Cellular Secretory Pathway Research Articles

Identification of Human T Cell Leukemia Virus Type 1 Tax Amino Acid Signals and Cellular Factors Involved in Secretion of the Viral Oncoprotein

Human T cell leukemia virus type 1 (HTLV-1) is the etiologic agent of a number of pathologic abnormalities, including adult T cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The viral oncoprotein Tax has been implicated in the pathogenesis of these diseases. Recently, cell-free Tax was detected in the cerebrospinal fluid of HAM/TSP patients, implying that extracellular Tax may be relevant to neurologic disease. Additionally, the presence of a nuclear export signal within Tax and its active secretion has been demonstrated in vitro. However, the mechanism of Tax secretion remains to be established. Studies reported herein elucidate the process of Tax secretion and identify domains of Tax critical to its subcellular localization and secretion. Tax was shown to interact with a number of cellular secretory pathway proteins in both the model cell line BHK (baby hamster kidney)-21 and an HTLV-1-infected T cell line, C8166, physiologically relevant to HTLV-1-induced disease. Silencing of selected components of the secretory pathway affected Tax secretion, further confirming regulated secretion of Tax. Additionally, mutations in two putative secretory signals within Tax DHE and YTNI resulted in aberrant subcellular localization of Tax and significantly altered protein secretion. Together, these studies demonstrate that Tax secretion is a regulated event facilitated by its interactions with proteins of the cellular secretory pathway and the presence of secretory signals within the carboxyl-terminal domain of the protein.

The Cytoplasmic Tail of Glycoprotein M (gpUL100) Expresses Trafficking Signals Required for Human Cytomegalovirus Assembly and Replication

The virion envelope of human cytomegalovirus (HCMV) is complex and consists of an incompletely defined number of glycoproteins. The gM/gN protein complex is the most abundant protein component of the envelope. Studies have indicated that deletion of the viral gene encoding either gM or gN is a lethal mutation. Analysis of the amino acid sequence of gM disclosed a C-terminal acidic cluster of amino acids and a tyrosine-containing trafficking motif, both of which are well-described trafficking/sorting signals in the cellular secretory pathway. To investigate the roles of these signals in the trafficking of the gM/gN complex during virus assembly, we made a series of gM (UL100 open reading frame) mutants in the AD169 strain of HCMV. Mutant viruses that lacked the entire C-terminal cytoplasmic tail of gM were not viable, suggesting that the cytoplasmic tail of gM is essential for virus replication. In addition, the gM mutant protein lacking the cytoplasmic domain exhibited decreased protein stability. Mutant viruses with a deletion of the acidic cluster or alanine substitutions in tyrosine-based motifs were viable but exhibited a replication-impaired phenotype suggestive of a defect in virion assembly. Analysis of these mutant gMs using static immunofluorescence and fluorescence recovery after photobleaching demonstrated delayed kinetics of intracellular localization of the gM/gN protein to the virus assembly compartment compared to the wild-type protein. These data suggest an important role of the glycoprotein gM during virus assembly, particularly in the dynamics of gM trafficking during viral-particle assembly.

Effects of overexpression of X‐box binding protein 1 on recombinant protein production in Chinese hamster ovary and NS0 myeloma cells

X-box binding protein 1 (XBP-1) is a key regulator of the cellular secretory pathway and unfolded protein response (UPR). It has been shown that the spliced form of XBP-1, XBP-1S, functions as a transcription activator and up-regulates many genes associated with protein secretion and biosynthesis of endoplasmic reticula. Since the production of some recombinant proteins is widely believed to be limited by the secretory capacity of the host cell, an increase in protein production may be achieved by overexpressing XBP-1S. In this study, the effects of XBP-1S on the productivity of monoclonal antibody (MAb), interferon gamma (IFNgamma), and erythropoietin (EPO) are examined in Chinese hamster ovary (CHO) and NS0 cell lines. Results show that XBP-1S may become a determinative factor only when accumulation of recombinant proteins exceeds the secretory capacity of the host cell. In transient transfection systems where a bottleneck in protein secretion was achieved, overexpression of XBP-1S improved protein titers by up to 2.5-fold. In contrast, overexpression of XBP-1S had no detectable effects on protein productivity of stable cell lines that did not exhibit any secretory bottleneck. We conclude that overexpression of XBP-1S is an effective strategy in enhancing recombinant protein production when the secretory pathway of the host cell is saturated by high-level synthesis of recombinant proteins.

