scholarly journals Genetic Analysis of Determinants for Spike Glycoprotein Assembly into Murine Coronavirus Virions: Distinct Roles for Charge-Rich and Cysteine-Rich Regions of the Endodomain

2004 ◽  
Vol 78 (18) ◽  
pp. 9904-9917 ◽  
Author(s):  
Rong Ye ◽  
Cynthia Montalto-Morrison ◽  
Paul S. Masters
Keyword(s):  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Hepatitis Virus ◽  
Transmembrane Protein ◽  
Terminal Segment ◽  
Type I ◽  
Viral Fusion ◽  
S Protein ◽  
Infected Cells ◽  
Carboxy Terminal

ABSTRACT The coronavirus spike protein (S) forms the distinctive virion surface structures that are characteristic of this viral family, appearing in negatively stained electron microscopy as stems capped with spherical bulbs. These structures are essential for the initiation of infection through attachment of the virus to cellular receptors followed by fusion to host cell membranes. The S protein can also mediate the formation of syncytia in infected cells. The S protein is a type I transmembrane protein that is very large compared to other viral fusion proteins, and all except a short carboxy-terminal segment of the S molecule constitutes the ectodomain. For the prototype coronavirus mouse hepatitis virus (MHV), it has previously been established that S protein assembly into virions is specified by the carboxy-terminal segment, which comprises the transmembrane domain and the endodomain. We have genetically dissected these domains in the MHV S protein to localize the determinants of S incorporation into virions. Our results establish that assembly competence maps to the endodomain of S, which was shown to be sufficient to target a heterologous integral membrane protein for incorporation into MHV virions. In particular, mutational analysis indicated a major role for the charge-rich carboxy-terminal region of the endodomain. Additionally, we found that the adjacent cysteine-rich region of the endodomain is critical for fusion of infected cells, confirming results previously obtained with S protein expression systems.

scholarly journals Atypical Fusion Peptide of Nelson Bay Virus Fusion-Associated Small Transmembrane Protein

2005 ◽  
Vol 79 (3) ◽  
pp. 1853-1860 ◽  
Author(s):  
LiTing T. Cheng ◽  
Richard K. Plemper ◽  
Richard W. Compans
Keyword(s):  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Transmembrane Protein ◽  
Syncytium Formation ◽  
Surface Expression ◽  
Fusion Peptide ◽  
Cell Surface Expression ◽  
Type I ◽  
Fusion Activity ◽  
Hydrophobic Domain

ABSTRACT A 10-kDa nonstructural transmembrane protein (p10) encoded by a reovirus, Nelson Bay virus, has been shown to induce syncytium formation (34). Sequence analysis and structural studies identified p10 as a type I membrane protein with a central transmembrane domain, a cytoplasmic basic region, and an N-terminal hydrophobic domain (HD) that was hypothesized to function as a fusion peptide. We performed mutational analysis on this slightly hydrophobic motif to identify possible structural requirements for fusion activity. Bulky aliphatic residues were found to be essential for optimal fusion, and an aromatic or highly hydrophobic side chain was found to be required at position 12. The requirement for hydrophilic residues within the HD was also examined: substitution of 10-Ser or 14-Ser with hydrophobic residues was found to reduce cell surface expression of p10 and delayed the onset of syncytium formation. Nonconservative substitutions of charged residues in the HD did not have an effect on fusion activity. Taken together, our results suggest that the HD is involved in both syncytium formation and in determining p10 transport and surface expression.

scholarly journals A trimeric hydrophobic zipper mediates the intramembrane assembly of SARS-CoV-2 spike

2021 ◽  
Author(s):  
Qingshan Fu ◽  
James J Chou
Keyword(s):  
Transmembrane Domain ◽  
Structural Information ◽  
Heptad Repeat ◽  
Host Immune System ◽  
Type I ◽  
Fold Axis ◽  
Viral Fusion ◽  
S Protein ◽  
Viral Fusion Protein ◽  
Exact Function

