Targeting of a heterodimeric membrane protein complex to the Golgi: rubella virus E2 glycoprotein contains a transmembrane Golgi retention signal.

Rubella virus (RV) envelope glycoproteins, E2 and E1, form a heterodimeric complex that is targeted to medial/trans-Golgi cisternae. To identify the Golgi targeting signal(s) for the E2/E1 spike complex, we constructed chimeric proteins consisting of domains from RV glycoproteins and vesicular stomatitis virus (VSV) G protein. The location of the chimeric proteins in stably transfected Chinese hamster ovary cells was determined by immunofluorescence, immunoelectron microscopy, and by the extent of processing of their N-linked glycans. A trans-dominant Golgi retention signal was identified within the C-terminal region of E2. When the transmembrane (TM) and cytoplasmic (CT) domains of VSV G were replaced with those of RV E2, the hybrid protein (G-E2TMCT+) was retained in the Golgi. Transport of G-E2TMCT+ to the Golgi was rapid (t1/2 = 10-20 min). The G-E2TMCT+ protein was determined to be distal to or within the medial Golgi based on acquisition of endo H resistance but proximal to the trans-Golgi network since it lacked sialic acid. Deletion analysis revealed that only the TM domain of E2 was required for Golgi targeting. Although the cytoplasmic domain of E2 was not necessary for Golgi retention, it was required for efficient transport of VSV G-RV chimeras out of the endoplasmic reticulum. When assayed in sucrose velocity sedimentations gradients, the Golgi-retained G-E2TMCT+ protein behaved as a dimer. Unlike virtually all other Golgi targeting signals, the E2 TM domain does not contain any polar amino acids. The TM and CT domains of E1 were not required for targeting of E2 and E1 to the Golgi indicating that a heterodimer of two integral membrane proteins can be retained in the Golgi by a single retention signal.     

Targeting of a Short Peptide Derived from the Cytoplasmic Tail of the G1 Membrane Glycoprotein of Uukuniemi Virus (Bunyaviridae) to the Golgi Complex

Members of the Bunyaviridae family acquire an envelope by budding through the lipid bilayer of the Golgi complex. The budding compartment is thought to be determined by the accumulation of the two heterodimeric membrane glycoproteins G1 and G2 in the Golgi. We recently mapped the retention signal for Golgi localization in one Bunyaviridae member (Uukuniemi virus) to the cytoplasmic tail of G1. We now show that a myc-tagged 81-residue G1 tail peptide expressed in BHK21 cells is efficiently targeted to the Golgi complex and retained there during a 3-h chase. Green-fluorescence protein tagged at either end with this peptide or with a C-terminally truncated 60-residue G1 tail peptide was also efficiently targeted to the Golgi. The 81-residue peptide colocalized with mannosidase II (a medial Golgi marker) and partially with p58 (an intermediate compartment marker) and TGN38 (a trans-Golgi marker). In addition, the 81-residue tail peptide induced the formation of brefeldin A-resistant vacuoles that did not costain with markers for other membrane compartments. Removal of the first 10 N-terminal residues had no effect on the Golgi localization but abolished the vacuolar staining. The shortest peptide still able to become targeted to the Golgi encompassed residues 10 to 40. Subcellular fractionation showed that the 81-residue tail peptide was associated with microsomal membranes. Removal of the two palmitylation sites from the tail peptide did not affect Golgi localization and had only a minor effect on the association with microsomal membranes. Taken together, the results provide strong evidence that Golgi retention of the heterodimeric G1-G2 spike protein complex of Uukuniemi virus is mediated by a short region in the cytoplasmic tail of the G1 glycoprotein.     

