Altered cytoplasmic domains affect intracellular transport of the vesicular stomatitis virus glycoprotein

We have altered the structure of the COOH-terminus of the vesicular stomatitis virus (VSV) glycoprotein (G) by introducing deletions into a cDNA clone encoding G protein. We examined the effects of these deletions on intracellular transport of G protein after expression of the deleted genes in eucaryotic cells under control of the SV40 late promoter. To prevent readthrough of translation into vector sequences, we introduced synthetic DNA linkers containing translation stop codons at the site of the deletion. G proteins that lacked the cytoplasmic domain and most of the transmembrane domain were secreted slowly from the cells. Deletion mutants affecting the structure of the cytoplasmic domain fell into two classes. The first class completely arrested transport of the protein to the cell surface at a stage prior to acquisition of complex oligosaccharides. The second class showed severely reduced rates of complex sugar addition although the proteins were eventually transported to the cell surface. Indirect immunofluorescence microscopy suggested that mutant proteins in both classes may accumulate in the rough endoplasmic reticulum.     

Mutations of the Rous sarcoma virus env gene that affect the transport and subcellular location of the glycoprotein products

The envelope glycoproteins of Rous sarcoma virus (RSV), gp85 and gp37, are anchored in the membrane by a 27-amino acid, hydrophobic domain that lies adjacent to a 22-amino acid, cytoplasmic domain at the carboxy terminus of gp37. We have altered these cytoplasmic and transmembrane domains by introducing deletion mutations into the molecularly cloned sequences of a proviral env gene. The effects of the mutations on the transport and subcellular localization of the Rous sarcoma virus glycoproteins were examined in monkey (CV-1) cells using an SV40 expression vector. We found, on the one hand, that replacement of the nonconserved region of the cytoplasmic domain with a longer, unrelated sequence of amino acids (mutant C1) did not alter the rate of transport to the Golgi apparatus nor the appearance of the glycoprotein on the cell surface. Larger deletions, extending into the conserved region of the cytoplasmic domain (mutant C2), resulted in a slower rate of transport to the Golgi apparatus, but did not prevent transport to the cell surface. On the other hand, removal of the entire cytoplasmic and transmembrane domains (mutant C3) did block transport and therefore did not result in secretion of the truncated protein. Our results demonstrate that the C3 polypeptide was not transported to the Golgi apparatus, although it apparently remained in a soluble, nonanchored form in the lumen of the rough endoplasmic reticulum; therefore, it appears that this mutant protein lacks a functional sorting signal. Surprisingly, subcellular localization by internal immunofluorescence revealed that the C3 protein (unlike the wild type) did not accumulate on the nuclear membrane but rather in vesicles distributed throughout the cytoplasm. This observation suggests that the wild-type glycoproteins (and perhaps other membrane-bound or secreted proteins) are specifically transported to the nuclear membrane after their biosynthesis elsewhere in the rough endoplasmic reticulum.     

Membrane integration and intracellular transport of the coronavirus glycoprotein E1, a class III membrane glycoprotein

The E1-glycoprotein (Mr = 26,014; 228 amino acids) of mouse hepatitis virus A59 is a class III membrane glycoprotein which has been used in this study as a model system in the study of membrane integration and protein transport. The protein lacks an NH2-terminal cleavable signal sequence and spans the viral membrane three times. Hydrophobic domains I and III could serve as signal sequences for cotranslational membrane integration. Domain I alone was sufficient to translocate the hydrophilic NH2 terminus of E1 across the membranes as evidenced by glycosylation of a newly introduced N-glycosylation site. The COOH-terminal part of E1 involving amino acids Leu124 to Thr228 was found to associate tightly with membranes at the post-translational level, although this part of the molecule lacks pronounced hydrophobic sequences. Membrane protection assays with proteinase K showed that a 2-kDa hydrophilic fragment was removed from the COOH terminus of E1 indicating that the protein is largely embedded into the membrane. Microinjection of in vitro transcribed capped and polyadenylated mRNA into CV-1 cells or into secretory AtT20 pituitary tumor cells showed that the E1-protein accumulated in the Golgi but was not detectable at the plasma membrane or in secretory granules. The 28 NH2-terminal hydrophilic amino acid residues play no role in membrane assembly or in intracellular targeting. Various NH2-terminal portions of E1 were fused to Ile145 of the cytoplasmic N-protein of mouse hepatitis virus. The resulting hybrid proteins were shown to assemble into membranes in vitro and were detected either in the rough endoplasmic reticulum or transient vesicles of microinjected cells.     

