The endoplasmic reticulum (ER)-associated degradation system regulates aggregation and degradation of mutant neuroserpin

Familial encephalopathy with neuroserpin inclusion bodies is a neurodegenerative disorder characterized by the accumulation of neuroserpin polymers in the endoplasmic reticulum (ER) of cortical and subcortical neurons in the CNS because of neuroserpin point mutations. ER-associated degradation (ERAD) is involved in mutant neuroserpin degradation. In this study, we demonstrate that two ER-associated E3 ligases, Hrd1 and gp78, are involved in the ubiquitination and degradation of mutant neuroserpin. Overexpression of Hrd1 and gp78 decreases the mutant neuroserpin protein level, whereas Hrd1 and gp78 knockdown increases mutant neuroserpin stability. Moreover, ERAD impairment by mutant valosin-containing protein increases the mutant neuroserpin protein level and aggregate formation. Thus, these findings identify mutant neuroserpin as an ERAD target and show that Hrd1 and gp78 mediate mutant neuroserpin turnover through the ERAD pathway.     

Different p97/VCP complexes function in retrotranslocation step of mammalian ER-associated degradation (ERAD)

Studies in yeast indicate that three specialized endoplasmic reticulum-associated degradation (ERAD) pathways, namely ERAD-L, -M, or -C, dispose substrates with structural lesions in the lumenal, transmembrane, or cytosolic domains, respectively. The ubiquitin ligase (E3) Hrd1p and its cooperating partners are required for ERAD-L and -M pathways, whereas Doa10p complex is required for the ERAD-C pathway. We investigated these pathways in mammalian cells by assessing the requirements of the mammalian ERAD E3s, gp78 and Hrd1, in degradation of four substrates each with different type of structural lesions: CD3δ, Z-variant α1-antitrypsin, tyrosinase (C89R) and mutant cystic fibrosis transmembrane conductance regulator (CFTRΔF508). We demonstrated that tyrosinase (C89R) is a substrate for Hrd1 while all others are gp78 substrates. Knockdown of Hrd1 diminished gp78 substrate levels, but silencing of gp78 had no effect on Hrd1's substrate, suggesting that the functional interaction between Hrd1 and gp78 is unidirectional. Furthermore, while Ufd1 is dispensable for gp78-mediated ERAD, it is essential for Hrd1-mediated ERAD. Interestingly, Npl4 was found to be a key component for both pathways. These results suggest that the Hrd1-mediated ERAD requires a well-established retrotranslocation machinery, the p97/VCP-Ufd1-Npl4 complex, whereas the gp78 pathway needs only p97/VCP and Npl4. In addition, the three distinct ERAD pathways described in yeast may not be strictly conserved in mammalian cells as gp78 can function on three substrates with different structural lesions.     

Membrane-associated ubiquitin ligase complex containing gp78 mediates sterol-accelerated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase

The endoplasmic reticulum (ER)-associated degradation (ERAD) pathway in the yeast Saccharomyces cerevisiae is mediated by two membrane-bound ubiquitin ligases, Doa10 and Hrd1. These enzymes are found in distinct multiprotein complexes that allow them to recognize and target a variety of substrates for proteasomal degradation. Although multiprotein complexes containing mammalian ERAD ubiquitin ligases likely exist, they have yet to be identified and characterized in detail. Here, we identify two ER membrane proteins, SPFH2 and TMUB1, as associated proteins of mammalian gp78, a membrane-bound ubiquitin ligase that bears significant sequence homology with mammalian Hrd1 and mediates sterol-accelerated ERAD of the cholesterol biosynthetic enzyme HMG-CoA reductase. Co-immunoprecipitation studies indicate that TMUB1 bridges SPFH2 to gp78 in ER membranes. The functional significance of these interactions is revealed by the observation that RNA interference (RNAi)-mediated knockdown of SPFH2 and TMUB1 blunts both the sterol-induced ubiquitination and degradation of endogenous reductase in HEK-293 cells. These studies mark the initial steps in the characterization of the mammalian gp78 ubiquitin ligase complex, the further elucidation of which may yield important insights into mechanisms underlying gp78-mediated ERAD.     

Intracellular degradation of the HIV-1 envelope glycoprotein. Evidence for, and some characteristics of, an endoplasmic reticulum degradation pathway.