Mapping the Golgi targeting and retention signal of Bunyamwera virus glycoproteins

The membrane glycoproteins (Gn and Gc) of Bunyamwera virus (BUN; family Bunyaviridae) accumulate in the Golgi complex, where virion maturation occurs. The Golgi targeting and retention signal has previously been shown to reside within the Gn protein. A series of truncated Gn and glycoprotein precursor cDNAs were constructed by progressively deleting the coding region of the transmembrane domain (TMD) and the cytoplasmic tail. We also constructed chimeric proteins of BUN Gc, enhanced green fluorescent protein (EGFP), and human respiratory syncytial virus (HRSV) fusion (F) protein that contain the Gn TMD with various lengths of its adjacent cytoplasmic tails. The subcellular localization of mutated BUN glycoproteins and chimeric proteins was investigated by double-staining immunofluorescence with antibodies against BUN glycoproteins or the HRSV F protein and with antibodies specific for the Golgi complex. The results revealed that Gn and all truncated Gn proteins that contained the intact TMD (residues 206 to 224) were able to translocate to the Golgi complex and also rescued the Gc protein, which is retained in the endoplasmic reticulum when expressed alone, to this organelle. The rescued Gc proteins acquired endo-beta-N-acetylglucosaminidase H resistance. The Gn TMD could also target chimeric EGFP to the Golgi and retain the F protein, which is characteristically expressed on the surface of HRSV-infected cells, in the Golgi. However, chimeric BUN Gc did not translocate to the Golgi, suggesting that an interaction with Gn is involved in Golgi retention of the Gc protein. Collectively, these data demonstrate that the Golgi targeting and retention signal of BUN glycoproteins resides in the TMD of the Gn protein.     

The membrane-proximal stem region of vesicular stomatitis virus G protein confers efficient virus assembly

In this report, we show that the glycoprotein of vesicular stomatitis virus (VSV G) contains within its extracellular membrane-proximal stem (GS) a domain that is required for efficient VSV budding. To determine a minimal sequence in GS that provides for high-level virus assembly, we have generated a series of recombinant DeltaG-VSVs which express chimeric glycoproteins having truncated stem sequences. The recombinant viruses having chimeras with 12 or more membrane-proximal residues of the G stem, and including the G protein transmembrane-cytoplasmic tail domains, produced near-wild-type levels of particles. In contrast, viruses encoding chimeras with shorter or no G-stem sequences produced approximately 10- to 20-fold less. This budding domain when present in chimeric glycoproteins also promoted their incorporation into the VSV envelope. We suggest that the G-stem budding domain promotes virus release by inducing membrane curvature at sites where virus budding occurs or by recruiting condensed nucleocapsids to sites on the plasma membrane which are competent for efficient virus budding.     

Uukuniemi virus glycoproteins accumulate in and cause morphological changes of the Golgi complex in the absence of virus maturation.

We have studied the transport of the Uukuniemi virus membrane glycoproteins in baby hamster kidney and chick embryo cells by using a temperature-sensitive mutant (ts12). Uukuniemi virus assembles in the Golgi complex, where both glycoproteins G1 and G2 and nucleocapsid protein N accumulate (E. Kuismanen, B. B?ng, M. Hurme, and R. F. Pettersson, J. Virol. 51:137-146, 1984). At the restrictive temperature (39 degrees C), the glycoproteins of ts12 were transported to the Golgi complex as in wild-type, virus-infected cells, whereas the nucleocapsid protein failed to accumulate there. Pulse-chase labeling followed by immunoprecipitation and treatment with endo-beta-N-acetylglucosaminidase H showed that G1 synthesized at 39 degrees C in ts12-infected cells had an altered mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting a lack of terminal glycosylation. The typical Uukuniemi virus-induced vacuolization and expansion of the Golgi complex could be seen also in ts12-infected cells at 39 degrees C, although no virus particles were formed. This suggests that the morphological changes were induced by the Uukuniemi virus glycoproteins. In wild-type virus- or ts12-infected cells, G1 and G2 could not be chased out from the Golgi complex even after 6 h of treatment with cycloheximide. The glycoproteins were thus retained in the Golgi even under conditions when no virus maturation took place and when nucleocapsids did not accumulate in the Golgi region. Accordingly, the glycoproteins of Uukuniemi virus were found to have properties resembling those of Golgi-specific proteins. This virus model system may be useful in studying the synthesis and transport of membrane proteins that are transported to and retained in the Golgi.     