Protein sorting to the vacuolar membrane.

The vacuolar membrane (tonoplast) of plant cells contains a polytopic integral membrane protein with six membrane-spanning domains and cytoplasmically oriented amino-terminal and carboxy-terminal domains. This protein, tonoplast intrinsic protein (TIP), is a member of the membrane intrinsic protein (MIP) family of proteins, a family of channel proteins found in a variety of organisms. In bean seeds, alpha-TIP is synthesized on the rough endoplasmic reticulum and its transport to the tonoplast is mediated by the secretory system. In this study, we report that a polypeptide segment that includes the sixth membrane domain and the cytoplasmic tail of 18 amino acids of alpha-TIP is sufficient to target the reporter protein phosphinotricine acetyltransferase to the tonoplast of stably transformed tobacco cells. To determine if the carboxy-terminal cytoplasmic tail of alpha-TIP contains important tonoplast targeting information, a deletion construct lacking the 15 carboxy-terminal amino acids was introduced for transient expression in tobacco cells; we found that the slightly truncated protein still accumulated in the tonoplast. From these results, we concluded that a transmembrane domain of a tonoplast protein probably contains sufficient information for transport to the tonoplast. Whether such transport occurs by bulk flow or involves specific cellular machinery remains to be determined.     

Defective assembly and intracellular transport of mutant paramyxovirus hemagglutinin-neuraminidase proteins containing altered cytoplasmic domains.

The hemagglutinin-neuraminidase (HN) integral membrane protein of paramyxoviruses is expressed at the cell surface as a tetramer consisting of a pair of disulfide-linked dimers. HN has a large C-terminal ectodomain, a 19-residue uncleaved signal-anchor domain, and a 17-residue N-terminal cytoplasmic tail. Various mutant HN genes were constructed to examine the role of residues flanking the signal-anchor domain, including the cytoplasmic tail, on assembly and intracellular transport of the HN glycoprotein. Expression of the altered genes showed that by 90 min after synthesis the majority of the mutant HN proteins were in a conformationally mature form as assayed by their reactivity with conformation-specific monoclonal antibodies. However, the mutant proteins showed varied endoplasmic reticulum-to-Golgi apparatus transport rates, ranging from that of wild-type HN (t1/2 approximately 90 min) to slowly transported molecules (t1/2 approximately 5 h) and to molecules in which transport was not detected. Pulse-chase experiments indicated that the altered HN molecules had a specific and transient interaction with the resident endoplasmic reticulum protein GRP78-BiP, and thus the altered HN molecules were not retained in the endoplasmic reticulum by a prolonged interaction with GRP78-BiP. Sucrose density gradient sedimentation analysis of the mutant HN molecules indicated that they all had an oligomeric form that differed from that of wild-type HN; most of the molecules were found as disulfide-linked dimers rather than as tetramers. These data suggest that the HN cytoplasmic tail may function in the assembly of the final transport-competent oligomeric form of HN and that mutant HN molecules with seemingly properly folded ectodomains are retained in the endoplasmic reticulum by an as yet unidentified mechanism. The possible role of the HN cytoplasmic tail as a signal for intracellular transport is discussed.     

Role of the nuclear envelope in synthesis, processing, and transport of membrane glycoproteins