Analysis of the fate of HIV-1 envelope protein gp160 (Env) has shown that newly synthesized proteins may be degraded within the biosynthetic pathway and that this degradation may take place in compartments other than the lysosomes. The fate of newly synthesized Env was studied in living BHK-21 cells with the recombinant vaccinia virus expression system. We found that gp160 not only undergoes physiological endoproteolytic cleavage, producing gp120, but is also degraded, producing proteolytic fragments of 120 kDa to 26 kDa in size, as determined by SDS/PAGE in non reducing conditions. Analysis of the 120-kDa proteolytic fragment, and comparison with gp120, showed that it is composed of peptides linked by disulfides bonds and lacks the V3-loop epitope and the C-terminal domain of gp120 (amino acids 506-516). A permeabilized cell system, with impaired transport of labeled Env from the endoplasmic reticulum (ER) to Golgi compartments, was developed to determine the site of degradation and to define some biochemical characteristics of the intracellular degradation process. In the semipermeable BHK-21 cells, there was: (a) no gp120 production (b), a progressive decrease in the amount of newly synthesized gp160 and a concomitant increase in the amount of a 120-kDa proteolytic fragment. This fragment had the same biochemical characteristics as the 120-kDa proteolytic fragment found in living nonpermeabilized cells, and (c) susceptibility of the V3 loop. This degradation process occurred in the ER, as shown by both biochemical and indirect immunofluorescence analysis. Furthermore, there was evidence that changes in redox state are involved in the ER-dependent envelope degradation pathway because adding reducing agents to permeabilized cells caused dose-dependent degradation of the 120-kDa proteolytic fragment and of the remaining gp160 glycoprotein. Thus our results provide direct evidence that regulated degradation of the HIV-1 envelope glycoprotein may take place in the ER of infected cells.     

AAA ATPase p97/valosin-containing protein interacts with gp78, a ubiquitin ligase for endoplasmic reticulum-associated degradation

Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control mechanism that eliminates unwanted proteins from the endoplasmic reticulum (ER) through a ubiquitin-dependent proteasomal degradation pathway. gp78 is a previously described ER membrane-anchored ubiquitin ligase (E3) involved in ubiquitination of ER proteins. AAA ATPase (ATPase associated with various cellular activities) p97/valosin-containing protein (VCP) subsequently dislodges the ubiquitinated proteins from the ER and chaperones them to the cytosol, where they undergo proteasomal degradation. We now report that gp78 physically interacts with p97/VCP and enhances p97/VCP-polyubiquitin association. The enhanced association correlates with decreases in ER stress-induced accumulation of polyubiquitinated proteins. This effect is abolished when the p97/VCP-interacting domain of gp78 is removed. Further, using ERAD substrate CD3delta, gp78 consistently enhances p97/VCP-CD3delta binding and facilitates CD3delta degradation. Moreover, inhibition of endogenous gp78 expression by RNA interference markedly increases the levels of total polyubiquitinated proteins, including CD3delta, and abrogates VCP-CD3delta interactions. The gp78 mutant with deletion of its p97/VCP-interacting domain fails to increase CD3delta degradation and leads to accumulation of polyubiquitinated CD3delta, suggesting a failure in delivering ubiquitinated CD3delta for degradation. These data suggest that gp78-p97/VCP interaction may represent one way of coupling ubiquitination with retrotranslocation and degradation of ERAD substrates.     

Gp78 cooperates with RMA1 in endoplasmic reticulum-associated degradation of CFTRDeltaF508

Misfolded or improperly assembled proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded via the ubiquitin–proteasome pathway, a process termed ER-associated degradation (ERAD). Saccharomyces cerevisiae Hrd1p/Der3p is an ER membrane-spanning ubiquitin ligase that participates in ERAD of the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is exogenously expressed in yeast cells. Two mammalian orthologues of yeast Hrd1p/Der3p, gp78 and HRD1, have been reported. Here, we demonstrate that gp78, but not HRD1, participates in ERAD of the CFTR mutant CFTRΔF508, by specifically promoting ubiquitylation of CFTRΔF508. Domain swapping experiments and deletion analysis revealed that gp78 binds to CFTRΔF508 through its ubiquitin binding region, the so-called coupling of ubiquitin to ER degradation (CUE) domain. Gp78 polyubiquitylated in vitro an N-terminal ubiquitin-glutathione-S-transferase (GST)-fusion protein, but not GST alone. This suggests that gp78 recognizes the ubiquitin that is already conjugated to CFTRΔF508 and catalyzes further polyubiquitylation of CFTRΔF508 in a manner similar to that of a multiubiquitin chain assembly factor (E4). Furthermore, we revealed by small interfering RNA methods that the ubiquitin ligase RMA1 functioned as an E3 enzyme upstream of gp78. Our data demonstrates that gp78 cooperates with RMA1 with E4-like activity in the ERAD of CFTRΔF508.     