The stop transfer sequence of the human UDP-glucuronosyltransferase 1A determines localization to the endoplasmic reticulum by both static retention and retrieval mechanisms

Human UDP-glucuronosyltransferase 1A (UGT1A) isoforms are endoplasmic reticulum (ER)-resident type I membrane proteins responsible for the detoxification of a broad range of toxic phenolic compounds. These proteins contain a C-terminal stop transfer sequence with a transmembrane domain (TMD), which anchors the protein into the membrane, followed by a short cytosolic tail (CT). Here, we investigated the mechanism of ER residency of UGT1A mediated by the stop transfer sequence by analysing the subcellular localization and sensitivity to endoglycosidases of chimeric proteins formed by fusion of UGT1A stop transfer sequence (TMD/CT) with the ectodomain of the plasma membrane CD4 reporter protein. We showed that the stop transfer sequence, when attached to C-terminus of the CD4 ectodomain was able to prevent it from being transported to the cell surface. The protein was retained in the ER indicating that this sequence functions as an ER localization signal. Furthermore, we demonstrated that ER localization conferred by the stop transfer sequence was mediated in part by the KSKTH retrieval signal located on the CT. Interestingly, our data indicated that UGT1A TMD alone was sufficient to retain the protein in ER without recycling from Golgi compartment, and brought evidence that organelle localization conferred by UGT1A TMD was determined by the length of its hydrophobic core. We conclude that both retrieval mechanism and static retention mediated by the stop transfer sequence contribute to ER residency of UGT1A proteins.     

The transmembrane domain of hepatitis C virus glycoprotein E1 is a signal for static retention in the endoplasmic reticulum

Hepatitis C virus (HCV) glycoproteins E1 and E2 assemble to form a noncovalent heterodimer which, in the cell, accumulates in the endoplasmic reticulum (ER). Contrary to what is observed for proteins with a KDEL or a KKXX ER-targeting signal, the ER localization of the HCV glycoprotein complex is due to a static retention in this compartment rather than to its retrieval from the cis-Golgi region. A static retention in the ER is also observed when E2 is expressed in the absence of E1 or for a chimeric protein containing the ectodomain of CD4 in fusion with the transmembrane domain (TMD) of E2. Although they do not exclude the presence of an intracellular localization signal in E1, these data do suggest that the TMD of E2 is an ER retention signal for HCV glycoprotein complex. In this study chimeric proteins containing the ectodomain of CD4 or CD8 fused to the C-terminal hydrophobic sequence of E1 were shown to be localized in the ER, indicating that the TMD of E1 is also a signal for ER localization. In addition, these chimeric proteins were not processed by Golgi enzymes, indicating that the TMD of E1 is responsible for true retention in the ER, without recycling through the Golgi apparatus. Together, these data suggest that at least two signals (TMDs of E1 and E2) are involved in ER retention of the HCV glycoprotein complex.     

The cytoplasmic tail of the human respiratory syncytial virus F protein plays critical roles in cellular localization of the F protein and infectious progeny production

