Biochemical and virological analysis of the 18-residue C-terminal tail of HIV-1 integrase

Background

Mouse mammary tumor virus (MMTV) encodes the Rem protein, an HIV Rev-like protein that enhances nuclear export of unspliced viral RNA in rodent cells. We have shown that Rem is expressed from a doubly spliced RNA, typical of complex retroviruses. Several recent reports indicate that MMTV can infect human cells, suggesting that MMTV might interact with human retroviruses, such as human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV), and human endogenous retrovirus type K (HERV-K). In this report, we test whether the export/regulatory proteins of human complex retroviruses will increase expression from vectors containing the Rem-responsive element (RmRE).

Results

MMTV Rem, HIV Rev, and HTLV Rex proteins, but not HERV-K Rec, enhanced expression from an MMTV-based reporter plasmid in human T cells, and this activity was dependent on the RmRE. No RmRE-dependent reporter gene expression was detectable using Rev, Rex, or Rec in HC11 mouse mammary cells. Cell fractionation and RNA quantitation experiments suggested that the regulatory proteins did not affect RNA stability or nuclear export in the MMTV reporter system. Rem had no demonstrable activity on export elements from HIV, HTLV, or HERV-K. Similar to the Rem-specific activity in rodent cells, the RmRE-dependent functions of Rem, Rev, or Rex in human cells were inhibited by a dominant-negative truncated nucleoporin that acts in the Crm1 pathway of RNA and protein export.

Conclusion

These data argue that many retroviral regulatory proteins recognize similar complex RNA structures, which may depend on the presence of cell-type specific proteins. Retroviral protein activity on the RmRE appears to affect a post-export function of the reporter RNA. Our results provide additional evidence that MMTV is a complex retrovirus with the potential for viral interactions in human cells.     

Proper protein folding in the endoplasmic reticulum is required for attachment of a glycosylphosphatidylinositol anchor in plants

Endoplasmic reticulum (ER) quality control processes recognize and eliminate misfolded proteins to maintain cellular protein homeostasis and prevent the accumulation of defective proteins in the secretory pathway. Glycosylphosphatidylinositol (GPI)-anchored proteins carry a glycolipid modification, which provides an efficient ER export signal and potentially prevents the entry into ER-associated degradation (ERAD), which is one of the major pathways for clearance of terminally misfolded proteins from the ER. Here, we analyzed the degradation routes of different misfolded glycoproteins carrying a C-terminal GPI-attachment signal peptide in Arabidopsis thaliana. We found that a fusion protein consisting of the misfolded extracellular domain from Arabidopsis STRUBBELIG and the GPI-anchor attachment sequence of COBRA1 was efficiently targeted to hydroxymethylglutaryl reductase degradation protein 1 complex-mediated ERAD without the detectable attachment of a GPI anchor. Non-native variants of the GPI-anchored lipid transfer protein 1 (LTPG1) that lack a severely misfolded domain, on the other hand, are modified with a GPI anchor and targeted to the vacuole for degradation. Impaired processing of the GPI-anchoring signal peptide by mutation of the cleavage site or in a GPI-transamidase-compromised mutant caused ER retention and routed the non-native LTPG1 to ERAD. Collectively, these results indicate that for severely misfolded proteins, ER quality control processes are dominant over ER export. For less severely misfolded proteins, the GPI anchor provides an efficient ER export signal resulting in transport to the vacuole.

Severely misfolded proteins carrying a glycosylphosphatidylinositol (GPI)-anchor attachment sequence undergo a stringent quality control process in the endoplasmic reticulum that prevents GPI anchoring.     