Role of the endoplasmic reticulum-associated degradation (ERAD) pathway in degradation of hepatitis C virus envelope proteins and production of virus particles

Viral infections frequently cause endoplasmic reticulum (ER) stress in host cells leading to stimulation of the ER-associated degradation (ERAD) pathway, which subsequently targets unassembled glycoproteins for ubiquitylation and proteasomal degradation. However, the role of the ERAD pathway in the viral life cycle is poorly defined. In this paper, we demonstrate that hepatitis C virus (HCV) infection activates the ERAD pathway, which in turn controls the fate of viral glycoproteins and modulates virus production. ERAD proteins, such as EDEM1 and EDEM3, were found to increase ubiquitylation of HCV envelope proteins via direct physical interaction. Knocking down of EDEM1 and EDEM3 increased the half-life of HCV E2, as well as virus production, whereas exogenous expression of these proteins reduced the production of infectious virus particles. Further investigation revealed that only EDEM1 and EDEM3 bind with SEL1L, an ER membrane adaptor protein involved in translocation of ERAD substrates from the ER to the cytoplasm. When HCV-infected cells were treated with kifunensine, a potent inhibitor of the ERAD pathway, the half-life of HCV E2 increased and so did virus production. Kifunensine inhibited the binding of EDEM1 and EDEM3 with SEL1L, thus blocking the ubiquitylation of HCV E2 protein. Chemical inhibition of the ERAD pathway neither affected production of the Japanese encephalitis virus (JEV) nor stability of the JEV envelope protein. A co-immunoprecipitation assay showed that EDEM orthologs do not bind with JEV envelope protein. These findings highlight the crucial role of the ERAD pathway in the life cycle of specific viruses.     

Huntingtin Interacts with the Cue Domain of gp78 and Inhibits gp78 Binding to Ubiquitin and p97/VCP

Huntington''s disease (HD) is caused by polyglutamine expansion in huntingtin (htt) protein, but the exact mechanism of HD pathogenesis remains uncertain. Recent evidence suggests that htt proteins with expanded polyglutamine tracts induce endoplasmic reticulum (ER) stress, probably by interfering with ER-associated degradation (ERAD). Here we report that mutant htt interacts and interferes with the function of gp78, an ER membrane-anchored ubiquitin ligase (E3) involved in ERAD. Mapping studies showed that the HEAT repeats 2&3 of htt interact with the cue domain of gp78. The interaction competitively reduces polyubiquitinated protein binding to gp78 and also sterically blocks gp78 interaction of p97/VCP, a molecular chaperone that is essential for ERAD. These effects of htt negatively regulate the function of gp78 in ERAD and are aggravated by polyglutamine expansion. Paradoxically, gp78 is still able to ubiquitinate and facilitate degradation of htt proteins with expanded polyglutamine. The impairment of ERAD by mutant htt proteins is associated with induction of ER stress. Our studies provide a novel molecular mechanism that supports the involvement of ER stress in HD pathogenesis.     

Differential regulation of CFTRΔF508 degradation by ubiquitin ligases gp78 and Hrd1

The most common mutation associated with cystic fibrosis is the deletion of phenylalanine 508 of cystic fibrosis transmembrane conductance regulator (CFTRΔF508). This mutation renders otherwise functional protein susceptible to ER-associated degradation (ERAD) and prevents CFTR from exiting the ER and trafficking to the plasma membrane. In this study, we demonstrate that RNAi-mediated silencing of gp78, an established ubiquitin ligase (E3) involved in ERAD, leads to accumulation of CFTRΔF508 protein in cells. gp78 facilitates the degradation of CFTRΔF508 by enhancing both its ubiquitination and interaction with p97/VCP. SVIP, which is the inhibitor of gp78, causes accumulation of CFTRΔF508. We showed that endogenous gp78 co-immunoprecipitates with Hrd1. Furthermore, the results indicate that silencing the expression of another ERAD E3, Hrd1, leads to stabilization of gp78 and decline in gp78 ubiquitination; thereby enhancing CFTRΔF508 degradation. The results support that gp78 is an E3 targeting CFTRΔF508 for degradation and Hrd1 inhibits CFTRΔF508 degradation by acting as an E3 for gp78.     

