Optimal Replication of Human Cytomegalovirus Correlates with Endocytosis of Glycoprotein gpUL132

Abstract

Envelopment of a herpesvirus particle is a complex process of which much is still to be learned. We previously identified the glycoprotein gpUL132 of human cytomegalovirus (HCMV) as an envelope component of the virion. In its carboxy-terminal portion, gpUL132 contains at least four motifs for sorting of transmembrane proteins to endosomes; among them are one dileucine-based signal and three tyrosine-based signals of the YXXØ and NPXY (where X stands for any amino acid, and Ø stands for any bulky hydrophobic amino acid) types. To investigate the role of each of these trafficking signals in intracellular localization and viral replication, we constructed a panel of expression plasmids and recombinant viruses in which the signals were rendered nonfunctional by mutagenesis. In transfected cells wild-type gpUL132 was mainly associated with the trans-Golgi network. Consecutive mutation of the trafficking signals resulted in increasing fractions of the protein localized at the cell surface, with gpUL132 mutated in all four trafficking motifs predominantly associated with the plasma membrane. Concomitant with increased surface expression, endocytosis of mutant gpUL132 was reduced, with a gpUL132 expressing all four motifs in mutated form being almost completely impaired in endocytosis. The replication of recombinant viruses harboring mutations in single trafficking motifs was comparable to replication of wild-type virus. In contrast, viruses containing mutations in three or four of the trafficking signals showed pronounced deficits in replication with a reduction of approximately 100-fold. Moreover, recombinant viruses expressing gpUL132 with three or four trafficking motifs mutated failed to incorporate the mutant protein into the virus particle. These results demonstrate a role of endocytosis of an HCMV envelope glycoprotein for incorporation into the virion and optimal virus replication.Morphogenesis of herpesviruses is a complex multistep process which requires the assembly of an infectious viral particle containing Ͼ70 proteins. The proteins are organized into distinct regions in the particle, including the capsid containing viral DNA, the tegument, and the envelope of the virion, suggesting an ordered addition of viral proteins during assembly.Most data in the literature favor a model in which the final virion is assembled by multiple budding and fusion events of the nascent particle. According to this model, genome-containing capsids acquire a first envelope by budding at the inner nuclear membrane in a step termed primary envelopment (34).

A Tyrosine-Based Trafficking Motif of the Tegument Protein pUL71 Is Crucial for Human Cytomegalovirus Secondary Envelopment

“…The importance of these signals has been analyzed in many cases. Mutation of such trafficking signals, e.g., in HCMV gpUL132 (14), results in accumulation of gpUL132 at the plasma membrane in infection, similarly to what is shown in this study for pUL71 in YXX⌽ mutant virus infection. In contrast to studies of glycoproteins, which have a transmembrane domain for membrane anchoring, relatively few studies of endocytosis have been carried out with proteins peripherally associated with the cytoplasmic side of membranes.…”

Regulation of Human Cytomegalovirus Secondary Envelopment by a C-Terminal Tetralysine Motif in pUL71

“…Sequence examination revealed two motifs within the 14 endmost C-terminal amino acids: a dileucine motif ( 357 LL 358 ) and a positively charged tetralysine motif ( 348 KKKK 351 ). Dileucine motifs are known trafficking motifs that regulate sorting of transmembrane proteins to endosomes and lysosomes (36,37). The function of positively charged amino acids is less clear.…”

The Polymorphic HCMV Glycoprotein UL20 Is Targeted for Lysosomal Degradation by Multiple Cytoplasmic Dileucine Motifs

“…For example, the mouse cytomegalovirus (MCMV)-encoded m06 uses dileucine motifs to target MHC class I for degradation (38). HCMV-encoded proteins like the envelope components glycoprotein B and UL132 also harbor functionally relevant dileucine-based or tyrosine-based sorting signals, respectively (39,40). Furthermore, the chemokine receptor-like proteins US27, US28 and UL33 were found to localize in endosomal compartments with US27 and US33 incorporating into viral envelopes as shown by electron microscopy (41,42).…”