From Adipose Tissue Protein Secretion to Adipopharmacology of Disease

An extensive research in the last few years has identified about hundred adipose tissue-secreted proteins, named adipokines or adipocytokines. However, our knowledge of the structures and molecules involved in the intracellu- lar secretory pathway (synthesis, translocation, folding, targeting, sorting, storage, and exocytosis) of adipokines is at pre- sent limited. Relatively more is known about insulin-responsive trafficking of glucose transporters (GLUTs). Adipokines have multiple biological functions beyond lipid and carbohydrate metabolism, whereas GLUT4 is the major glucose transporter of adipocytes and skeletal muscles. Adipokines play an important role in the pathogenesis of a wide variety of diseases, and dysregulation of GLUT4 transport is implicated in insulin resistance and related disorders. Conceptually, adipobiology of disease has emerged as a novel field of studies in basic and clinical medicine. Here we present a state-of- the-science on some aspects of these studies with a special reference to the intracellular secretory pathway, focusing on adiponectin, GLUT4, and nerve growth factor (NGF), and suggesting that each step of this pathway may be a potential drug target. Given the beneficial effects of adiponectin, NGF and GLUT4 on various metabolic, vascular and inflamma- tory processes, a hypothesis of metabotrophic factor deficit in the pathogenesis of adipose-linked diseases is discussed. Adipopharmacological evaluation of this hypothesis may provide novel targets for drug development. W e respectfully ded ic ate th is rev ie w t o Professor Rita Lev i- M ontalc in i on oc cas ion of her 97 th birthday (born 22 April, 1909) cellular secretory pathways. We propose that, at a mechanis- tic level, each step of these pathways may provide a novel target for drug development against adipose tissue-linked diseases. Given the beneficial effects of adiponectin, NGF and GLUT4 on various metabolic, vascular and inflamma- tory processes, a hypothesis of metabotrophic factor deficit in the pathogenesis of these diseases is discussed.

Hijacking Components of the Cellular Secretory Pathway for Replication of Poliovirus RNA

Infection of cells with poliovirus induces a massive intracellular membrane reorganization to form vesicle-like structures where viral RNA replication occurs. The mechanism of membrane remodeling remains unknown, although some observations have implicated components of the cellular secretory and/or autophagy pathways. Recently, we showed that some members of the Arf family of small GTPases, which control secretory trafficking, became membrane-bound after the synthesis of poliovirus proteins in vitro and associated with newly formed membranous RNA replication complexes in infected cells. The recruitment of Arfs to specific target membranes is mediated by a group of guanine nucleotide exchange factors (GEFs) that recycle Arf from its inactive, GDP-bound state to an active GTP-bound form. Here we show that two different viral proteins independently recruit different Arf GEFs (GBF1 and BIG1/2) to the new structures that support virus replication. Intracellular Arf-GTP levels increase approximately 4-fold during poliovirus infection. The requirement for these GEFs explains the sensitivity of virus growth to brefeldin A, which can be rescued by the overexpression of GBF1. The recruitment of Arf to membranes via specific GEFs by poliovirus proteins provides an important clue toward identifying cellular pathways utilized by the virus to form its membranous replication complex.

Protein-protein Interaction Between HTLV-1 Tax Protein and the Components of the Cellular Secretory Pathway

Tax protein has been shown to play an integral role in HTLV-1-induced diseases including adult T cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Extracellular Tax has been detected in the cerebrospinal fluid of HAM/TSP patients, suggesting that cell-free Tax may be physiologically involved in the progression of neurologic disease. We have previously demonstrated the secretion of full-length Tax and its co-localization with the cytoplasmic organelles relevant to secretion. The present study elucidates the mechanism of Tax secretion. To identify Tax interacting proteins within cell, we have used an antibody array spotted with antibodies directed against cellular secretory pathway proteins. Upon reaction with protein extracts from Tax-treated cells, antibody array analyses have suggested the interaction of Tax with SCAMP1, SCAMP2, SNAP23, and COPII; proteins that facilitate transport between nucleus and cytoplasm, and between endoplasmic reticulum, Golgi complex, and plasma membrane. Subsequently, these specific protein-protein interactions have been confirmed by co-immunoprecipitation and GST pull-down assays. Collectively, these studies have demonstrated the interaction of Tax with multiple proteins in the secretory pathway and that Tax may be secreted and act as an extracellular effector molecule.

