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.     

Roles for the cytoplasmic tails of the fusion and hemagglutinin-neuraminidase proteins in budding of the paramyxovirus simian virus 5

The efficient release of many enveloped viruses from cells involves the coalescence of viral components at sites of budding on the plasma membrane of infected cells. This coalescence is believed to require interactions between the cytoplasmic tails of surface glycoproteins and the matrix (M) protein. For the paramyxovirus simian virus 5 (SV5), the cytoplasmic tail of the hemagglutinin-neuraminidase (HN) protein has been shown previously to be important for normal virus budding. To investigate a role for the cytoplasmic tail of the fusion (F) protein in virus assembly and budding, we generated a series of F cytoplasmic tail-truncated recombinant viruses. Analysis of these viruses in tissue culture indicated that the cytoplasmic tail of the F protein was dispensable for normal virus replication and budding. To investigate further the requirements for assembly and budding of SV5, we generated two double-mutant recombinant viruses that lack 8 amino acids of the predicted 17-amino-acid HN protein cytoplasmic tail in combination with truncation of either 10 or 18 amino acids from the predicted 20-amino-acid F protein cytoplasmic tail. Both of the double mutant recombinant viruses displayed a replication defect in tissue culture and a budding defect, the extent of which was dependent on the length of the remaining F cytoplasmic tail. Taken together, this work and our earlier data on virus-like particle formation (A. P. Schmitt, G. P. Leser, D. L. Waning, and R. A. Lamb, J. Virol. 76:3953-3964, 2002) suggest a redundant role for the cytoplasmic tails of the HN and F proteins in virus assembly and budding.     

Down-regulation of paramyxovirus hemagglutinin-neuraminidase glycoprotein surface expression by a mutant fusion protein containing a retention signal for the endoplasmic reticulum.

The human parainfluenza virus type 3 (HPIV3) fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins are the principal components involved in virion receptor binding, membrane penetration, and ultimately, syncytium formation. While the requirement for both F and HN in this process has been determined from recombinant expression studies, stable physical association of these proteins in coimmunoprecipitation studies has not been observed. In addition, coexpression of other heterologous paramyxovirus F or HN glycoproteins with either HPIV3 F or HN does not result in the formation of syncytia, suggesting serotype-specific protein differences. In this study, we report that simian virus 5 and Sendai virus heterologous HN proteins and measles virus hemagglutinin (H) were found to be down-regulated when coexpressed with HPIV3 F. As an alternative to detecting physical associations of these proteins by coimmunoprecipitation, further studies were performed with a mutant HPIV3 F protein (F-KDEL) lacking a transmembrane anchor and cytoplasmic tail and containing a carboxyl-terminal retention signal for the endoplasmic reticulum (ER). F-KDEL was defective for transport to the cell surface and could down-regulate surface expression of HPIV3 HN and heterologous HN/H proteins from simian virus 5, Sendai virus, and measles virus in coexpression experiments. HN/H down-regulation appeared to result, in part, from an early block to HPIV3 HN synthesis, as well as an instability of the heterologous HN/H proteins within the ER. In contrast, coexpression of F-KDEL with HPIV3 wild-type F or the heterologous receptor-binding proteins, respiratory syncytial virus glycoprotein (G) and vesicular stomatitis virus glycoprotein (G), were not affected in transport to the cell surface. Together, these results support the notion that the reported serotype-specific restriction of syncytium formation may involve, in part, down-regulation of heterologous HN expression.     

Role of the membrane anchoring and cytoplasmic domains in intracellular transport and localization of viral glycoproteins.

The role of the transmembrane and the cytoplasmic regions of viral glycoproteins namely, the envelope glycoprotein gD of herpes simplex virus and the integral membrane glycoprotein E3-11.6 K of the nonenveloped adenovirus that are localized in the nuclear envelope has been studied. Chimeras of the cell surface glycoprotein G of vesicular stomatitis virus containing the transmembrane and (or) the cytoplasmic-tail domains of either herpes simplex virus gD or adenovirus E3-11.6 K protein were examined for their intracellular transport and localization. The results show that hybrids containing the membrane anchoring and (or) the cytoplasmic tail domains of either herpes simplex virus gD or adenovirus E3-11.6 K glycoprotein were localized in the nuclear envelope as well as in the endoplasmic reticulum and the Golgi complex. These results suggest that the membrane anchoring and the cytoplasmic domains of herpes simplex virus glycoproteins gD, as well as the adenovirus integral membrane protein E3-11.6 K, were necessary for localization in the nuclear envelope and could influence retention in the endoplasmic reticulum and the Golgi complex.     