The importance of the F protein cytoplasmic tail (CT) for replication of human respiratory syncytial virus (HRSV) was examined by monitoring the behavior of viruses expressing F proteins with a modified COOH terminus. The F protein mutant viruses were recovered and amplified under conditions where F protein function was complemented by expression of a heterologous viral envelope protein. The effect of the F protein modifications was then examined in the context of a viral infection in standard cell types (Vero and HEp-2). The F protein modifications consisted of a deletion of the predicted CT or a replacement of the CT with the CT of the vesicular stomatitis virus (VSV) G protein. In addition, engineered HRSVs that lacked all homologous glycoprotein genes (SH, G, and F) and expressed instead either the authentic VSV G protein or a VSV G containing the HRSV F protein CT were examined. We found that deletion or replacement of the F protein CT seriously impaired the production of infectious progeny. Cells infected with viruses bearing CT modifications displayed increased F protein surface expression and increased syncytium formation. The distribution of F protein in the plasma membrane of infected cells was altered, resulting in an F protein that was evenly distributed rather than localized predominantly to virus-induced surface filaments. CT deletion or exchange also abrogated interaction of F protein with Triton-insoluble lipid rafts. Addition of the F protein CT to the VSV G protein, expressed as the only viral glycoprotein in an HRSV genome, had the opposite effects: the number of infectious progeny was higher, the surface distribution was changed from relatively even to localized, and the proportion of VSV G protein associated with lipid rafts was higher. Together, these results show that the HRSV F protein CT plays a critical role in F protein cellular localization and production of infectious virus and suggest that the function provided by the CT is independent of the F protein ectodomain and transmembrane domain and is mediated by F protein-lipid raft interaction.     

Uukuniemi virus maturation: accumulation of virus particles and viral antigens in the Golgi complex.

We studied the maturation of Uukuniemi virus and the localization of the viral surface glycoproteins and nucleocapsid protein in infected cells by electron microscopy, indirect immunofluorescence, and immunoelectron microscopy with specific antisera prepared in rabbits against the two glycoproteins G1 and G2 and the nucleocapsid protein N. Electron microscopy of thin sections from infected cells showed virus particles maturing at smooth-surfaced membranes close to the nucleus. Localization of the G1/G2 and N proteins by indirect immunofluorescence at different stages after infection showed the antigens to be present throughout the cell interior but concentrated in the juxtanuclear region. The G1/G2 antiserum also appeared to stain the nuclear and plasma membranes. Double staining with tetramethylrhodamine isothiocyanate-conjugated wheat germ agglutinin, which preferentially stains the Golgi complex, and fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin G, which stained the G1/G2 or N proteins, showed that the staining of the juxtanuclear region coincided. Similarly, double staining for thiamine pyrophosphatase, an enzyme activity specific for the Golgi complex, showed the fluorescence and the cytochemical stain to coincide in the juxtanuclear region. Immunoperoxidase electron microscopy of cells permeabilized with saponin revealed that the viral glycoproteins were present in the rough endoplasmic reticulum and the nuclear and Golgi membranes; the latter was heavily stained. With this method, the N protein was localized to the cytoplasm, especially around smooth-surfaced vesicles in the Golgi region. Taken together, the results indicate that Uukuniemi virus and its structural proteins accumulate in the Golgi complex, supporting the idea that this compartment rather than the plasma membrane is the site of virus maturation. This raises the interesting possibility that deficient transport of the glycoproteins to the plasma membrane and hence their accumulation in the Golgi complex determines the site of virus maturation.     

Functional Role of Hepatitis C Virus Chimeric Glycoproteins in the Infectivity of Pseudotyped Virus

The putative envelope glycoproteins of hepatitis C virus (HCV) likely play an important role in the initiation of viral infection. Available information suggests that the genomic regions encoding the putative envelope glycoproteins, when expressed as recombinant proteins in mammalian cells, largely accumulate in the endoplasmic reticulum. In this study, genomic regions which include the putative ectodomain of the E1 (amino acids 174 to 359) and E2 (amino acids 371 to 742) glycoproteins were appended to the transmembrane domain and cytoplasmic tail of vesicular stomatitis virus (VSV) G protein. This provided a membrane anchor signal and the VSV incorporation signal at the carboxy termini of the E1 and E2 glycoproteins. The chimeric gene constructs exhibited expression of the recombinant proteins on the cell surface in a transient expression assay. When infected with a temperature-sensitive VSV mutant (ts045) and grown at the nonpermissive temperature (40.5°C), cells transiently expressing the E1 or E2 chimeric glycoprotein generated VSV/HCV pseudotyped virus. The resulting pseudotyped virus generated from E1 or E2 surprisingly exhibited the ability to infect mammalian cells and sera derived from chimpanzees immunized with the homologous HCV envelope glycoproteins neutralized pseudotyped virus infectivity. Results from this study suggested a potential functional role for both the E1 and E2 glycoproteins in the infectivity of VSV/HCV pseudotyped virus in mammalian cells. These observations further suggest the importance of using both viral glycoproteins in a candidate subunit vaccine and the potential for using a VSV/HCV pseudotyped virus to determine HCV neutralizing antibodies.     