HTLV-1 Tax nucleocytoplasmic shuttling, interaction with the secretory pathway, extracellular signaling, and implications for neurologic disease

The human T cell leukemia virus type 1 (HTLV-1) oncoprotein Tax interacts with numerous cellular pathways promoting both the survival and pathogenesis of the virus in the human population. Tax has been studied extensively with respect to its role in transcriptional transactivation and its involvement in the up-regulation of a number of cellular genes during the process of oncogenic transformation. These processes are dependent on Tax localization to the nucleus where it interacts with a number of cellular transcription factors during its course of nuclear action. However, there is mounting evidence suggesting that Tax may shuttle between the nucleus and cytoplasm, localize to several cytoplasmic organelles with subsequent secretion from both Tax-transfected cells as well as HTLV-1-infected cells. In addition, the presence of cell-free Tax in cerebral spinal fluid (CSF) was recently demonstrated to occur during all stages of HAM/TSP. This has brought about an increased interest in the cytoplasmic localization of Tax and the implications this localization may have with respect to the progression of HTLV-1-associated disease processes. This review addresses the functional implications relevant to the localization and accumulation of Tax in the cytoplasm including the Tax amino acid signals and cellular protein interactions that may regulate this process. Specifically, we have discussed three important processes associated with the cytoplasmic localization of Tax. First, the process of Tax shuttling between the nucleus and cytoplasm will be described and how this process may be involved in regulating different transcriptional activation pathways. Second, cytoplasmic localization of Tax will be discussed with relevance to Tax secretion and the interaction of Tax with proteins in the cellular secretory pathway. Finally, the secretion of Tax and the effects of extracellular Tax on HTLV-1 pathogenesis will be addressed.

Procollagen trafficking, processing and fibrillogenesis

Collagen fibrils in the extracellular matrix allow connective tissues such as tendon, skin and bone to withstand tensile forces. The fibrils are indeterminate in length, insoluble and form elaborate three-dimensional arrays that extend over numerous cell lengths. Studies of the molecular basis of collagen fibrillogenesis have provided insight into the trafficking of procollagen (the precursor of collagen) through the cellular secretory pathway, the conversion of procollagen to collagen by the procollagen metalloproteinases, and the directional deposition of fibrils involving the plasma membrane and late secretory pathway. Fibril-associated molecules are targeted to the surface of collagen fibrils, and these molecules play an important role in regulating the diameter and interactions between the fibrils.

Secretion of the Human T Cell Leukemia Virus Type I Transactivator Protein Tax

Human T cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T cell leukemia and HTLV-I-associated myelopathy/tropical spastic paraparesis. The HTLV-I protein Tax is well known as a transcriptional transactivator and inducer of cellular transformation. However, it is also known that extracellular Tax induces the production and release of cytokines, such as tumor necrosis factor-alpha and interleukin-6, which have adverse effects on cells of the central nervous system. The cellular process by which Tax exits the cell into the extracellular environment is currently unknown. In most cell types, Tax has been shown to localize primarily to the nucleus. However, Tax has also been found to accumulate in the cytoplasm. The results contained herein begin to characterize the process of Tax secretion from the cell. Specifically, cytoplasmic Tax was demonstrated to localize to organelles associated with the cellular secretory process including the endoplasmic reticulum and Golgi complex. Additionally, it was demonstrated that full-length Tax was secreted from both baby hamster kidney cells and a human kidney tumor cell line, suggesting that Tax enters the secretory pathway in a leaderless manner. Tax secretion was partially inhibited by brefeldin A, suggesting that Tax migrated from the endoplasmic reticulum to the Golgi complex. In addition, combined treatment of Tax-transfected BHK-21 cells with phorbol myristate acetate and ionomycin resulted in a small increase in the amount of Tax secreted, suggesting that a fraction of cytoplasmic Tax was present in the regulated secretory pathway. These studies begin to provide a link between Tax localization to the cytoplasm, the detection of Tax in the extracellular environment, its possible role as an extracellular effector molecule, and a potential role in neurodegenerative disease associated with HTLV-I infection.