Misfolded proteins traffic from the endoplasmic reticulum (ER) due to ER export signals

Most misfolded secretory proteins remain in the endoplasmic reticulum (ER) and are degraded by ER-associated degradation (ERAD). However, some misfolded proteins exit the ER and traffic to the Golgi before degradation. Using model misfolded substrates, with or without defined ER exit signals, we found misfolded proteins can depart the ER by continuing to exhibit the functional export signals present in the corresponding correctly folded proteins. Anterograde transport of misfolded proteins utilizes the same machinery responsible for exporting correctly folded proteins. Passive ER retention, in which misfolded proteins fail to exit the ER due to the absence of exit signals or the inability to functionally present them, likely contributes to the retention of nonnative proteins in the ER. Intriguingly, compromising ERAD resulted in increased anterograde trafficking of a misfolded protein with an ER exit signal, suggesting that ERAD and ER exit machinery can compete for binding of misfolded proteins. Disabling ERAD did not result in transport of an ERAD substrate lacking an export signal. This is an important distinction for those seeking possible therapeutic approaches involving inactivating ERAD in anticipation of exporting a partially active protein.     

Characterization of early EDEM1 protein maturation events and their functional implications

The endoplasmic reticulum (ER) quality control factor EDEM1 associates with a number of ER proteins and ER-associated degradation (ERAD) substrates; however, an understanding of its role in ERAD is unclear. The early maturation events for EDEM1 including signal sequence cleavage and glycosylation were analyzed, and their relationship to the function of EDEM1 was determined. EDEM1 has five N-linked glycosylation sites with the most C-terminal site recognized poorly cotranslationally, resulting in the accumulation of EDEM1 containing four or five glycans. The fifth site was modified post-translationally when bypassed cotranslationally. Signal sequence cleavage of EDEM1 was found to be a slow and inefficient process. Signal sequence cleavage produced a soluble form of EDEM1 that efficiently associated with the oxidoreductase ERdj5 and most effectively accelerated the turnover of a soluble ERAD substrate. In contrast, a type-II membrane form of EDEM1 was generated when the signal sequence was uncleaved, creating an N-terminal transmembrane segment. The membrane form of EDEM1 efficiently associated with the ER membrane protein SEL1L and accelerated the turnover of a membrane-associated ERAD substrate. Together, these results demonstrated that signal sequence cleavage functionally regulated the association of EDEM1-soluble and membrane-integrated isoforms with distinct ERAD machinery and substrates.     

Inhibition of endoplasmic reticulum-associated degradation rescues native folding in loss of function protein misfolding diseases

Lysosomal storage disorders are often caused by mutations that destabilize native folding and impair trafficking of secretory proteins. We demonstrate that endoplasmic reticulum (ER)-associated degradation (ERAD) prevents native folding of mutated lysosomal enzymes in patient-derived fibroblasts from two clinically distinct lysosomal storage disorders, namely Gaucher and Tay-Sachs disease. Prolonging ER retention via ERAD inhibition enhanced folding, trafficking, and activity of these unstable enzyme variants. Furthermore, combining ERAD inhibition with enhancement of the cellular folding capacity via proteostasis modulation resulted in synergistic rescue of mutated enzymes. ERAD inhibition was achieved by cell treatment with small molecules that interfere with recognition (kifunensine) or retrotranslocation (eeyarestatin I) of misfolded substrates. These different mechanisms of ERAD inhibition were shown to enhance ER retention of mutated proteins but were associated with dramatically different levels of ER stress, unfolded protein response activation, and unfolded protein response-induced apoptosis.     

Intramembrane proteolysis and endoplasmic reticulum retention of hepatitis C virus core protein

Hepatitis C virus (HCV) core protein is suggested to localize to the endoplasmic reticulum (ER) through a C-terminal hydrophobic region that acts as a membrane anchor for core protein and as a signal sequence for E1 protein. The signal sequence of core protein is further processed by signal peptide peptidase (SPP). We examined the regions of core protein responsible for ER retention and processing by SPP. Analysis of the intracellular localization of deletion mutants of HCV core protein revealed that not only the C-terminal signal-anchor sequence but also an upstream hydrophobic region from amino acid 128 to 151 is required for ER retention of core protein. Precise mutation analyses indicated that replacement of Leu(139), Val(140), and Leu(144) of core protein by Ala inhibited processing by SPP, but cleavage at the core-E1 junction by signal peptidase was maintained. Additionally, the processed E1 protein was translocated into the ER and glycosylated with high-mannose oligosaccharides. Core protein derived from the mutants was translocated into the nucleus in spite of the presence of the unprocessed C-terminal signal-anchor sequence. Although the direct association of core protein with a wild-type SPP was not observed, expression of a loss-of-function SPP mutant inhibited cleavage of the signal sequence by SPP and coimmunoprecipitation with unprocessed core protein. These results indicate that Leu(139), Val(140), and Leu(144) in core protein play crucial roles in the ER retention and SPP cleavage of HCV core protein.     