The paramyxovirus fusion protein C-terminal region: mutagenesis indicates an indivisible protein unit

Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Fusion is mediated by the viral fusion (F) protein, and it undergoes large irreversible conformational changes to cause membrane merger. The C terminus of PIV5 F contains a membrane-proximal 7-residue external region (MPER), followed by the transmembrane (TM) domain and a 20-residue cytoplasmic tail. To study the sequence requirements of the F protein C terminus for fusion, we constructed chimeras containing the ectodomain of parainfluenza virus 5 F (PIV5 F) and either the MPER, the TM domain, or the cytoplasmic tail of the F proteins of the paramyxoviruses measles virus, mumps virus, Newcastle disease virus, human parainfluenza virus 3, and Nipah virus. The chimeras were expressed, and their ability to cause cell fusion was analyzed. The chimeric proteins were variably expressed at the cell surface. We found that chimeras containing the ectodomain of PIV5 F with the C terminus of other paramyxoviruses were unable to cause cell fusion. Fusion could be restored by decreasing the activation energy of refolding through introduction of a destabilizing mutation (S443P). Replacing individual regions, singly or doubly, in the chimeras with native PIV5 F sequences restored fusion to various degrees, but it did not have an additive effect in restoring activity. Thus, the F protein C terminus may be a specific structure that only functions with its cognate ectodomain. Alanine scanning mutagenesis of MPER indicates that it has a regulatory role in fusion since both hyperfusogenic and hypofusogenic mutations were found.     

Retargeting of viral glycoproteins into a non-exporting compartment during the myogenic differentiation of rat L6 cells

We have analysed protein trafficking during the differentiation of rat L6 myoblasts into myotubes. Different proteins were found to lose different amounts of their processing by the Golgi apparatus during the myogenic differentiation, indicating that they were transported to this organelle with differing efficiencies. In order to investigate the destination of the nonprocessed glycoproteins we analysed the behaviour of vesicular stomatitis virus (VSV) and Semliki Forest virus glycoproteins in the presence of Brefeldin A, which returns the enzymes of the Golgi apparatus to the ER. Such experiments indicated that during myogenesis a fraction of both glycoproteins was shunted into a compartment that did not participate recycling with the Golgi apparatus. Immunofluorescence studies with the mutant VSV tsO45 G protein suggested that this compartment was diffusively distributed. We investigated whether the cytoplasmic tail had a role in the myogenic transport modulation by analysing the behaviour of recombinant VSV G proteins. Exchanging the cytoplasmic tail or the tail plus the membrane anchor had no effect, suggesting that the luminal portion was responsible for the diverted transport. Taken together, the results suggest that during the myogenesis of L6 myoblasts, varying fractions of different viral glycoproteins were sorted from the ER into a specific compartment that did not recycle with the Golgi apparatus.     

Uukuniemi virus maturation: immunofluorescence microscopy with monoclonal glycoprotein-specific antibodies.