Complex Formation by Glycoproteins M and N of Human Cytomegalovirus: Structural and Functional Aspects

The genomes of herpesviruses contain a number of genes which are conserved throughout the family of Herpesviridae, indicating that the proteins may serve important functions in the replication of these viruses. Among these are several envelope glycoproteins, including glycoprotein M (gM) and gN, which form a complex that is covalently linked via disulfide bonds in some herpesviruses. However, deletion of gM and/or gN from most alphaherpesviruses has limited effects on replication of the respective viruses in vitro. In contrast, insertional inactivation of the gM gene of the betaherpesvirus human cytomegalovirus (HCMV) results in a replication-incompetent virus. We have started to analyze the structural and functional aspects of the interaction between gM and gN of HCMV. We show that large parts of gM are dispensable for the formation of a gM/gN complex that is transported to distal parts of the cellular secretory pathway. In addition, we demonstrate that the disulfide bond is between the cysteine at position 44 in gM and cysteine 90 in gN. However, disulfide linkage is not a prerequisite for modification and transport of the gM/gN complex. Moreover, mutant viruses that lack a disulfide bridge between gM and gN replicate with efficiencies similar to that of wild-type viruses.

Expansion of the Golgi apparatus in rat cerebral cortex following intracerebroventricular injections

Streptozotocin (STZ) is a bacterial toxin which selectively damages both insulin-producing cells and insulin receptors. Injections of STZ into the cerebral ventricles of experimental animals are followed by sustained biochemical, metabolic and behavioral effects resembling those which are found in human brains afflicted by Alzheimer's disease. The aim of the present study was to assess the effects of double intracerebroventricular application of STZ on the ultrastructure of rat frontoparietal cortical neurons. The most prominent change, seen 3 weeks after STZ injection, was a significant enlargement of the Golgi apparatus caused by expansion of the trans-Golgi segment of the cellular protein secretory pathway. Morphometric analysis revealed that the area of the trans part of the Golgi complex in neuronal cells was increased more than two-fold (median values: 312 × 103 nm3 in 14 neurons from control animals, and 846 × 103 nm3 in 19 neurons from STZ-treated animals, P=0.0012), whereas that of the cis part did not significantly change. The effects of STZ did not resemble Golgi atrophy and fragmentation described in neurons from disease-prone brain structures of patients with Alzheimer's disease, but were similar to that observed after intravenous application of a non-metabolizable glucose analog 2-deoxyglucose. Considering that proamyloidogenic processing of beta-amyloid precursor protein may occur preferentially in the trans-Golgi segment, the observed early response of neuronal ultrastructure to desensitization of insulin receptors may predispose cells to form beta-amyloid deposits.

Fast anterograde transport of herpes simplex virus: role for the amyloid precursor protein of alzheimer's disease.

Anterograde transport of herpes simplex virus (HSV) from its site of synthesis in the neuronal cell body out the neuronal process to the mucosal membrane is crucial for transmission of the virus from one person to another, yet the molecular mechanism is not known. By injecting GFP-labeled HSV into the giant axon of the squid, we reconstitute fast anterograde transport of human HSV and use this as an assay to uncover the underlying molecular mechanism. HSV travels by fast axonal transport at velocities four-fold faster (0.9 microm/sec average, 1.2 microm/sec maximal) than that of mitochondria moving in the same axon (0.2 microm/sec) and ten-fold faster than negatively charged beads (0.08 microm/sec). Transport of HSV utilizes cellular transport mechanisms because it appears to be driven from inside cellular membranes as revealed by negative stain electron microscopy and by the association of TGN46, a component of the cellular secretory pathway, with GFP-labeled viral particles. Finally, we show that amyloid precursor protein (APP), a putative receptor for the microtubule motor, kinesin, is a major component of viral particles, at least as abundant as any viral encoded protein, while another putative motor receptor, JIP 1/2, is not detected. Conventional kinesin is also associated with viral particles. This work links fast anterograde transport of the common pathogen, HSV, with the neurodegenerative Alzheimer's disease. This novel connection should prompt new ideas for treatment and prevention strategies.

