Characterization of membrane topology and retention signal of pestiviral glycoprotein E1

Pestiviruses are members of the family Flaviviridae, a group of enveloped viruses that bud at intracellular membranes. Pestivirus particles contain three glycosylated envelope proteins, Erns, E1 and E2. Among them, E1 is the least characterized concerning both biochemical features and function. E1 from bovine viral diarrhea virus (BVDV) strain CP7 was analyzed with regard to its intracellular localization and membrane topology. Here, it is shown that even in the absence of other viral proteins, E1 is not secreted or expressed at the cell surface, but localizes predominantly in the ER. Using engineered chimeric TM domains with sequences from E1 and vesicular stomatitis virus G protein, the E1 ER-retention signal could be narrowed down to six fully conserved polar residues in the middle part of the transmembrane domain of E1. Retention was observed even when several of these polar residues were exchanged for alanine. Mutations with a strong impact on E1 retention prevented recovery of infectious viruses when tested in the viral context. Analysis of the membrane topology of E1 before and after the signal peptide cleavage via a selective permeabilization and an in vivo labelling approach revealed that mature E1 is a typical type I transmembrane protein with a single span transmembrane anchor at its C-terminus whereas it adopts a hairpin-like structure with the C-terminus located in the ER lumen when the pre-cleavage situation is mimicked by blocking the cleavage site between E1 and E2. Importance The shortage of specific antibodies against E1 making detection and further analysis of E1 difficult, resulted in a lack of knowledge on E1 compared to Erns and E2 with regard to biosynthesis, structure and function. It is known that pestiviruses bud intracellularly. Here, we show that E1 contains its own ER retention signal: six fully conserved polar residues in the middle part of the TM domain are shown to be the determinants for ER retention of E1. Moreover, those six polar residues could serve as a functional group that intensely affect the generation of infectious viral particles. In addition, the membrane topology of E1 has been determined. In this context, we also identified dynamic changes in membrane topology of E1 with the carboxy-terminus located on the luminal side of the ER in the pre-cleavage state and relocation of this sequence upon signal peptidase cleavage. Our work provides the first systematic analysis of the pestiviral E1 protein with regard to its biochemical and functional characteristics.

Unconventional ER to Endosomal Trafficking by a Retroviral Protein

ABSTRACTMouse mammary tumor virus (MMTV) encodes a Rem precursor protein that specifies both regulatory and accessory functions. Rem is cleaved at the ER membrane into a functional N-terminal signal peptide (SP) and the C-terminus (Rem-CT). Rem-CT lacks a membrane-spanning domain and a known ER retention signal, yet was not detectably secreted into cell supernatants. Inhibition of intracellular trafficking by the drug Brefeldin A (BFA), which interferes with the ER to Golgi secretory pathway, resulted in dramatically reduced intracellular Rem-CT levels. A Rem mutant lacking glycosylation sites was cleaved into SP and Rem-CT, but was insensitive to BFA, suggesting that unglycosylated Rem-CT does not exit the ER or reach a degradative compartment. BFA reduction of Rem-CT levels was not rescued by proteasome or lysosomal inhibitors. Rem-CT has simple glycans, which are necessary for Rem-CT stability and trafficking, but indicate that Rem-CT does not traffic through the Golgi. Analysis of wild-type Rem-CT and its glycosylation mutant by confocal microscopy revealed that both were primarily localized to the ER lumen. A small fraction of wild-type Rem-CT, but not the unglycosylated mutant, were co-localized with Rab5+ endosomes. Expression of a dominant-negative (DN) form of ADP ribosylation factor 1 (Arf1) (T31N) mimicked the effects of BFA by reducing Rem-CT levels, suggesting that Arf1 prevents Rem-CT localization to a degradative compartment. A DN form of the AAA ATPase, p97/VCP, rescued Rem-CT in the presence of BFA or DN Arf1. Thus, Rem-CT uses an unconventional trafficking scheme, perhaps to thwart innate immunity to MMTV infection.IMPORTANCEMouse mammary tumor virus is a complex retrovirus that encodes a regulatory/accessory protein, Rem. Rem is a precursor protein that is processed at the endoplasmic reticulum (ER) membrane by signal peptidase. The N-terminal SP eludes ER-associated degradation to traffic to the nucleus and serve a human immunodeficiency virus Rev-like function. In contrast, the function of the C-terminal glycosylated cleavage product (Rem-CT) is unknown. Since localization is critical for protein function, we used multiple methods to localize Rem-CT. Surprisingly, Rem-CT, which lacks a transmembrane domain or an ER retention signal, was detected primarily within the ER and required glycosylation for trafficking to endosomes. Blocking of retrograde trafficking through Arf1 reduced Rem-CT levels, but was not restored by lysosomal or proteasomal inhibitors. The unique trafficking of Rem-CT suggests a novel intracellular trafficking pathway, potentially impacting host anti-viral immunity.