Monoclonal antibodies directed against Uukuniemi virus glycoproteins G1 and G2 in combination with polyclonal antibodies against the nucleoprotein (N) were used to study the maturation of the virus in Golgi complexes of infected chicken embryo fibroblasts and BHK cells. Of 25 monoclonal antibodies obtained, 10 were shown to be G1 specific and 15 were shown to be G2 specific by immunoblotting and immunoprecipitation. In double-staining experiments, some of the monoclonal antibodies gave similar distributions of fluorescence as compared with the staining obtained from polyclonal rabbit anti-G1-G2 antibodies. Others, however, preferentially stained either the glycoproteins in the Golgi complex or those at the cell surface. This may indicate that the glycoproteins underwent conformational changes during their transport. Uukuniemi virus infection resulted in the vacuolization of the membranes of Golgi complexes where the maturation of the virus was taking place. Double-staining experiments with monoclonal antibodies which preferentially stained the Golgi-associated viral glycoproteins and with anti-N polyclonal rabbit antiserum showed a correlation between the progressive vacuolization of the Golgi complex and the accumulation of viral nucleoprotein in the Golgi region, suggesting that a morphological alteration of the Golgi complex may be a prerequisite for intracellular maturation of the virus. Treatment of Uukuniemi virus-infected cells with tunicamycin, a drug which inhibits N-linked glycosylation, resulted in the accumulation of both glycoproteins at an intracellular location, apparently representing the endoplasmic reticulum. Double-staining experiments showed a parallel accumulation of nucleoprotein at these sites, indicating that local accumulation of glycoproteins is required for nucleoprotein binding to intracellular membranes.     

The transmembrane domain of the severe acute respiratory syndrome coronavirus ORF7b protein is necessary and sufficient for its retention in the Golgi complex

The severe acute respiratory syndrome coronavirus (SARS-CoV) ORF7b (also called 7b) protein is an integral membrane protein that is translated from a bicistronic open reading frame encoded within subgenomic RNA 7. When expressed independently or during virus infection, ORF7b accumulates in the Golgi compartment, colocalizing with both cis- and trans-Golgi markers. To identify the domains of this protein that are responsible for Golgi localization, we have generated a set of mutant proteins and analyzed their subcellular localizations by indirect immunofluorescence confocal microscopy. The N- and C-terminal sequences are dispensable, but the ORF7b transmembrane domain (TMD) is essential for Golgi compartment localization. When the TMD of human CD4 was replaced with the ORF7b TMD, the resulting chimeric protein localized to the Golgi complex. Scanning alanine mutagenesis identified two regions in the carboxy-terminal portion of the TMD that eliminated the Golgi complex localization of the chimeric CD4 proteins or ORF7b protein. Collectively, these data demonstrate that the Golgi complex retention signal of the ORF7b protein resides solely within the TMD.     

A Golgi retention signal in a membrane-spanning domain of coronavirus E1 protein

The E1 glycoprotein from an avian coronavirus is a model protein for studying retention in the Golgi complex. In animal cells expressing the protein from cDNA, the E1 protein is targeted to cis Golgi cisternae (Machamer, C. E., S. A. Mentone, J. K. Rose, and M. G. Farquhar. 1990. Proc. Natl. Acad. Sci. USA. 87:6944-6948). We show that the first of the three membrane-spanning domains of the E1 protein can retain two different plasma membrane proteins in the Golgi region of transfected cells. Both the vesicular stomatitis virus G protein and the alpha-subunit of human chorionic gonadotropin (anchored to the membrane by fusion with the G protein membrane-spanning domain and cytoplasmic tail) were retained in the Golgi region of transfected cells when their single membrane-spanning domains were replaced with the first membrane-spanning domain from E1. Single amino acid substitutions in this sequence released retention of the chimeric G protein, as well as a mutant E1 protein which lacks the second and third membrane-spanning domains. The important feature of the retention sequence appears to be the uncharged polar residues which line one face of a predicted alpha helix. This is the first retention signal to be defined for a resident Golgi protein. The fact that it is present in a membrane-spanning domain suggests a novel mechanism of retention in which the membrane composition of the Golgi complex plays an instrumental role in retaining its resident proteins.     