Mutants of the Rous sarcoma virus envelope glycoprotein that lack the transmembrane anchor and cytoplasmic domains: analysis of intracellular transport and assembly into virions.

The envelope glycoprotein complex of Rous sarcoma virus consists of a knoblike, receptor-binding gp85 polypeptide that is linked through disulfide bonds to a membrane-spanning gp37 spike. We used oligonucleotide-directed mutagenesis to assess the role of the hydrophobic transmembrane region and hydrophilic cytoplasmic domain of gp37 in intracellular transport and assembly into virions. Early termination codons were introduced on either side of the hydrophobic transmembrane region, and the mutated env genes were expressed from the late promoter of simian virus 40. This resulted in the synthesis of glycoprotein complexes composed of a normal gp85 and a truncated gp37 molecule that lacked the cytoplasmic domain alone or both the cytoplasmic and transmembrane domains. The biosynthesis and intracellular transport of the truncated proteins were not significantly different from those of the wild-type glycoproteins, suggesting that any protein signals for biosynthesis and intracellular transport of this viral glycoprotein complex must reside in its extracellular domain. The glycoprotein complex lacking the cytoplasmic domain of gp37 is stably expressed on the cell surface in a manner similar to that of the wild type. In contrast, the complex lacking both the transmembrane and cytoplasmic domains is secreted as a soluble molecule into the media. It can be concluded, therefore, that the transmembrane domain alone is essential for anchoring the RSV env complex in the cell membrane and that the cytoplasmic domain is not required for anchor function. Insertion of the mutated genes into an infectious proviral genome allowed us to assess the ability of the truncated gene products to be assembled into virions and to determine whether such virions were infectious. Viral genomes encoding the secreted glycoprotein were noninfectious, whereas those encoding a glycoprotein complex lacking only the cytoplasmic domain of gp37 were infectious. Virions produced from these mutant-infected cells contained normal levels of glycoprotein. The cytoplasmic tail of gp37 is thus not required for the assembly of envelope glycoproteins into virions. It is unlikely, therefore, that this region of gp37 interacts with viral core proteins during the selective incorporation of viral glycoproteins into the viral envelope.     

Pediococcus cerevisiae mutant with altered transport of folates.

A Pediococcus cerevisiae mutant that actively accumulated folate (PteGlu), in contrast to the wild-type, was also found to exhibit changes in the pattern of uptake of 5-methyl-tetrahydrofolate (5-CH3-H4PteGlu) and amethopterin. Most of the 5-CH3-H4PteGlue accumulated through a glucose- and temperature-dependent process, and a concentrative uptake was also found in gluocse-starved cells and in cells incubated at OC. About 75% of the accumulated 5-CH3-H4PteGlu exchanged with amethopterin. In contrast to the wild type, the mutant accumulated both diastereoisomers of 5-CH3-H4PteGlue by glucose-dependent and glucose-independent processes. Amethopterin and PteGlue competitively inhibited the uptake in both processes, with an apparent lower affinity of the carrier for PteGlu than for the analogue. p-Chloromercuribenzoate strongly inhibited the uptake (75%). The p-chloromercuribenzoate-nonsusceptible and temperature-independent uptake was also competed by amethopterin. Metabolic poisons like sodium azide, potassium fluoride, iodoacetate, and 2,4-dimitrophenol inhibited the glucose-dependent process. Uptake, in the absence of glucose, was enhanced by sodium azide and potassium fluoride.     

Analysis of connexin intracellular transport and assembly

Central to understanding the establishment and regulation of gap junction-mediated intercellular communication is a detailed knowledge of how these complex structures are assembled. In this article, we describe methods to modulate and/or monitor the transport of connexins from the endoplasmic reticulum to the Golgi complex and from the trans Golgi network to the cell surface. We also present techniques that we have developed to assay assembly of newly synthesized connexin monomers into connexons and gap junctional plaques.     

Sequence and structure alignment of paramyxovirus hemagglutinin-neuraminidase with influenza virus neuraminidase.

A model is proposed for the three-dimensional structure of the paramyxovirus hemagglutinin-neuraminidase (HN) protein. The model is broadly similar to the structure of the influenza virus neuraminidase and is based on the identification of invariant amino acids among HN sequences which have counterparts in the enzyme-active center of influenza virus neuraminidase. The influenza virus enzyme-active site is constructed from strain-invariant functional and framework residues, but in this model of HN, it is primarily the functional residues, i.e., those that make direct contact with the substrate sialic acid, which have identical counterparts in neuraminidase. The framework residues of the active site are different in HN and in neuraminidase and appear to be less strictly conserved within HN sequences than within neuraminidase sequences.     

