Identification of Glycoprotein gpTRL10 as a Structural Component of Human Cytomegalovirus

Abstract

Human cytomegalovirus (HCMV) has a coding capacity for glycoproteins which far exceeds that of other herpesviruses. Few of these proteins have been characterized. We have investigated the gene product(s) of reading frame 10, which is present in both the internal and terminal repeat regions of HCMV strain AD169 and only once in clinical isolates. The putative protein product is a 171-amino-acid glycoprotein with a theoretical mass of 20.5 kDa. We characterized the protein encoded by this reading frame in the laboratory strain AD169 and a recent isolate, TB40E. The results from both strains were comparable. Northern blot analyses showed that the gene was transcribed with early/late kinetics. Two proteins of 22 and 23.5-kDa were detected in virus-infected cells and in cells transiently expressing recombinant TRL10. Both forms contained only high-mannose-linked carbohydrate modifications. In addition, virus-infected cells expressed small amounts of the protein modified with complex N-linked sugars. Image analysis localized transiently expressed TRL10 to the endoplasmic reticulum. Immunoblot analyses as well as immunoelectron microscopy of purified virions demonstrated that TRL10 represents a structural component of the virus particle. Immunoblot analysis in the absence of reducing agents indicated that TRL10, like the other HCMV envelope glycoproteins, is present in a disulfide-linked complex. Sequence analysis of the TRL10 coding region in nine low-passage clinical isolates revealed strain-specific variation. In summary, the protein product of the TRL10 open reading frame represents a novel structural glycoprotein of HCMV and was termed gpTRL10.

Deletion of gpUL132, a Structural Component of Human Cytomegalovirus, Results in Impaired Virus Replication in Fibroblasts

“…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 (22,26,39). 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.…”

“…The products of the readings frames UL4 (gp48) and TRL10 (gpTRL10) have been reported to be structural components of the virion (11,39). Both proteins are dispensable for replication of HCMV in fibroblasts, but no function has been attributed to either one (17,40).…”

Complex Formation by Glycoproteins M and N of Human Cytomegalovirus: Structural and Functional Aspects

“…Next we examined the possibility of nonspecific interaction between gM and unrelated viral glycoproteins by coexpression of gM and gpTRL10, an HCMV virion glycoprotein whose expression is restricted to the ER in the absence of additional viral functions (36). When coexpressed with gM, gpTRL10 remained localized to the ER, indicating that gM did not provide a chaperone function for this HCMV glycoprotein (Fig.…”

Human Cytomegalovirus Infection Elicits a Glycoprotein M (gM)/gN-Specific Virus-Neutralizing Antibody Response

“…At 48 h posttransfection, the cells were washed twice with DPBS, fixed with 4% (wt/vol) paraformaldehyde, washed three times with DPBS, permeabilized with permeabilizing solution (0.1% NP-40, 0.01% SDS in DPBS) for 5 min, washed again, and blocked for 1 hour with DPBS containing 10% (vol/vol) goat serum. To evaluate protein expression, cells were incubated with primary mouse MAbs 65-8 (anti-pp65), 7-17 (anti-gB), IMP-91 (anti-gM), 14-16A (anti-gM/gN complex), 36-14 (antipp150), and 14-4b (anti-gH), and with anti-myc MAb 9E10 (for detection of myc-tagged gpTRL10) as described in previous studies (1,5,6,8,28,40,43). Primary incubations were performed at 37°C for 1 h. After the slides were washed with DPBS, they were incubated with FITC-conjugated goat anti-mouse IgG plus IgM at a 1:40 dilution for 1 h at 37°C.…”