Golgi localization of ERManI defines spatial separation of the mammalian glycoprotein quality control system

The Golgi complex has been implicated as a possible component of endoplasmic reticulum (ER) glycoprotein quality control, although the elucidation of its exact role is lacking. ERManI, a putative ER resident mannosidase, plays a rate-limiting role in generating a signal that targets misfolded N-linked glycoproteins for ER-associated degradation (ERAD). Herein we demonstrate that the endogenous human homologue predominantly resides in the Golgi complex, where it is subjected to O-glycosylation. To distinguish the intracellular site where the glycoprotein ERAD signal is generated, a COPI-binding motif was appended to the N terminus of the recombinant protein to facilitate its retrograde translocation back to the ER. Partial redistribution of the modified ERManI was observed along with an accelerated rate at which N-linked glycans of misfolded α1-antitrypsin variant NHK were trimmed. Despite these observations, the rate of NHK degradation was not accelerated, implicating the Golgi complex as the site for glycoprotein ERAD substrate tagging. Taken together, these data provide a potential mechanistic explanation for the spatial separation by which glycoprotein quality control components operate in mammalian cells.     

Dual loss of ER export and endocytic signals with altered melanosome morphology in the silver mutation of Pmel17

Pmel17 is a pigment cell-specific integral membrane protein that participates in the formation of the intralumenal fibrils upon which melanins are deposited in melanosomes. The Pmel17 cytoplasmic domain is truncated by the mouse silver mutation, which is associated with coat hypopigmentation in certain strain backgrounds. Here, we show that the truncation interferes with at least two steps in Pmel17 intracellular transport, resulting in defects in melanosome biogenesis. Human Pmel17 engineered with the truncation found in the mouse silver mutant (hPmel17si) is inefficiently exported from the endoplasmic reticulum (ER). Localization and metabolic pulse-chase analyses with site-directed mutants and chimeric proteins show that this effect is due to the loss of a conserved C-terminal valine that serves as an ER exit signal. hPmel17si that exits the ER accumulates abnormally at the plasma membrane due to the loss of a di-leucine-based endocytic signal. The combined effects of reduced ER export and endocytosis significantly deplete Pmel17 within endocytic compartments and delay proteolytic maturation required for premelanosome-like fibrillogenesis. The ER export delay and cell surface retention are also observed for endogenous Pmel17si in melanocytes from silver mice, within which Pmel17 accumulation in premelanosomes is dramatically reduced. Mature melanosomes in these cells are larger, rounder, more highly pigmented, and less striated than in control melanocytes. These data reveal a dual sorting defect in a natural mutant of Pmel17 and support a requirement of endocytic trafficking in Pmel17 fibril formation.     

Applicability of green fluorescence protein in the study of endothelin converting enzyme‐1c trafficking

Endothelin‐1 (ET‐1) is one of the most potent peptide vasoconstrictors known. It is produced upon the cleavage of its precursor big endothelin‐1 by endothelin converting enzyme‐1 (ECE‐1). Production of ET‐1 is thought to be dependent upon the expression of ECE‐1 at the cell surface. Therefore, mechanisms inducing the trafficking of ECE‐1 to the cell surface have been the focus of recent research. This research has identified phosphorylation of the cytoplasmic region of ECE‐1 as a main cellular signal inducing its trafficking to the cell surface. Previous studies have used green fluorescent protein (GFP) tagged ECE‐1 to monitor phosphorylation induced trafficking of ECE‐1 to the cell surface. However, it has been speculated that the addition of the GFP tag can itself alter enzyme activity and phosphorylation of ECE‐1, and hence the suitability of GFP or any other protein tag in studying ECE‐1 distribution and trafficking. ECE‐1c is the most widely expressed isoform in endothelial cells. We therefore expressed ECE‐1c with a GFP tag either at the N or C‐terminus of ECE‐1c. Catalytic activity and effect on protein kinase C (PKC) induced phosphorylation was compared between the two chimeras and wild‐type ECE‐1c. Our results indicate that positioning of the GFP tag on the C‐terminus abrogates activity without effecting PKC‐induced phosphorylation. However, GFP tag on the N‐terminus has the opposite effect. Results of this study shed light on the applicability of GFP or perhaps other protein tags in studying ECE‐1c distribution and trafficking.     