Molecular physiology of the SERCA and SPCA pumps

Intracellular Ca 2+-transport ATPases exert a pivotal role in the endoplasmic reticulum and in the compartments of the cellular secretory pathway by maintaining a sufficiently high lumenal Ca 2+ (and Mn 2+) concentration in these compartments required for an impressive number of vastly different cell functions. At the same time this lumenal Ca 2+ represents a store of releasable activator Ca 2+ controlling an equally impressive number of cytosolic functions. This review mainly focuses on the different Ca 2+-transport ATPases found in the intracellular compartments of mainly animal non-muscle cells: the sarco(endo)plasmic reticulum Ca 2+-ATPase (SERCA) pumps. Although it is not our intention to treat the ATPases of the specialized sarcoplasmic reticulum in depth, we can hardly ignore the SERCA1 pump of fast-twitch skeletal muscle since its structure and function is by far the best understood and it can serve as a guide to understand the other members of the family. In a second part of this review we describe the relatively novel family of secretory pathway Ca 2+/Mn 2+ ATPases (SPCA), which in eukaryotic cells are primarily found in the Golgi compartment.

The trans Golgi network is lost from cells infected with African swine fever virus.

The cellular secretory pathway is important during the assembly and envelopment of viruses and also controls the transport of host proteins, such as cytokines and major histocompatibility proteins, that function during the elimination of viruses by the immune system. African swine fever virus (ASFV) encodes at least 26 proteins with stretches of hydrophobic amino acids suggesting entry into the secretory pathway (R. J. Yanez, J. M. Rodriguez, M. L. Nogal, L. Yuste, C. Enriquez, J. F. Rodriguez, and E. Vinuela, Virology 208:249-278, 1995). To predict how and where these potential membrane proteins function, we have studied the integrity of the secretory pathway in cells infected with ASFV. Remarkably, ASFV caused complete loss of immunofluorescence signal for the trans Golgi network (TGN) marker protein TGN46 and dispersed the AP1 TGN adapter complex. Loss of TGN46 signal was not due to degradation of TGN46, suggesting redistribution of TGN46 to other membrane compartments. ASFV markedly slowed transport of cathepsin D to lysosomes, demonstrating that loss of TGN structure correlated with loss of TGN function. ASFV shows a tropism for macrophages, and it is possible that ASFV compromises TGN function to augment the activity of viral membrane proteins or to suppress the function of host immunoregulatory proteins.

Cross-talk between the p42/p44 MAP Kinase and Smad Pathways in Transforming Growth Factor β1-induced Furin Gene Transactivation

Furin, a predominant convertase of the cellular constitutive secretory pathway, is known to be involved in the maturation of a number of growth/differentiation factors, but the mechanisms governing its expression remain elusive. We have previously demonstrated that transforming growth factor (TGF) beta 1, through the activation of Smad transducers, regulates its own converting enzyme, furin, creating a unique activation/regulation loop of potential importance in a variety of cell fate and functions. Here we studied the involvement of the p42/p44 MAPK pathway in such regulation. Using HepG2 cells transfected with fur P1 LUC (luciferase) promoter construct, we observed that forced expression of a dominant negative mutant form of the small G protein p21(ras) (RasN17) inhibited TGF beta 1-induced fur gene transcription, suggesting the involvement of the p42/p44 MAPK cascade. In addition, TGF beta induced sustained activation/phosphorylation of endogenous p42/p44 MAPK. Further-more, the role of MAPK cascade in fur gene transcription was highlighted by the use of the MEK1/2 inhibitors, PD98059 or U0126, or co-expression of a p44 antisense construct that repressed the induction of fur promoter transactivation. Conversely, overexpression of a constitutively active form of MEK1 increased unstimulated, TGF beta 1-stimulated, and Smad2-stimulated promoter P1 transactivation, and the universal Smad inhibitor, Smad7, inhibited this effect. Activation of Smad2 by MEK1 or TGF beta 1 resulted in an enhanced nuclear localization of Smad2, which was inhibited upon blocking MEK1 activity. Our findings clearly show that the activation of the p42/p44 MAPK pathway is involved in fur gene expression and led us to propose a co-operative model whereby TGF beta 1-induced receptor activation stimulates not only a Smad pathway but also a parallel p42/p44 MAPK pathway that targets Smad2 for an increased nuclear translocation and enhanced fur gene transactivation. Such an uncovered mechanism may be a key determinant for the regulation of furin in embryogenesis and growth-related physiopathological conditions.