A disease-associated frameshift mutation in caveolin-1 disrupts caveolae formation and function through introduction of a de novo ER retention signal

Caveolin-1 (CAV1) is an essential component of caveolae and is implicated in numerous physiological processes. Recent studies have identified heterozygous mutations in the CAV1 gene in patients with pulmonary arterial hypertension (PAH), but the mechanisms by which these mutations impact caveolae assembly and contribute to disease remain unclear. To address this question, we examined the consequences of a familial PAH-associated frameshift mutation in CAV1, P158PfsX22, on caveolae assembly and function. We show that C-terminus of the CAV1 P158 protein contains a functional ER-retention signal that inhibits ER exit and caveolae formation and accelerates CAV1 turnover in Cav1–/– MEFs. Moreover, when coexpressed with wild-type (WT) CAV1 in Cav1–/– MEFs, CAV1-P158 functions as a dominant negative by partially disrupting WT CAV1 trafficking. In patient skin fibroblasts, CAV1 and caveolar accessory protein levels are reduced, fewer caveolae are observed, and CAV1 complexes exhibit biochemical abnormalities. Patient fibroblasts also exhibit decreased resistance to a hypo-osmotic challenge, suggesting the function of caveolae as membrane reservoir is compromised. We conclude that the P158PfsX22 frameshift introduces a gain of function that gives rise to a dominant negative form of CAV1, defining a new mechanism by which disease-associated mutations in CAV1 impair caveolae assembly.

Golgi targeting of Drosophila melanogaster β4GalNAcTB requires a DHHC protein family–related protein as a pilot

Drosophila melanogaster β4GalNAcTB mutant flies revealed that this particular N-acetylgalactosaminyltransferase is predominant in the formation of lacdiNAc (GalNAcβ1,4GlcNAc)-modified glycolipids, but enzymatic activity could not be confirmed for the cloned enzyme. Using a heterologous expression cloning approach, we isolated β4GalNAcTB together with β4GalNAcTB pilot (GABPI), a multimembrane-spanning protein related to Asp-His-His-Cys (DHHC) proteins but lacking the DHHC consensus sequence. In the absence of GABPI, inactive β4GalNAcTB is trapped in the endoplasmic reticulum (ER). Coexpression of β4GalNAcTB and GABPI generates the active enzyme that is localized together with GABPI in the Golgi. GABPI associates with β4GalNAcTB and, when expressed with an ER retention signal, holds active β4GalNAcTB in the ER. Importantly, treatment of isolated membrane vesicles with Triton X-100 disturbs β4GalNAcTB activity. This phenomenon occurs with multimembrane-spanning glycosyltransferases but is normally not a property of glycosyltransferases with one membrane anchor. In summary, our data provide evidence that GABPI is required for ER export and activity of β4GalNAcTB.

An orderly inactivation of intracellular retention signals controls surface expression of the T cell antigen receptor

Exit from the endoplasmic reticulum (ER) is an important checkpoint for proper assembly of multimeric plasma membrane receptors. The six subunits of the T cell receptor (TCR; TCRα, TCRβ, CD3γ, CD3δ, CD3ε, and CD3ζ) are each endowed with ER retention/retrieval signals, and regulation of its targeting to the plasma membrane is therefore especially intriguing. We have studied the importance of the distinct ER retention signals at different stages of TCR intracellular assembly. To this end, we have characterized first the presence of ER retention signals in CD3γ. Despite the presence of multiple ER retention signals in CD3γ, εγ dimers reach the cell surface when the single CD3ε ER retention signal is deleted. Furthermore, inclusion of this CD3ε mutant promoted plasma membrane expression of incomplete αβγε and αβδε complexes without CD3ζ. It therefore appears that the CD3ε ER retention signal is dominant and that it is only overridden upon the incorporation of CD3ζ. We propose that the stepwise assembly of the TCR complex guarantees that all assembly intermediates have at least one functional ER retention signal and that only a full signaling-competent TCR complex is expressed on the cell surface.