Variants of vaccinia virus hemagglutinin altered in intracellular transport.

Two classes of revertants were isolated from a vaccinia virus mutant whose hemagglutinins (HAs) accumulate on nuclear envelopes and rough endoplasmic reticulums. The HAs of one of the revertants had the same phenotype as the wild type, i.e., rapid and efficient movement to the cell surface. The HAs of the second class had biphasic transport: rapid export to the cell surface as in the wild type and slow movement to the medial cisternae of the Golgi apparatus. Biochemical and nucleotide sequence analyses showed that the HAs of all the mutants examined that have defects in transport from the rough endoplasmic reticulum to the Golgi apparatus have altered cytoplasmic domains and that the HAs of the second class of revertants lack the whole cytoplasmic domain, while the HAs of the first class of revertants have a wild-type cytoplasmic domain.     

Intracellular transport, assembly, and degradation of wild-type and disease-linked mutant gap junction proteins

More than 130 different mutations in the gap junction integral plasma membrane protein connexin32 (Cx32) have been linked to the human peripheral neuropathy X-linked Charcot-Marie-Tooth disease (CMTX). How these various mutants are processed by the cell and the mechanism(s) by which they cause CMTX are unknown. To address these issues, we have studied the intracellular transport, assembly, and degradation of three CMTX-linked Cx32 mutants stably expressed in PC12 cells. Each mutant had a distinct fate: E208K Cx32 appeared to be retained in the endoplasmic reticulum (ER), whereas both the E186K and R142W mutants were transported to perinuclear compartments from which they trafficked either to lysosomes (R142W Cx32) or back to the ER (E186K Cx32). Despite these differences, each mutant was soluble in nonionic detergent but unable to assemble into homomeric connexons. Degradation of both mutant and wild-type connexins was rapid (t(1/2) < 3 h) and took place at least in part in the ER by a process sensitive to proteasome inhibitors. The mutants studied are therefore unlikely to cause disease by accumulating in degradation-resistant aggregates but instead are efficiently cleared from the cell by quality control processes that prevent abnormal connexin molecules from traversing the secretory pathway.     

Aberrant membrane insertion of a cytoplasmic tail deletion mutant of the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus.

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a type II glycoprotein oriented in the plasma membrane with its amino terminus in the cytoplasm and its carboxy terminus external to the cell. We have previously shown that the membrane insertion of HN protein requires signal recognition particle SRP, occurs cotranslationally, and utilizes the same GTP-dependent step that has been described for secretory proteins, type I proteins, and multispanning proteins (C. Wilson, R. Gilmore, and T. Morrison, Mol. Cell. Biol. 7:1386-1392, 1987; C. Wilson, T. Connolly, T. Morrison, and R. Gilmore, J. Cell Biol. 107:69-77, 1988). The role of the amino-terminal cytoplasmic domain in the faithful membrane insertion of this type II protein was explored by characterizing the membrane integration of a mutant lacking 23 of the 26 amino acids of the cytoplasmic domain. The mutant protein was able to interact with SRP, resulting in translation inhibition, membrane targeting, and membrane translocation, but the efficiency of translocation was considerably lower than for the wild-type HN protein. In addition, a significant proportion of the mutant protein synthesized in the presence of SRP and microsomal membranes was associated with the membrane in an EDTA- and alkali-insensitive manner yet integrated into membranes with its carboxy-terminal domain on the cytoplasmic side of membrane vesicles. Membrane-integrated molecules with this reverse orientation were not detected when the mutant protein was synthesized in the absence of SRP or a functional SRP receptor. Truncated mRNAs encoding amino-terminal segments of the wild-type and mutant proteins were translated to prepare ribosomes bearing arrested nascent chains. The arrested mutant nascent chain, in contrast to the wild-type nascent chain, was also able to insert into membranes in a GTP- and SRP-independent manner. Results suggest that the cytoplasmic domain plays a role in the proper membrane insertion of this type II glycoprotein.     