Intramembrane proteolysis promotes trafficking of hepatitis C virus core protein to lipid droplets

Hepatitis C virus (HCV) is the major causative pathogen associated with liver cirrhosis and hepatocellular carcinoma. The virus has a positive-sense RNA genome encoding a single polyprotein with the virion components located in the N-terminal portion. During biosynthesis of the polyprotein, an internal signal sequence between the core protein and the envelope protein E1 targets the nascent polypeptide to the endoplasmic reticulum (ER) membrane for translocation of E1 into the ER. Following membrane insertion, the signal sequence is cleaved from E1 by signal peptidase. Here we provide evidence that after cleavage by signal peptidase, the signal peptide is further processed by the intramembrane-cleaving protease SPP that promotes the release of core protein from the ER membrane. Core protein is then free for subsequent trafficking to lipid droplets. This study represents an example of a potential role for intramembrane proteolysis in the maturation of a viral protein.     

Cell surface expression of GluR5 kainate receptors is regulated by an endoplasmic reticulum retention signal

Kainate receptors (KARs) are mediators of excitatory neurotransmission in the mammalian central nervous system, and their efficient targeting and trafficking is critical for normal synaptic function. A key step in the delivery of KARs to the neuronal plasma membrane is the exit of newly assembled receptors from the endoplasmic reticulum (ER). Here we report the identification of a novel ER retention signal in the alternatively spliced C-terminal domain of the GluR5-2b subunit, which controls receptor trafficking in both heterologous cells and neurons. The ER retention motif consists of a critical arginine (Arg-896) and surrounding amino acids, disruption of which promotes ER exit and surface expression of the receptors, as well as altering their physiological properties. The Arg-896-mediated ER retention of GluR5 is regulated by a mutation that mimics phosphorylation of Thr-898, but not by PDZ interactions. Furthermore, two positively charged residues (Arg-900 and Lys-901) in the C terminus were also found to regulate ER export of the receptors. Taken together, our results identify novel trafficking signals in the C-terminal domain of GluR5-2b and demonstrate that alternative splicing is an important mechanism regulating KAR function.     

US2, a human cytomegalovirus-encoded type I membrane protein, contains a non-cleavable amino-terminal signal peptide.

The human cytomegalovirus US2 gene product targets major histocompatibility class I molecules for degradation in a proteasome-dependent fashion. Degradation requires interaction between the endoplasmic reticulum (ER) lumenal domains of US2 and class I. While ER insertion of US2 is essential for US2 function, US2 lacks a cleavable signal peptide. Radiosequence analysis of glycosylated US2 confirms the presence of the NH(2) terminus predicted on the basis of the amino acid sequence, with no evidence for processing by signal peptidase. Despite the absence of cleavage, the US2 NH(2)-terminal segment constitutes its signal peptide and is sufficient to drive ER translocation of chimeric reporter proteins, again without further cleavage. The putative US2 signal peptide c-region is responsible for the absence of cleavage, despite the presence of a suitable -3,-1 amino acid motif for signal peptidase recognition. In addition, the US2 signal peptide affects the early processing events of the nascent polypeptide, altering the efficiency of ER insertion and subsequent N-linked glycosylation. To our knowledge, US2 is the first example of a membrane protein that does not contain a cleavable signal peptide, yet otherwise behaves like a type I membrane glycoprotein.     

Role of the TonB amino terminus in energy transduction between membranes.

