Envelopment of varicella-zoster virus: targeting of viral glycoproteins to the trans-Golgi network.

Previous studies suggested that varicella-zoster virus derives itsfinal envelope from thetrans-Golgi network (TGN) and that envelope glycoproteins (gps) are transported to the TGN independently of nucleocapsids. We tested the hypothesis that gpI is targeted to the TGN as a result of a signal sequence or patch encoded in its cytosolic domain. cDNAs encoding gpI wild type (wt) and a truncated mutant gpI(trc) lacking transmembrane andcytosolicdomainswereclonedbyusingthePCR.CellstransfectedwithcDNAencodinggpI(wt)orgpI(trc) synthesizedandNglycosylatedtheproteins.gpI(wt)accumulatedintheTGN,somereachedtheplasmalemma, butnonewassecreted.Incontrast,gpI(trc)wasretainedandprobablydegradedintheendoplasmicreticulum; nonewasfoundoncellsurfaces,butsomewassecreted.ThedistributionofgpI(trc)wasnotaffectedbydeletion of potential glycosylation sites. To locate a potential gpI-targeting sequence, cells were transfected with cDNA encoding chimeric proteins in which the ectodomain of a plasmalemmal marker, the interleukin-2 receptor (tac),wasfusedtodifferentdomainsofgpI.Achimericproteininwhichtacwasfusedwiththetransmembrane andcytoplasmicdomainsofgpIwastargetedtotheTGN.Incontrast,achimericproteininwhichtacwasfused only with the gpI transmembrane domain passed through the TGN and concentrated in endosomes. We conclude that gpI is targeted to the TGN as a result of a targeting sequence or patch in its cytosolic domain. Studies of the transport and modification of viral envelope proteins have made important contributions to the understanding of the pathways and mechanisms of viral maturation (11, 25). Insight into the steps by which a virus is assembled from its components can provide information that may ultimately lead to antiviral therapies. A critical element in the maturation of a virus is the mechanism by which the viral nucleocapsid becomes enclosed in an infection-competent membrane.Thesiteofthisenvelopmentmaybedeterminedby the targeting of the viral envelope glycoproteins (gps). For example, the E1 and E2 gps of rubella virus are transported to the Golgi region, which is believed to be the site of its envelopment(13,14),whereasthegpsofcoronavirus,flavivirus,and rotavirus accumulate in the endoplasmic reticulum (ER), which is thought to provide the envelope for these virions (23). The most extensively investigated of the alphaherpesviruses has been herpes simplex virus; however, the site at which herpes simplex viruses are enveloped is controversial. Electron microscopic observations first suggested the hypothesis that the viruses acquire an envelope while budding through the inner nuclear membrane (5, 20, 21). This idea has received considerable support; thus, herpes simplex virus gps have been localized to the nuclear membrane by immunofluorescence, immunoelectron microscopy, and subcellular fractionation (3, 5, 25). Unfortunately, however, studies of the localization of herpes simplex virus gps have not been able definitively to exclude the possibility that the gps are located in the outer, rather than the inner, nuclear membrane. Since the outer nuclear membrane is continuous with the ER, the localization of gps in the nuclear envelope could reflect only their site of biosynthesis and not targeting related to viral envelopment. A second alphaherpesvirus, pseudorabies virus, has been re