The outer nuclear membrane is morphologically similar to rough endoplasmic reticulum. The presence of ribosomes bound to its cytoplasmic surface suggests that it could be a site of synthesis of membrane glycoproteins. We have examined the biogenesis of the vesicular stomatitis virus G protein in the nuclear envelope as a model for the biogenesis of membrane glycoproteins. G protein was present in nuclear membranes of infected Friend erythroleukemia cells immediately following synthesis and was transported out of nuclear membranes to cytoplasmic membranes with a time course similar to transport from rough endoplasmic reticulum (t 1/2 = 5-7 min). Temperature-sensitive mutations in viral membrane proteins which block transport of G protein from endoplasmic reticulum also blocked transport of G protein from the nuclear envelope. Friend erythroleukemia cells and NIH 3T3 cells differed in the fraction of newly synthesized G protein found in nuclear membranes, apparently reflecting the relative amount of nuclear membrane compared to endoplasmic reticulum available for glycoprotein synthesis. Nuclear membranes from erythroleukemia cells appeared to have the enzymatic activities necessary for cleavage of the signal sequence and core glycosylation of newly synthesized G protein. Signal peptidase activity was detected by the ability of detergent-solubilized membranes of isolated nuclei to correctly remove the signal sequence of human preplacental lactogen. RNA isolated from the nuclear envelope was highly enriched for G protein mRNA, suggesting that G protein was synthesized on the outer nuclear membrane rather than redistributing to nuclear membranes from endoplasmic reticulum before or during cell fractionation. These results suggest a mechanism for incorporation of membrane glycoproteins into the nuclear envelope and suggest that in some cell types the nuclear envelope is a major source of newly synthesized membrane glycoproteins.     

The carboxy-terminal 10 amino acid residues of cytochrome b5 are necessary for its targeting to the endoplasmic reticulum.

Cytochrome b5 is an integral membrane protein located on the outer surface of the endoplasmic reticulum (ER). This cytochrome is considered to be synthesized on free ribosomes and to be inserted post-translationally into the ER membrane, without participation of a signal recognition particle. To elucidate the signal responsible for targeting of cytochrome b5 to the ER membrane in vivo, DNAs encoding various derivatives of the cytochrome were constructed and introduced into cultured mammalian COS cells, and the subcellular distributions of the derivatives expressed in the cells were then analyzed. The deletion of more than 11 amino acid residues at the carboxy-terminal end of cytochrome b5 abolished the targeting and anchoring of the cytochrome to the ER membrane. Fusion proteins consisting of the carboxy-terminal 10 amino acid residues of cytochrome b5 and passenger proteins with the hydrophobic portion could be localized in the ER membrane. Thus, the last 10 amino acid residues of cytochrome b5 carry information necessary for the cytochrome to be targeted to the ER membrane.     

MHC class II-associated invariant chain contains a sorting signal for endosomal compartments

The invariant chain (Ii) is a transmembrane protein that associates with the MHC class II molecules in the endoplasmic reticulum. Expression of Ii in MHC class II-negative CV1 cells showed that it acquired complex-type oligosaccharide side chains and was retained in endosomal compartments. To search for a sorting signal, we made progressive deletions from the cytoplasmic N-terminus of Ii. Deleting 11 amino acid residues resulted in a protein that was still sorted and retained in endosomal vesicles, whereas deletion of 15 or more amino acid residues resulted in a protein that became resident in the plasma membrane. Amino acids 12-15 are thus essential for intracellular transport to endosomal compartments. As Ii is intracellularly associated with the MHC class II molecules, it is proposed that Ii determines the intracellular transport route of these molecules.     

The amino-terminal structures that determine topological orientation of cytochrome P-450 in microsomal membrane.

We previously showed that the amino-terminal region of P-450 is responsible not only for targeting to endoplasmic reticulum (ER) membrane but also for stable anchoring to the membrane. In the present study, we introduced several mutations or deletions into the signal-anchor region of the chimeric proteins in which the amino-terminal regions of two forms of cytochrome P-450 were fused to the mature portion of interleukin 2. The amino-terminal acidic amino acid residues were replaced with basic amino acid residues or the hydrophobic core sequences were partially deleted, and these mutant proteins were assayed in vitro for their capacity to be inserted into or translocated across the ER membrane. The proteins that received the former manipulations were processed and the IL-2 portion was translocated across the membrane. In one case, the processing did not occur, thereby enabling the chimeric protein to anchor on the luminal side of the ER. Those that received the latter manipulation were also processed and the IL-2 portion translocated across the ER. These results strongly suggest that the signal-anchor function is determined both by the amino-terminal charged amino acid residues and by the length of the hydrophobic stretch.     

Deletions and insertions within an amino-terminal domain of pp60v-src inactivate transformation and modulate membrane stability.