The pathway and targeting signal for delivery of the integral membrane glycoprotein LEP100 to lysosomes

A complete set of chimeras was made between the lysosomal membrane glycoprotein LEP100 and the plasma membrane-directed vesicular stomatitis virus G protein, combining a glycosylated lumenal or ectodomain, a single transmembrane domain, and a cytosolic carboxyl-terminal domain. These chimeras, the parent molecules, and a truncated form of LEP100 lacking the transmembrane and cytosolic domains were expressed in mouse L cells. Only LEP100 and chimeras that included the cytosolic 11 amino acid carboxyl terminus of LEP100 were targeted to lysosomes. The other chimeras accumulated in the plasma membrane, and truncated LEP100 was secreted. Chimeras that included the extracellular domain of vesicular stomatitis G protein and the carboxyl terminus of LEP100 were targeted to lysosomes and very rapidly degraded. Therefore, in chimera-expressing cells, virtually all the chimeric molecules were newly synthesized and still in the biosynthesis and lysosomal targeting pathways. The behavior of one of these chimeras was studied in detail. After its processing in the Golgi apparatus, the chimera entered the plasma membrane/endosome compartment and rapidly cycled between the plasma membrane and endosomes before going to lysosomes. In pulse-expression experiments, a large population of chimeric molecules was observed to appear transiently in the plasma membrane by immunofluorescence microscopy. Soon after protein synthesis was inhibited, this surface population disappeared. When lysosomal proteolysis was inhibited, chimeric molecules accumulated in lysosomes. These data suggest that the plasma membrane/early endosome compartment is on the pathway to the lysosomal membrane. This explains why mutations that block endocytosis result in the accumulation of lysosomal membrane proteins in the plasma membrane.     

Characterization of an endoplasmic reticulum retention signal in the rubella virus E1 glycoprotein.

Rubella virus contains three structural proteins, capsid, E2, and E1. E2 and E1 are type I membrane glycoproteins that form a heterodimer in the endoplasmic reticulum (ER) before they are transported to and retained in the Golgi complex, where virus assembly occurs. The bulk of unassembled E2 and E1 subunits are not transported to the Golgi complex. We have recently shown that E2 contains a Golgi-targeting signal that mediates retention of the E2-E1 complex (T. C. Hobman, L. Woodward, and M. G. Farquhar, Mol. Biol. Cell 6:7-20, 1995). The focus of this study was to determine if E1 glycoprotein also contains intracellular targeting information. We constructed a series of chimeric reporter proteins by fusing domains from E1 to the ectodomains of two other type I membrane proteins which are normally transported to the cell surface, vesicular stomatitis virus G protein (G) and CD8. Fusion of the E1 transmembrane and cytoplasmic regions, but not analogous domains from two control membrane proteins, to the ectodomains of G and CD8 proteins caused the resulting chimeras to be retained in the ER. Association of the ER-retained chimeras with known ER chaperone proteins was not detected. ER localization required both the transmembrane and cytoplasmic regions of E1, since neither of these domains alone was sufficient to retain the reporter proteins. Increasing the length of the E1 cytoplasmic domain by 10 amino acids completely abrogated ER retention. This finding also indicated that the chimeras were not retained as a result of misfolding. In summary, we have identified a new type of ER retention signal that may function to prevent unassembled E1 subunits and/or immature E2-E1 dimers from reaching the Golgi complex, where they could interfere with viral assembly. Accordingly, assembly of E2 and E1 would mask the signal, thereby allowing transport of the heterodimer from the ER.     

Membrane anchoring domain of herpes simplex virus glycoprotein gB is sufficient for nuclear envelope localization.

We have used the glycoprotein gB of herpes simplex virus type 1 (gB-1), which buds from the inner nuclear membrane, as a model protein to study localization of membrane proteins in the nuclear envelope. To determine whether specific domains of gB-1 glycoprotein are involved in localization in the nuclear envelope, we have used deletion mutants of gB-1 protein as well as chimeric proteins constructed by replacing the domains of the cell surface glycoprotein G of vesicular stomatitis virus with the corresponding domains of gB. Mutant and chimeric proteins expressed in COS cells were localized by immunoelectron microscopy. A chimeric protein (gB-G) containing the ectodomain of gB and the transmembrane and cytoplasmic domains of G did not localize in the nuclear envelope. When the ectodomain of G was fused to the transmembrane and cytoplasmic domains of gB, however, the resulting chimeric protein (G-gB) was localized in the nuclear envelope. Substitution of the transmembrane domain of G with the 69 hydrophobic amino acids containing the membrane anchoring domain of gB allowed the hybrid protein (G-tmgB) to be localized in the nuclear envelope, suggesting that residues 721 to 795 of gB can promote retention of proteins in the nuclear envelope. Deletion mutations in the hydrophobic region further showed that a transmembrane segment of 21 hydrophobic amino acids, residues 774 to 795 of gB, was sufficient for localization in the nuclear envelope. Since wild-type gB and the mutant and chimeric proteins that were localized in the nuclear envelope were also retained in the endoplasmic reticulum, the membrane spanning segment of gB could also influence retention in the endoplasmic reticulum.     