Expression and function of transplantation antigens with altered or deleted cytoplasmic domains

Two mutants of the class I gene encoding the H-2L^d transplantation antigen have been constructed. In one mutant the cytoplasmic domain of the class I molecule has been altered by deletion of 24 of the 31 C-terminal residues, and in the second the C-terminal 25 residues of the cytoplasmic domain have been replaced with a unique sequence of 19 amino acids. These mutant class I genes have been transferred into mouse L cells by DNA-mediated gene transfer. Both mutant genes are expressed at normal levels on the cell surface, and they have charge properties and sizes consistent with the introduced alterations. These mutant L^d molecules can serve as target antigens for allogeneic cytotoxic T cells and as restricting elements for virus-specific cytotoxic T cells. These results show that the 24 residues replaced or deleted from the carboxy terminus of the class I molecule are not required for its transport to or integration in the plasma membrane, nor for its function as a target antigen or a restricting element during T-cell-mediated cytotoxicity.     

Recognition domains in assembly of oligomeric membrane proteins

Many integral membrane proteins, particularly receptors on the cell surface, are made up of several polypeptide chains. After translation and insertion into the ER membrane, these subunits must assemble into the mature protein. However, the mechanisms controlling their faithful assembly are largely unknown. Recent evidence has shed some light on two cell surface receptors that use different strategies to assemble their subunits. Zach Hall discusses oligomerization of the T-cell receptor and the acetylcholine receptor.     

Mutations in the cytoplasmic domain of a paramyxovirus fusion glycoprotein rescue syncytium formation and eliminate the hemagglutinin-neuraminidase protein requirement for membrane fusion

SER virus is closely related to the paramyxovirus simian virus 5 (SV5) but is defective in syncytium formation. The SER virus F protein has a long cytoplasmic tail (CT) domain that has been shown to inhibit membrane fusion, and this inhibitory effect could be eliminated by truncation of the C-terminal sequence (S. Tong, M. Li, A. Vincent, R. W. Compans, E. Fritsch, R. Beier, C. Klenk, M. Ohuchi, and H.-D. Klenk, Virology 301:322-333, 2002). To study the sequence requirements for regulation of fusion, codons for SER virus F protein residues spanning amino acids 535 to 542 and 548 were mutated singly to alanines, and the two leucine residues at positions 539 and 548 were mutated doubly to alanines. We found that leu-539 and leu-548 in the CT domain played a critical role in the inhibition of fusion, as mutation of the two leucines singly to alanines partially rescued fusion, and the double mutation L539, 548A completely rescued syncytium formation. Mutation of charged residues to alanines had little effect on the suppression of fusion activity, whereas the mutation of serine residues to alanines enhanced fusion activity significantly. The L539, 548A mutant also showed extensive syncytium formation when expressed without the SER virus HN protein. By constructing a chimeric SV5-SER virus F CT protein, we also found that the inhibitory effect of the long CT of the SER virus F protein could be partially transferred to the SV5 F protein. These results demonstrate that an elongated CT of a paramyxovirus F protein interferes with membrane fusion in a sequence-dependent manner.     

Evolution of homologous domains of cytoplasmic intermediate filament proteins and lamins

The earliest gene duplications in the evolution of the intermediate filament proteins created the ancestors of acidic keratins, basic keratins, nonepithelial intermediate filament proteins, and lamins. Biochemistry and function of cytoplasmic intermediate filaments differ greatly from those of lamins. Cytoplasmic intermediate filament proteins have a different cellular location than lamins, form different types of supramolecular structures, and are missing a protein segment found in lamins; but the data presented here indicate that the cytoplasmic intermediate filaments do not have a common ancestor separate from the ancestor of lamins. In the non-epithelial intermediate filament branch, the ancestor of neurofilament proteins and the common ancestor of desmin, vimentin, and glial fibrillary acidic protein (GFAP) diverged first. By evolutionary criteria, the intermediate filament protein recently discovered in neuronal cells does not belong to the neurofilament family but is more closely related to desmin, vimentin, and GFAP. Sequences of different sub-domains yield different evolutionary trees, possibly indicating existence of sub- domain-specific functions.      

Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis.

The gene associated with cystic fibrosis (CF) encodes a membrane-associated, N-linked glycoprotein called CFTR. Mutations were introduced into CFTR at residues known to be altered in CF chromosomes and in residues believed to play a role in its function. Examination of the various mutant proteins in COS-7 cells indicated that mature, fully glycosylated CFTR was absent from cells containing delta F508, delta 1507, K464M, F508R, and S5491 cDNA plasmids. Instead, an incompletely glycosylated version of the protein was detected. We propose that the mutant versions of CFTR are recognized as abnormal and remain incompletely processed in the endoplasmic reticulum where they are subsequently degraded. Since mutations with this phenotype represent at least 70% of known CF chromosomes, we argue that the molecular basis of most cystic fibrosis is the absence of mature CFTR at the correct cellular location.