Targeting of gp78 for ubiquitin-mediated proteasomal degradation by Hrd1: Cross-talk between E3s in the endoplasmic reticulum

There are an increasing number of ubiquitin ligases (E3s) implicated in endoplasmic reticulum (ER)-associated degradation (ERAD) in mammals. The two for which the greatest amount of information exists are the RING finger proteins gp78 and Hrd1, which are the structural orthologs of the yeast ERAD E3 Hrd1p. We now report that Hrd1, also known as synoviolin, targets gp78 for proteasomal degradation independent of the ubiquitin ligase activity of gp78, without evidence of a reciprocal effect. This degradation is observed in mouse embryonic fibroblasts lacking Hrd1, as well as with acute manipulation of Hrd1. The significance of this is underscored by the diminished level of a gp78-specific substrate, Insig-1, when Hrd1 expression is decreased and gp78 levels are consequently increased. These finding demonstrate a previously unappreciated level of complexity of the ubiquitin system in ERAD and have potentially important ramifications for processes where gp78 is implicated including regulation of lipid metabolism, metastasis, cystic fibrosis and neurodegenerative disorders.     

Identification of SVIP as an endogenous inhibitor of endoplasmic reticulum-associated degradation

Misfolded proteins in the endoplasmic reticulum (ER) are eliminated by a process known as ER-associated degradation (ERAD), which starts with misfolded protein recognition, followed by ubiquitination, retrotranslocation to the cytosol, deglycosylation, and targeting to the proteasome for degradation. Actions of multisubunit protein machineries in the ER membrane integrate these steps. We hypothesized that regulation of the multisubunit machinery assembly is a mechanism by which ERAD activity is regulated. To test this hypothesis, we investigated the potential regulatory role of the small p97/VCP-interacting protein (SVIP) on the formation of the ERAD machinery that includes ubiquitin ligase gp78, AAA ATPase p97/VCP, and the putative channel Derlin1. We found that SVIP is anchored to microsomal membrane via myristoylation and co-fractionated with gp78, Derlin1, p97/VCP, and calnexin to the ER. Like gp78, SVIP also physically interacts with p97/VCP and Derlin1. Overexpression of SVIP blocks unassembled CD3delta from association with gp78 and p97/VCP, which is accompanied by decreases in CD3delta ubiquitination and degradation. Silencing SVIP expression markedly enhances the formation of gp78-p97/VCP-Derlin1 complex, which correlates with increased degradation of CD3delta and misfolded Z variant of alpha-1-antitrypsin, established substrates of gp78. These results suggest that SVIP is an endogenous inhibitor of ERAD that acts through regulating the assembly of the gp78-p97/VCP-Derlin1 complex.     

ER stress differentially regulates the stabilities of ERAD ubiquitin ligases and their substrates

Endoplasmic reticulum (ER) stress-induced accumulation of misfolded proteins in the ER stimulates the ER-associated degradation (ERAD) process. ERAD in turn eliminates those misfolded proteins. Upregulation of ubiquitination enzymes is an essential mechanism by which ER stress enhances ERAD. However, ectopic overexpression of ubiquitination enzymes often fails to increase, and sometimes, inhibits ERAD. To further understand how ER stress regulates ERAD, we studied the effects of ER stress on ubiquitin ligase (E3) gp78-mediated ERAD and on the stabilities of gp78 and another ERAD E3 Hrd1. The results showed that ER stress-inducing agent tunicamycin significantly enhanced ERAD in cells that either express endogenous or overexpress gp78. Importantly, ER stress could increase ERAD even when new protein synthesis was inhibited by cycloheximide. Surprisingly, tunicamycin treatment stabilized gp78, an established ERAD E3 and an ERAD substrate as well, for up to 8h. By contrast, ER stress had little effects on the stability of another E3 Hrd1 except that it reduced the total ubiquitination level of Hrd1. Our data suggest that ER stress differentially regulates the stabilities of ERAD E3s and their substrates, which may represent a novel mechanism by which ER stress increases ERAD.     

