (15
). Finally, gpUL132 contains a dileucine-based sorting signal (DEEAVNLL127
) which is potentially also involved in binding to cellular adaptor protein complexes. Corresponding motifs in alphaherpesviruses are responsible for direct transport of glycoproteins to membranes that are involved in cell-cell contact (28
). Thus, it seems likely that trafficking of gpUL132 involves transport to the plasma membrane and recycling from there to the endosomal compartment involved in virus envelopment. Future experiments will test this hypothesis.
In summary, we have shown that the protein product of ORF UL132 of HCMV represents a structural viral component and that deletion of the reading frame results in a drastic replication deficit in low-passage and laboratory-adapted strains of HCMV. Further studies will be aimed at clarifying the function of gpUL132 during the replication of HCMV.