Escherichia coli TonB protein is an energy transducer, coupling cytoplasmic membrane energy to active transport of vitamin B12 and iron-siderophores across the outer membrane. TonB is anchored in the cytoplasmic membrane by its hydrophobic amino terminus, with the remainder occupying the periplasmic space. In this report we establish several functions for the hydrophobic amino terminus of TonB. A G-26-->D substitution in the amino terminus prevents export of TonB, suggesting that the amino terminus contains an export signal for proper localization of TonB within the cell envelope. Substitution of the first membrane-spanning domain of the cytoplasmic membrane protein TetA for the TonB amino terminus eliminates TonB activity without altering TonB export, suggesting that the amino terminus contains sequence-specific information. Detectable TonB cross-linking to ExbB is also prevented, suggesting that the two proteins interact primarily through their transmembrane domains. In vivo cleavage of the amino terminus of TonB carrying an engineered leader peptidase cleavage site eliminates (i) TonB activity, (ii) detectable interaction with a membrane fraction having a density intermediate to those of the cytoplasmic and outer membranes, and (iii) cross-linking to ExbB. In contrast, the amino terminus is not required for cross-linking to other proteins with which TonB can form complexes, including FepA. Additionally, although the amino terminus clearly is a membrane anchor, it is not the only means by which TonB associates with the cytoplasmic membrane. TonB lacking its amino-terminal membrane anchor still remains largely associated with the cytoplasmic membrane.     

Core protein of pestiviruses is processed at the C terminus by signal peptide peptidase

The core protein of pestiviruses is released from the polyprotein by viral and cellular proteinases. Here we report on an additional intramembrane proteolytic step that generates the C terminus of the core protein. C-terminal processing of the core protein of classical swine fever virus (CSFV) was blocked by the inhibitor (Z-LL)(2)-ketone, which is specific for signal peptide peptidase (SPP). The same effect was obtained by overexpression of the dominant-negative SPP D(265)A mutant. The presence of (Z-LL)(2)-ketone reduced the viability of CSFV almost 100-fold in a concentration-dependent manner. Reduction of virus viability was also observed in infection experiments using a cell line that inducibly expressed SPP D(265)A. The position of SPP cleavage was determined by C-terminal sequencing of core protein purified from virions. The C terminus of CSFV core protein is alanine(255) and is located in the hydrophobic center of the signal peptide. The intramembrane generation of the C terminus of the CSFV core protein is almost identical to the processing scheme of the core protein of hepatitis C viruses.     

Receptor-mediated ER export of human MHC class I molecules is regulated by the C-terminal single amino acid

Major histocompatibility complex class I (MHC-I) molecules bind antigens in the endoplasmic reticulum (ER) and deliver them to the cell surface for immune surveillance of viruses and tumors. Whereas key steps of MHC-I assembly and its acquisition of peptides in the ER are relatively well defined, little is known about how MHC-I molecules leave the ER for cell surface expression. Here, we show that ER export of human classical MHC-I molecules (HLA-A/-B/-C) is regulated by their C-terminal single amino acid, valine or alanine. These amino acids, conserved in nearly all known human MHC-I alleles, serve as the ER export signal by binding to the Sec23/24 complex, a structural component of coat protein complex II (COPII) vesicles that mediate ER-to-Golgi trafficking. Together, our results strongly suggest that ER export of human classical MHC-I molecules can occur via a receptor-mediated process dictated by a highly conserved ER export signal.     

A New Type of Signal Peptidase Cleavage Site Identified in an RNA Virus Polyprotein

Pestiviruses, a group of enveloped positive strand RNA viruses belonging to the family Flaviviridae, express their genes via a polyprotein that is subsequently processed by proteases. The structural protein region contains typical signal peptidase cleavage sites. Only the site at the C terminus of the glycoprotein E^rns is different because it does not contain a hydrophobic transmembrane region but an amphipathic helix functioning as the E^rns membrane anchor. Despite the absence of a hydrophobic region, the site between the C terminus of E^rns and E1, the protein located downstream in the polyprotein, is cleaved by signal peptidase, as demonstrated by mutagenesis and inhibitor studies. Thus, E^rnsE1 is processed at a novel type of signal peptidase cleavage site showing a different membrane topology. Prevention of glycosylation or introduction of mutations into the C-terminal region of E^rns severely impairs processing, presumably by preventing proper membrane interaction or disturbing a conformation critical for the protein to be accepted as a substrate by signal peptidase.