The Cowpox Virus SPI-3 and Myxoma Virus SERP1 Serpins Are Not Functionally Interchangeable despite Their Similar Proteinase Inhibition Profiles in Vitro

The myxoma virus (MYX) serpin SERP1 is a secreted glycoprotein with anti-inflammatory activity that is required for full MYX virulence in vivo. The cowpox virus (CPV) serpin SPI-3 (vaccinia virus ORF K2L) is a nonsecreted glycoprotein that blocks cell–cell fusion, independent of serpin activity, and is not required for virulence of vaccinia virus or CPV in mice. Although SPI-3 has only 29% overall identity to SERP1, both serpins have arginine at the P1 position in the reactive center loop, and SPI-3 has a proteinase inhibitory profile strikingly similar to that of SERP1 [Turner, P. C., Baquero, M. T., Yuan, S., Thoennes, S. R., and Moyer, R. W. (2000) Virology 272, 267–280]. To determine whether SPI-3 and SERP1 were functionally equivalent, a CPV variant was constructed where the SPI-3 gene was deleted and replaced with the SERP1 gene regulated by the SPI-3 promoter. Cells infected with CPVΔSPI-3::SERP1 secrete SERP1 and show extensive fusion, suggesting that SERP1 is unable to functionally substitute for SPI-3 in fusion inhibition. In the reciprocal experiment, both copies of SERP1 were deleted from MYX and replaced with SPI-3 under the control of the SERP1 promoter. Cells infected with the MYXΔSERP1::SPI-3 recombinant unexpectedly secreted SPI-3, suggesting either that the cellular secretory pathway is enhanced by MYX or that CPV encodes a protein that prevents SPI-3 secretion. MYXΔSERP1::SPI-3 was as attenuated in rabbits as MYXΔSERP1::lacZ, indicating that SPI-3 cannot substitute for SERP1 in MYX pathogenesis.

Selective Accumulation of the Endoplasmic Reticulum–Golgi Intermediate Compartment Induced by the Antitumor Drug KRN5500

KRN5500 is a semisynthetic spicamycin analogue consisting of a seven-carbon amino sugar linked to a C14 unsaturated fatty acid through glycine and to the amino group of adenine. The drug inhibits cell growth potently and has antitumor activity in in vivo models. The mechanism of the antiproliferative effect of KRN5500 remains to be elucidated. We have found that acute exposure of drug-sensitive HT-29 colon adenocarcinoma cells to the drug results initially in swelling of the Golgi apparatus. Continuous exposure to the drug resulted in the emergence of a resistant population of cells characterized by numerous intracellular vacuoles. These KRN5500-resistant tumor cells exhibited increased staining with the Golgi stain NBD C6–ceramide and the ER–Golgi fluorescent dye BODIPY–brefeldin A, which, unlike the parental drug-sensitive cells, was dispersed throughout the cytoplasm. Marker enzymes associated with the ER (glucose 6-phosphatase) and cis-Golgi (GalNAc transferase) were elevated >2-fold and nearly 4-fold, respectively, in drug-resistant cell lines while the trans-Golgi marker enzyme, galactosyltransferase, was not. The additional findings that the KRN5500-resistant cells have a >2-fold elevation in ERGIC-53, a cis-Golgi marker protein of the ER–Golgi intermediate compartment (ERGIC), as well as increased 58K, a 58-kDa microtubule-binding protein with formiminotransferase cyclodeaminase activity, and tubulin indicate that the cellular secretory pathway is a primary determinant of sensitivity to KRN5500, as resistance to this agent corresponds with accumulation of several components relatable to ER and cis-Golgi function. Further support for this conclusion is provided by studies which demonstrate that KRN5500 alters the distribution of newly synthesized carcinoembryonic antigen within the secretory pathway, including arrest of this N-glycosylated protein in the Golgi of LS-174T colon carcinoma cells.

A single-amino-acid substitution of a tyrosine residue in the rubella virus E1 cytoplasmic domain blocks virus release.