We previously showed (V. W. Raymond and J. T. Parsons, Virology 160:400-410, 1987) that variants of the Prague A strain of Rous sarcoma virus containing large deletions impinging on a region of the src gene encoding amino acid residues 143 to 169 were defective for transformation of chicken cells in culture. Here we report that introduction of small (tri-and tetrapeptide) deletions into a region of pp60v-src containing amino acid residues 155 to 175 was found to inactivate transformation. In addition, insertion of four, but not one, amino acid residues at position 161 also inhibited transformation. Biochemical analysis of the src proteins encoded by individual transformation-defective variants revealed that the structural alterations introduced into this domain had only marginal effects upon src tyrosine-specific protein kinase activity. However, the src proteins encoded by defective variants exhibited a significantly shorter half-life within the cell, although these proteins efficiently and rapidly associated with cellular membranes. Our results suggest that the structural domain encompassing residues 155 to 177 may influence the stability of pp60src in the cellular membrane, possibly via the interaction of src with a cellular membrane component(s) or substrate(s).     

Structural and functional dissection of an MHC class I antigen-binding adenovirus glycoprotein.

The early transmembrane glycoprotein E19 of adenovirus-2 binds to class I antigens of the major histocompatibility complex (MHC). The association is initiated in the endoplasmic reticulum of infected cells and abrogates the intracellular transport of the class I molecules. To examine which parts of the E19 molecule are responsible for the association with the class I antigens and which parts confine the protein to the endoplasmic reticulum we have constructed a series of mutated E19 genes, which have been expressed in an improved mammalian expression vector. By various manipulations the membrane anchoring and the cytoplasmic domains were removed from the protein. The biosynthesis of the mutant protein was examined. All mutant proteins were secreted from the cells suggesting that the transmembrane and/or cytoplasmic portions of the E19 molecule are responsible for its confinement to the endoplasmic reticulum. The ability to associate with class I antigens was retained by the lumenal domain of the E19 protein.     

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.     

Analysis of bovine herpesvirus 1 glycoprotein gIV truncations and deletions expressed by recombinant vaccinia viruses.

Glycoprotein gIV is an envelope component of bovine herpesvirus type 1 and appears to be involved in attachment, penetration, and cell fusion. Four antigenic domains which include both continuous and discontinuous epitopes have been previously defined by competition binding assays using gIV-specific monoclonal antibodies (MAbs). Here we describe the construction of C-terminal truncations and internal deletions in the gIV-encoding gene and analyses of the effects of these mutations on the synthesis, processing, transport, and antigenicity of glycoprotein gIV as expressed by recombinant vaccinia viruses. Wild-type gIV expressed by recombinant vaccinia virus STgIV was indistinguishable from authentic gIV produced in bovine herpesvirus 1-infected cells with respect to molecular weight, processing, transport, and antigenicity. Analysis of the mutant proteins showed that the binding sites for MAbs 9D6 and 3D9S, which recognize linear epitopes, lie between amino acids 164 and 216 and amino acids 320 and 355, respectively. Discontinuous epitopes recognized by MAbs 3E7, 4C1, 2C8, and 3C1 were located between amino acids 19 and 320, whereas amino acids 320 to 355 were critical for binding of MAb 136. All mutant proteins containing amino acids 245 to 320 were processed, possess endo-beta-N-acetylglucosaminidase H-resistant oligosaccharides, and were transported to the cell surface or secreted into the medium. In contrast, mutant proteins missing amino acids 245 to 320 were retained in the rough endoplasmic reticulum. These findings suggest that residues 245 to 320 are important for proper processing and transport of gIV to the cell surface.     

Intracellular distribution of Sindbis virus membrane proteins in BHK-21 cells infected with wild-type virus and maturation-defective mutants.