Ubiquitin ligase gp78 increases solubility and facilitates degradation of the Z variant of alpha-1-antitrypsin

Deficiency of circulating alpha-1-antitrypsin (AAT) is the most widely recognized abnormality of a proteinase inhibitor that causes lung disease. AAT-deficiency is caused by mutations of the AAT gene that lead to AAT protein retention in the endoplasmic reticulum (ER). Moreover, the mutant AAT accumulated in the ER predisposes the homozygote to severe liver injuries, such as neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma. Despite the fact that mutant AAT protein is subject to ER-associated degradation (ERAD), yeast genetic studies have determined that the ubiquitination machinery, Hrd1/Der3p-cue1p-Ubc7/6p, which plays a prominent role in ERAD, is not involved in degradation of mutant AAT. Here we report that gp78, a ubiquitin ligase (E3) pairing with mammalian Ubc7 for ERAD, ubiquitinates and facilitates degradation of ATZ, the classic deficiency variant of AAT having a Z mutation (Glu 342 Lys). Unexpectedly, gp78 over-expression also significantly increases ATZ solubility. p97/VCP, an AAA ATPase essential for retrotranslocation of misfolded proteins from the ER during ERAD, is involved in gp78-mediated degradation of ATZ. Surprisingly, unlike other ERAD substrates that cause ER stress leading to apoptosis when accumulated in the ER, ATZ, in fact, increases cell proliferation when over-expressed in cells. This effect can be partially inhibited by gp78 over-expression. These data indicate that gp78 assumes multiple unique quality control roles over ATZ, including the facilitation of degradation and inhibition of aggregation of ATZ.     

Small Molecule Targets Env for Endoplasmic Reticulum-Associated Protein Degradation and Inhibits Human Immunodeficiency Virus Type 1 Propagation

Human immunodeficiency virus type 1 (HIV-1) is dependent on its envelope glycoprotein (Env) to bind, fuse, and subsequently infect a cell. We show here that treatment of HIV-1-infected cells with glycyl-prolyl-glycine amide (GPG-NH₂), dramatically reduced the infectivity of the released viral particles by decreasing their Env incorporation. The mechanism of GPG-NH₂ was uncovered by examining Env expression and maturation in treated cells. GPG-NH₂ treatment was found to affect Env by significantly decreasing its steady-state levels, its processing into gp120/gp41, and its mass by inducing glycan removal in a manner dependent on its native signal sequence and the proteasome. Therefore, GPG-NH₂ negatively impacts Env maturation, facilitating its targeting for endoplasmic reticulum-associated protein degradation, where Env is deglycosylated en route to its degradation. These findings illustrate that nontoxic drugs such as GPG-NH₂, which can selectively target glycoproteins to existing cellular degradation pathways, may be useful for pathogen therapy.The endoplasmic reticulum (ER) contains a number of molecular chaperones and folding factors that aid in the maturation of proteins that traverse the secretory pathway. This process is strictly monitored by the ER quality control system, which selects properly folded proteins for export to the Golgi (16) and targets misfolded proteins for destruction through the ER-associated protein degradation pathway (ERAD) (4, 28). Once an ER protein is selected as a substrate for ERAD, it is translocated from the ER lumen to the cytosol through an ER translocon. This retrotranslocation process is thought to be driven by either the cytosolic AAA-ATPase p97 (39) or the 19S proteasome cap (23). Upon entrance into the cytosol, the ERAD substrate is ubquitinated, and its glycans are removed by an N-glycanase to prepare it for proteasomal degradation (11, 28).Viral envelope glycoproteins utilize the host cell secretory pathway for their proper maturation and trafficking to the site of viral assembly. The human immunodeficiency virus type 1 (HIV-1) encodes the envelope glycoprotein (Env), which initiates HIV-1 infections by mediating attachment and fusion of the viral envelope with the host cell membrane (17). Therefore, infectious HIV-1 particle production relies on the ability of Env to pass the rigorous ER quality control system.Env is initially synthesized as a type I membrane precursor glycoprotein termed gp160, which is cotranslationally targeted to the ER by its 30-amino-acid N-terminal signal sequence (24). Within the ER, gp160 receives ∼30 N-linked glycans and is assisted in its maturation by the chaperones BiP, calnexin, and calreticulin as it undergoes extensive disulfide bond formations (15, 21, 31). Once gp160 has reached its native state with ten disulfide bonds and its signal sequence has been cleaved posttranslationally (21, 25), it assembles into trimers (26) and is exported to the Golgi. Within the Golgi, gp160 is cleaved by cellular endoproteases, yielding the transmembrane protein gp41 and the noncovalently associated surface protein gp120 (27). Thereafter, this complex is transported to the plasma membrane, where it is incorporated into the envelope of assembling HIV-1 particles.We have previously shown that a tripeptide amide corresponding to a conserved motif of the HIV-1 Env, glycyl-prolyl-glycine amide (GPG-NH₂), suppressed the replication of all 47 HIV-1 laboratory strains and clinical isolates examined with a 50% inhibitory concentration of ∼10 μM, a concentration that is 200- to 2,000-fold less than what affected cell growth or had other toxic effects on peripheral blood mononuclear cells (35). However, this suppression was not, as we had anticipated, due to interactions of the peptide with the early events of the HIV-1 replication cycle, such as attachment or entry (36). In the present study, we demonstrate that GPG-NH₂ reduced Env incorporation into HIV-1 particles during replication by targeting Env toward the ERAD pathway. The ability of GPG-NH₂ to target Env for degradation was dependent on the presence of functional proteasomes and required the full-length Env signal sequence. These findings illustrate that small molecules may be utilized therapeutically to specifically target unwanted pathogenic proteins for degradation by the existing cellular machinery.     