Retention of a cis Golgi protein requires polar residues on one face of a predicted alpha-helix in the transmembrane domain.

The first membrane-spanning domain (m1) of the model cis Golgi protein M (formerly called E1) from the avian coronavirus infectious bronchitis virus is required for targeting to the Golgi complex. When inserted in place of the membrane-spanning domain of a plasma membrane protein (vesicular stomatitis virus G protein), the chimeric protein ("Gm1") is retained in the Golgi complex of transfected cells. To determine the precise features of the m1 domain responsible for Golgi targeting, we produced single amino acid substitutions in m1 and analyzed their effects on localization of Gm1. Expression at the plasma membrane was used as the criterion for loss of Golgi retention. Rates of oligosaccharide processing were used as a measure of rate and efficiency of transport through the Golgi complex. We identified four uncharged polar residues that are critical for Golgi retention of Gm1 (Asn465, Thr469, Thr476, and Gln480). These residues line one face of a predicted alpha-helix. Interestingly, when the m1 domain of the homologous M protein from mouse hepatitis virus is inserted into the G protein reporter, the chimeric protein is not efficiently retained in the Golgi complex, but transported to the cell surface. Although it possesses three of the four residues we identified as important in the avian m1 sequence, other residues in the membrane-spanning domain from the mouse protein must prevent efficient recognition of the polar face within the lipid bilayer of the cis Golgi.     

Processing and membrane topology of the spike proteins G1 and G2 of Uukuniemi virus.

The membrane glycoproteins G1 and G2 of the members of the Bunyaviridae family are synthesized as a precursor from a single open reading frame. Here, we have analyzed the processing and membrane insertion of G1 and G2 of a member of the Phlebovirus genus, Uukuniemi virus. By expressing C-terminally truncated forms of the p10 precursor containing the whole of G1 and decreasing portions of G2, we found that processing in BHK21 cells occurred with an efficiency of about 50% if G1 was followed by 50 residues of G2, while complete processing occurred if 98, 150, or 200 residues of G2 were present. Surprisingly, processing of all truncated G2 forms was less efficient in HeLa cells. Proteinase K treatment of microsomes isolated from infected cells indicated that the C terminus of G1 is exposed on the cytoplasmic face. Using G1 tail peptide antisera, the tail was likewise found by immunofluorescence to be exposed on the cytoplasmic face in streptolysin O-permeabilized cells. By introducing stop codons at various positions of the G1 tail and at the natural cleavage site between G1 and G2 and expressing these mutants in BHK cells, we found that no further processing of the G1 C terminus occurred following cleavage of G2 by the signal peptidase. This was also supported by the finding that an antiserum raised against a peptide corresponding to the region immediately upstream from the G2 signal sequence reacted in immunoblotting with G1 from virions. Finally, we show that both G1 and G2 are palmitylated. Taken together, these results show that processing of p10 of Uukuniemi virus occurs cotranslationally at only one site, i.e., downstream of the internal G2 signal sequence. G1 and G2 are inserted as type I proteins into the lipid bilayer, leaving the G1 tail exposed on the cytoplasmic face of the membrane. Since the G2 tail is only 5 residues long, the G1 tail is likely to be responsible for the interaction with the nucleoproteins during the budding process, in addition to harboring a Golgi localization signal.