The association of Sindbis virus proteins with cellular membranes during virus maturation was examined by utilizing a technique for fractionating the membranes of BHK-21 cells into three subcellular classes, which were enriched for rough endoplasmic reticulum, smooth endoplasmic reticulum, and plasma membrane. Pulse-chase experiments with wild-type (strain SVHR) virus-infected cells showed that virus envelope proteins were incorporated initially into membranes of the rough endoplasmic reticulum and subsequently migrated to the smooth and plasma membrane fractions. Large amounts of capsid protein were associated with the plasma membrane fraction even at the earliest times postpulse, and relatively little was found associated with the other membranes, suggesting a rapid and preferential association of nucleocapsids with the plasma membrane. We also examined the intracellular processing of the proteins of two temperature-sensitive Sindbis virus mutants in pulse-chase experiments at the nonpermissive temperature. Labeled virus proteins of mutant ts-20 (complementation group E) first appeared in the rough endoplasmic reticulum and were then transported to the smooth and plasma membrane fractions, as in wild-type (strain SVHR) virus-infected cells. In cells infected with ts-23 (complementation group D), the pulse-labeled virus proteins appeared initially in the rough membrane fraction and were transported to the smooth membrane fraction, but only limited amounts reached the plasma membrane. Thus, in ts-23-infected cells, the transport of the virus-encoded proteins from the smooth membranes seemed to be defective. In both ts-20- and ts-23-infected cells the envelope precursor polypeptide PE2 was not processed to E2, and no label was incorporated into free virus at the nonpermissive temperature.     

Transient expression and mutational analysis of the rotavirus intracellular receptor: the C-terminal methionine residue is essential for ligand binding.

Maturation of rotavirus involves an intracellular membrane budding event in which the single-shelled icosahedral particle interacts with a virus-encoded receptor glycoprotein, NS28, that is located in the rough endoplasmic reticulum membrane. The receptor is a tetramer and is oriented with the C-terminal 131 amino acids on the cytoplasmic side of the membrane (A.R. Bellamy and G.W. Both, Adv. Virus Res. 38:1-48, 1990). We have used the T7-vaccinia virus transient expression system to deliver mutant variants of the NS28 gene to CV1 cells in order to assess the effects of site-specific modifications on receptor function. Three types of mutant proteins have been constructed by altering the extreme C-terminal methionine, cysteine residues within the third hydrophobic domain, and internal residues located within the cytoplasmic portion of the receptor, respectively. Deletion or conservative substitution of the C-terminal methionine completely abolishes receptor activity. Substitution of cysteine residues has no effect on receptor activity or on the ability of the receptor to adopt its native oligomeric state. Internal deletions result only in a reduction in the level of binding. An N-terminally truncated form of the receptor, containing only the cytoplasmic domain, retains full receptor activity and can form membrane-associated tetramers.     

Export of honeybee prepromelittin in Escherichia coli depends on the membrane potential but does not depend on proteins secA and secY

Honeybee prepromelittin (70 amino acid residues), the precursor of an eukaryotic secretory protein, and a hybrid protein between prepromelittin and mouse dihydrofolate reductase (257 amino acid residues) were expressed in Escherichia coli and characterized with respect to their requirements for transport across the plasma membrane. Both precursor proteins are posttranslationally processed and exported into the periplasm, and they both depend on the membrane potential for this to occur. With respect to dependence on components of the export machinery, however, the two precursor proteins show striking differences: the small precursor protein prepromelittin does not require the function of proteins secA and secY; the large precursor protein prepromelittin-dihydrofolate reductase, on the other hand, depends on both components. The implications of these observations with respect to the mechanisms of protein export in E. coli and of protein import into the endoplasmic reticulum are discussed.     

The relationship between the morphology of cell organelles and kinetics of the secretory process in male sex accessory glands of mice

Summary Two male sex accessory glands of the mouse, seminal vesicle and coagulating gland, were compared with the aim of relating differences in the morphology of organelles to the kinetics of the secretory process. The epithelial cells of the two glands were assessed by morphometric analysis, cytochemical staining, and electron-microscopic autoradiography after administration of a labeled amino acid. The rough endoplasmic reticulum of the seminal vesicle comprised narrow parallel cisternae, while that of the coagulating gland was greatly distended and occupied a much larger percentage of the cytoplasmic volume. Radioactively labeled products were secreted much more rapidly in the seminal vesicle than in the coagulating gland. The primary point of difference in kinetics of intracellular transport between the two glands was in exit of material from the rough endoplasmic reticulum. The more rapid drainage of the rough endoplasmic reticulum may be related to its relatively greater membrane surface density and lesser internal volume. In contrast, similarities in size and cytochemical staining in the Golgi apparatus of the two glands were accompanied by similar kinetics of intracellular transport of secretory protein through this organelle.     