The E3 Ubiquitin Ligase gp78 Protects against ER Stress in Zebrafish Liver

Enhanced endoplasmic reticulum （ER）-associated protein degradation （ERAD） activity by the unfolded protein response （UPR） represents one of the mechanisms for restoring ER homeostasis. In vitro evidence indicates that the mammalian gp78 protein is an E3 ubiquitin ligase that facilitates ERAD by polyubiquitinating and targeting proteins for proteasomal degradation under both physiologic and stress conditions. However, the in vivo function of gp78 in maintaining ER protein homeostasis remains untested. Here we show that like its mammalian counterpart, the zebrafish gp78 is also an E3 ubiquitin ligase as revealed by in vitro ubiquitination assays. Expression analysis uncovered that gp78 is highly expressed in several organs, including liver and brain, of both larval and adult fish. Treatment of larvae or adult fish with tunicamycin induces ER stress and upregulates the expression of several key components of the gp78 ERAD complex in the liver. Moreover, liver-specific overexpression of the dominant-negative form of gp78 （gp78-R2M） renders liver more sensitive to tunicamycin-induced ER stress and enhances the expression of sterol response element binding protein （Srebp）-target genes, which was largely suppressed in fish overexpressing wild-type gp78. Together, these data indicate that gp78 plays a critical role in protecting against ER stress in liver.     

Gp78, a membrane-anchored ubiquitin ligase, associates with Insig-1 and couples sterol-regulated ubiquitination to degradation of HMG CoA reductase

Sterol-regulated ubiquitination is an obligatory step in ER-associated degradation (ERAD) of HMG CoA reductase, a rate-limiting enzyme in cholesterol synthesis. Accelerated degradation of reductase, one of several strategies animal cells use to limit production of cholesterol, requires sterol-induced binding of the enzyme to ER membrane proteins called Insigs. Once formed, the reductase-Insig complex is recognized by a putative membrane-associated ubiquitin ligase (E3) that mediates the reductase ubiquitination reaction. Here, we show that gp78, a membrane bound E3, binds to Insig-1 and is required for sterol-regulated ubiquitination of reductase. In addition, gp78 couples regulated ubiquitination to degradation of reductase by binding to VCP, an ATPase that plays a key role in recognition and degradation of ERAD substrates. The current results identify gp78 as the E3 that initiates sterol-accelerated degradation of reductase, and Insig-1 as a bridge between gp78/VCP and the reductase substrate.     

