ABSTRACTHerpes simplex virus (HSV) and varicella-zoster virus (VZV) are two pathogenic human alphaherpesviruses whose intracellular assembly is thought to follow different pathways. VZV presumably acquires its envelope in thetrans-Golgi network (TGN), and it has recently been shown that its major envelope glycoprotein, VZV-gE, accumulates in this compartment when expressed alone. In contrast, the envelopment of HSV has been proposed to occur at the inner nuclear membrane, although to which compartment the gE homolog (HSV-gE) is transported is unknown. For this reason, we have studied the intracellular traffic of HSV-gE and have found that this glycoprotein accumulates at steady state in the TGN, both when expressed from cloned cDNA and in HSV-infected cells. In addition, HSV-gE cycles between the TGN and the cell surface and requires a conserved tyrosine-containing motif within its cytoplasmic tail for proper trafficking. These results show that VZV-gE and HSV-gE have similar intracellular trafficking pathways, probably reflecting the presence of similar sorting signals in the cytoplasmic domains of both molecules, and suggest that the respective viruses, VZV and HSV, could use the same subcellular organelle, the TGN, for their envelopment.

In mammalian cells, the mannose 6-phosphate receptors (MPRs) and the lysosomal glycoproteins, lysosomal-associated membrane protein (LAMP) I, lysosomal integral membrane protein (LIMP) II, are directly transported from the trans-Golgi network to endosomes and lysosomes. While MPR traffic relies on the AP-1 adaptor complex, we report that proper targeting of LAMP I and LIMP II to lysosomes requires the AP-3 adaptor-like complex. Overexpression of these proteins, which contain either a tyrosine- or a di-leucine-based-sorting motif, promotes AP-3 recruitment on membranes. Inhibition of AP-3 function using antisense oligonucleotides leads to a selective misrouting of both LAMP I and LIMP II to the cell surface without affecting MPR trafficking. These results provide evidence that AP-3 functions in the intracellular targeting of transmembrane glycoproteins to lysosomes.

The varicella-zoster virus (VZV) is the etiological agent of two different human pathologies, chickenpox (varicella) and shingles (zoster). This alphaherpesvirus is believed to acquire its lipidic envelope in the trans-Golgi network (TGN). This is consistent with previous data showing that the most abundant VZV envelope glycoprotein gE accumulates at steady-state in this organelle when expressed from cloned cDNA. In the present study, we have investigated the intracellular trafficking of gI, another VZV envelope glycoprotein. In transfected cells, this protein shows a very slow biosynthetic transport to the cell surface where it accumulates. However, upon co-expression of gE, gI experiences a dramatic increase in its exit rate from the endoplasmic reticulum, it accumulates in a sialyltransferase-positive compartment, presumably the TGN, and cycles between this compartment and the cell surface. This differential behavior results from the ability of gE and gI to form a complex in the early stages of the biosynthetic pathway whose intracellular traffic is exclusively determined by the sorting information in the tail of gE. Thus, gI provides the first example of a molecule localized to the TGN by means of its association with another TGN protein. We also show that, during the early stages of VZV infection, both proteins are also found in the TGN of the host cell. This suggests the existence of an intermediate stage during VZV biogenesis in which the envelope glycoproteins, transiently arrested in the TGN, could promote the envelopment of newly synthesized nucleocapsids into this compartment and, therefore, the assembly of infective viruses.

In polarized Madin-Darby canine kidney (MDCK) cells, sorting of membrane proteins in the trans-Golgi network for basolateral delivery depends on the presence of cytoplasmic determinants that are related or unrelated to clathrin-coated pit localization signals. Whether these signals mediate basolateral protein sorting through common or distinct pathways is unknown. The cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor (CD-MPR) contains clathrin-coated pit localization signals that are necessary for endocytosis and lysosomal enzyme targeting. In this study, we have addressed the function of these signals in polarized sorting of the CD-MPR. A chimeric protein, made of the luminal domain of the influenza virus hemagglutinin fused to the transmembrane and cytoplasmic domains of the CD-MPR was stably expressed in MDCK cells. This chimera (HCD) is able to interact with the AP-1 Golgi-specific assembly proteins and is detected on the basolateral plasma membrane of MDCK cells where it is endocytosed. Deletion analysis and site-directed mutagenesis of the cytoplasmic domain of the CD-MPR indicate that HCD chimeras devoid of clathrin-coated pit localization signals are still transported to the basolateral membrane where they accumulate. A HCD chimera containing only the transmembrane domain and the 12 membrane-proximal amino acids of the CD-MPR cytoplasmic tail is also found on the basolateral membrane but is unable to interact with the AP-1 assembly proteins. However, the overexpression of this mutant results in partial apical delivery. It is concluded, therefore, that the basolateral transport of this chimera requires a saturable sorting machinery distinct from AP-1.