Rubella virus particles, consisting of a nucleocapsid surrounded by a lipid envelope in which two virus-encoded glycoproteins E1 and E2 are embedded, assemble on intracellular membranes and are secreted from cells, possibly via the cellular secretory pathway. We have recently demonstrated that the cytoplasmic domain of E1 (residues 469 to 481, KCLYYLRGAIAPR) is required for virus release. Alteration of cysteine 470 to alanine did not affect virus release, whereas mutation of leucine 471 to alanine reduced virus production by 90%. In the present study, substitutions of remaining amino acids in the E1 cytoplasmic domain were made in order to investigate the role of each amino acid in regulating rubella virus release. Generated mutants were analyzed in the context of infectious full-length cDNA clone and virus-like particles using combined genetic, biochemical, and electron microscopic approaches. Substitution of a single residue of tyrosine 472 to alanine or tyrosine 473 to serine resulted in a block in virus release without affecting protein transport and virus budding into the lumen of the Golgi complexes. Infectious RNA transcripts bearing these mutations were incapable of forming plaques. Mutants with substitutions at the amino-terminal region (leucine 474, arginine 475, and glycine 476) in the E1 cytoplasmic domain had reduced virus release and small-plaque phenotype, while mutants with substitutions at the carboxy-terminal region (alanine 477, isoleucine 478, alanine 479, proline 480, and arginine 481) had only marginal defects in virus release. Plaque-forming revertants could be isolated from mutants Y472A and Y473S. Sequencing analysis revealed that the substituted serine residue in mutant Y473S reverted to the original tyrosine residue, whereas the substituted alanine residue in mutant Y472A was retained. These results indicate that the E1 cytoplasmic domain modulates virus release in a sequence-dependent manner and that the tyrosine residues are critical for this function. We postulate that residues YYLRG constitute a domain in the E1 tail that may interact with other proteins and this interaction is involved in regulating virus release.

Brome mosaic virus helicase- and polymerase-like proteins colocalize on the endoplasmic reticulum at sites of viral RNA synthesis.

The helicase-like 1a and polymerase-like 2a proteins of brome mosaic virus (BMV) are required for viral RNA replication in vivo, are present in membrane-bound viral RNA polymerase extracts, and share conservation with the many other members of the alphavirus-like superfamily. To better understand BMV RNA replication and BMV-host interactions, we used confocal microscopy and double-label immunofluorescence to determine and compare the sites of 1a, 2a, and nascent viral RNA accumulation in BMV-infected barley protoplasts. 1a and 2a showed nearly complete colocalization throughout infection, accumulating in defined cytoplasmic spots usually adjacent to or surrounding the nucleus. These spots grew throughout infection and by 16 h postinoculation often assumed a vesicle-like appearance. The BMV RNA replication complex incorporated 5-bromouridine 5'-triphosphate into RNA in vitro and in vivo, allowing immunofluorescent detection of nascent RNA. The cytoplasmic sites of BMV-specific RNA synthesis coincided with the sites of 1a and 2a accumulation, and at the resolution of confocal microscopy, all sites of 1a and 2a accumulation were sites of BMV RNA synthesis. Double-label immunofluorescence detection of selected subcellular markers and 1a or 2a showed that BMV replication complexes were tightly associated with markers for the endoplasmic reticulum but not the medial Golgi or later compartments of the cellular secretory pathway. Defining this association of BMV RNA replication complexes with endoplasmic reticulum markers should assist in identifying and characterizing host factors involved in BMV RNA replication.

The HTLV-I envelope glycoproteins: structure and functions.

The human T-cell lymphotropic virus type I (HTLV-I) envelope has a structural organization shared by all retroviral envelopes, which contain two mature viral glycoproteins deriving from a common precursor: an external surface protein (SU), associated with a transmembrane protein (TM) responsible for anchoring the SU-TM complex at the cell surface or in the viral envelope. Our understanding of the tertiary structure of these proteins is extremely poor. The intracellular maturation follows the normal cellular secretory pathway, resulting in expression of the mature glycoproteins at the cell surface. The five potential N-glycosylation sites are glycosylated. Most mutations artificially introduced into the glycoproteins result in loss of function, mostly due to abnormal intracellular maturation. This probably indicates a very compact structure of these proteins, where the entire structure is involved in correct conformation. Studies using neutralizing antibodies or mutagenesis have defined functional domains in the SU protein, which is responsible for receptor binding. These domains occur throughout the SU glycoprotein. Sequence analysis of the HTLV-I TM predicts a structure, and probably functions, similar to other retrovirus TMs: involvement of this glycoprotein in the different oligomerization steps leading to a fusogenic SU-TM complex and in the fusion process itself. These features remain to be proven, and it is not yet understood why the free HTLV-I viral particle is not infectious.