Unconventional roles of nonlipidated LC3 in ERAD tuning and coronavirus infection

Secretory and membrane proteins attain their native structure in the endoplasmic reticulum (ER). Folding-defective polypeptides are selected for degradation by processes collectively defined as ER-associated degradation (ERAD). Enhanced ERAD activity may interfere with protein biogenesis by inappropriately targeting not-yet-native protein folding intermediates for disposal. The regulation of ERAD is therefore crucial to maintain cellular proteostasis. At steady-state, select ERAD regulators are constitutively removed from the ER in a series of processes collectively defined as ERAD tuning. This sets the ERAD activity at levels that do not interfere with completion of ongoing folding programs. Our latest work highlights a crucial, autophagy-independent role of nonlipidated LC3 (LC3-I) as part of a membrane-bound receptor that insures the vesicle-mediated clearance of at least two ERAD regulators from the ER, EDEM1 and OS9. This pathway is hijacked by coronaviruses (CoV), and silencing of LC3 substantially inhibits viral replication.     

Human Ubc9 Is Involved in Intracellular HIV-1 Env Stability after Trafficking out of the Trans-Golgi Network in a Gag Dependent Manner

The cellular E2 Sumo conjugase, Ubc9 interacts with HIV-1 Gag, and is important for the assembly of infectious HIV-1 virions. In the previous study we demonstrated that in the absence of Ubc9, a defect in virion assembly was associated with decreased levels of mature intracellular Envelope (Env) that affected Env incorporation into virions and virion infectivity. We have further characterized the effect of Ubc9 knockdown on HIV Env processing and assembly. We found that gp160 stability in the endoplasmic reticulum (ER) and its trafficking to the trans-Golgi network (TGN) were unaffected, indicating that the decreased intracellular mature Env levels in Ubc9-depleted cells were due to a selective degradation of mature Env gp120 after cleavage from gp160 and trafficked out of the TGN. Decreased levels of Gag and mature Env were found to be associated with the plasma membrane and lipid rafts, which suggest that these viral proteins were not trafficked correctly to the assembly site. Intracellular gp120 were partially rescued when treated with a combination of lysosome inhibitors. Taken together our results suggest that in the absence of Ubc9, gp120 is preferentially degraded in the lysosomes likely before trafficking to assembly sites leading to the production of defective virions. This study provides further insight in the processing and packaging of the HIV-1 gp120 into mature HIV-1 virions.     

A mechanism of restricted human immunodeficiency virus type 1 expression in human glial cells.

We characterized in detail the life cycle of human immunodeficiency virus type 1 (HIV-1) in human glioma H4/CD4 cells which stably express transfected CD4 DNA (B. Volsky, K. Sakai, M. Reddy, and D. J. Volsky, Virology 186:303-308, 1992). Infection of cloned H4/CD4 cells with the N1T strain of cell-free HIV-1 (HIV-1/N1T) was rapid and highly productive as measured by the initial expression of viral DNA, RNA, and protein, but all viral products declined to low levels by 14 days after infection. Chronically infected, virus-producing H4/CD4 cells could be obtained by cell cloning, indicating that HIV-1 DNA can integrate and remain expressed in these cells. The HIV-1 produced in H4/CD4 cells was noninfectious to glial cells, but it could be transmitted with low efficiency to CEM cells. Examination of viral protein composition by immunoprecipitation with AIDS serum or anti-gp120 antibody revealed that HIV-1/N1T-infected H4/CD4 cells produced all major viral proteins including gp160, but not gp120. Deglycosylation experiments with three different glycosidases determined that the absence of gp120 was not due to aberrant glycosylation of gp160, indicating a defect in gp160 proteolytic processing. Similar results were obtained in acutely and chronically infected H4/CD4 cells. To determine the generality of this HIV-1 replication phenotype in H4/CD4 cells, nine different viral clones were tested for replication in H4/CD4 cells by transfection. Eight were transiently productive like N1T, but one clone, NL4-3, established a long-lived productive infection in H4/CD4 cells, produced infectious progeny virus, and produced both gp160 and gp120. We conclude that for most HIV-1 strains tested, HIV-1 infection of H4/CD4 is restricted to a single cycle because of the defective processing of gp160, resulting in the absence of gp120 on progeny virus.