Effects of altered cytoplasmic domains on transport of the vesicular stomatitis virus glycoprotein are transferable to other proteins.

Alterations of the cytoplasmic domain of the vesicular stomatitis virus glycoprotein (G protein) were shown previously to affect transport of the protein from the endoplasmic reticulum, and recent studies have shown that this occurs without detectable effects on G protein folding and trimerization (R. W. Doms et al., J. Cell Biol., in press). Deletions within this domain slowed exit of the mutant proteins from the endoplasmic reticulum, and replacement of this domain with a foreign 12-amino-acid sequence blocked all transport out of the endoplasmic reticulum. To extend these studies, we determined whether such effects of cytoplasmic domain changes were transferable to other proteins. Three different assays showed that the effects of the mutations on transport of two membrane-anchored secretory proteins were the same as those observed with vesicular stomatitis virus G protein. In addition, possible effects on oligomerization were examined for both transported and nontransported forms of membrane-anchored human chorionic gonadotropin-alpha. These membrane-anchored forms, like the nonanchored human chorionic gonadotropin-alpha, had sedimentation coefficients consistent with a monomeric structure. Taken together, our results provide strong evidence that these cytoplasmic mutations affect transport by affecting interactions at or near the cytoplasmic side of the membrane.     

Effects of Mutations in the Rubella Virus E1 Glycoprotein on E1-E2 Interaction and Membrane Fusion Activity

Rubella virus (RV) virions contain two glycosylated membrane proteins, E1 and E2, that exist as a heterodimer and form the viral spike complexes on the virion surface. Formation of an E1-E2 heterodimer is required for transport of E1 out of the endoplasmic reticulum lumen to the Golgi apparatus and plasma membrane. To investigate the nature of the E1-E2 interaction, we have introduced mutations in the internal hydrophobic region (residues 81 to 109) of E1. Substitution of serine at Cys82 (mutant C82S) or deletion of this hydrophobic domain (mutant dt) of E1 resulted in a disruption of the E1 conformation that ultimately affected E1-E2 heterodimer formation and cell surface expression of both E1 and E2. Substitution of either aspartic acid at Gly93 (G93D) or glycine at Pro104 (P104G) was found to impair neither E1-E2 heterodimer formation nor the transport of E1 and E2 to the cell surface. Fusion of RV-infected cells is induced by a brief treatment at a pH below 6.0. To test whether this internal hydrophobic domain is involved in the membrane fusion activity of RV, transformed BHK cell lines expressing either wild-type or mutant spike proteins were exposed to an acidic pH and polykaryon formation was measured. No fusion activity was observed in the C82S, dt, and G93D mutants; however, the wild type and the P104G mutant exhibited fusogenic activities, with greater than 60% and 20 to 40% of the cells being fused, respectively, at pH 4.8. These results suggest that it is likely that the region of E1 between amino acids 81 and 109 is involved in the membrane fusion activity of RV and that it may be important for the interaction of that protein with E2 to form the E1-E2 heterodimer.     

Nucleotide sequences of the mRNA''s encoding the vesicular stomatitis virus G and M proteins determined from cDNA clones containing the complete coding regions.

The complete nucleotide sequences of the vesicular stomatitis virus mRNA's encoding the glycoprotein (G) and the matrix protein (M) have been determined from cDNA clones that contain the complete coding sequences from each mRNA. The G protein mRNA is 1,665 nucleotides long, excluding polyadenylic acid, and encodes a protein of 511 amino acids including a signal peptide of 16 amino acids. G protein contains two large hydrophobic domains, one in the signal peptide and the other in the transmembrane segment near the COOH terminus. Two sites of glycosylation are predicted at amino acid residues 178 and 335. The close correspondence of the positions of these sites with the reported timing of the addition of the two oligosaccharides during synthesis of G suggests that glycosylation occurs as soon as the appropriate asparagine residues traverse the membrane of the rough endoplasmic reticulum. The mRNA encoding the vesicular stomatitis virus M protein is 831 nucleotides long, excluding polyadenylic acid, and encodes a protein of 229 amino acids. The predicted M protein sequence does not contain any long hydrophobic or nonpolar domains that might promote membrane association. The protein is rich in basic amino acids and contains a highly basic amino terminal domain. Details of construction of the nearly full-length cDNA clones are presented.