The S protein of the SARS-CoV-2 is a Type I membrane protein that mediates membrane fusion and viral entry. A vast amount of structural information is available for the ectodomain of S, a primary target by the host immune system, but much less is known regarding its transmembrane domain (TMD) and its membrane-proximal regions. Here, we determined the nuclear magnetic resonance (NMR) structure of the S protein TMD in bicelles that closely mimic a lipid bilayer. The TMD structure is a transmembrane α-helix (TMH) trimer that assembles spontaneously in membrane. The trimer structure shows an extensive hydrophobic core along the 3-fold axis that resembles that of a trimeric leucine/isoleucine zipper, but with tetrad, not heptad, repeat. The trimeric core is strong in bicelles, resisting hydrogen-deuterium exchange for weeks. Although highly stable, structural guided mutagenesis identified single mutations that can completely dissociate the TMD trimer. Multiple studies have shown that the membrane anchor of viral fusion protein can form highly specific oligomers, but the exact function of these oligomers remain unclear. Our findings should guide future experiments to address the above question for SARS coronaviruses.

scholarly journals The First Transmembrane Domain of the Hepatitis B Virus Large Envelope Protein Is Crucial for Infectivity

2009 ◽  
Vol 83 (22) ◽  
pp. 11819-11829 ◽  
Author(s):  
Charlotte Lepère-Douard ◽  
Maud Trotard ◽  
Jacques Le Seyec ◽  
Philippe Gripon
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Fusion Peptide ◽  
S Protein ◽  
Pest Sequence ◽  
B Virus ◽  
Infected Cells ◽  
Infection Processes

ABSTRACT The early steps of the hepatitis B virus (HBV) life cycle are still poorly understood. Indeed, neither the virus receptor at the cell surface nor the mechanism by which nucleocapsids are delivered to the cytosol of infected cells has been identified. Extensive mutagenesis studies in pre-S1, pre-S2, and most of the S domain of envelope proteins revealed the presence of two regions essential for HBV infectivity: the 77 first residues of the pre-S1 domain and a conformational motif in the antigenic loop of the S domain. In addition, at the N-terminal extremity of the S domain, a putative fusion peptide, partially overlapping the first transmembrane (TM1) domain and preceded by a PEST sequence likely containing several proteolytic cleavage sites, was identified. Since no mutational analysis of these two motifs potentially implicated in the fusion process was performed, we decided to investigate the ability of viruses bearing contiguous deletions or substitutions in the putative fusion peptide and PEST sequence to infect HepaRG cells. By introducing the mutations either in the L and M proteins or in the S protein, we demonstrated the following: (i) that in the TM1 domain of the L protein, three hydrophobic clusters of four residues were necessary for infectivity; (ii) that the same clusters were critical for S protein expression; and, finally, (iii) that the PEST sequence was dispensable for both assembly and infection processes.

scholarly journals Assembly of Spikes into Coronavirus Particles Is Mediated by the Carboxy-Terminal Domain of the Spike Protein

2000 ◽  
Vol 74 (3) ◽  
pp. 1566-1571 ◽  
Author(s):  
Gert-Jan Godeke ◽  
Cornelis A. M. de Haan ◽  
John W. A. Rossen ◽  
Harry Vennema ◽  
Peter J. M. Rottier
Keyword(s):  
Hepatitis Virus ◽  
Noncovalent Interactions ◽  
Terminal Segment ◽  
Type I ◽  
New Host ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
The One ◽  
Carboxy Terminal ◽  
S Proteins

ABSTRACT The type I glycoprotein S of coronavirus, trimers of which constitute the typical viral spikes, is assembled into virions through noncovalent interactions with the M protein. Here we demonstrate that incorporation is mediated by the short carboxy-terminal segment comprising the transmembrane and endodomain. To this aim, we used the virus-like particle (VLP) system that we developed earlier for the mouse hepatitis virus strain A59 (MHV-A59) and which we describe now also for the unrelated coronavirus feline infectious peritonitis virus (FIPV; strain 79-1146). Two chimeric MHV-FIPV S proteins were constructed, consisting of the ectodomain of the one virus and the transmembrane and endodomain of the other. These proteins were tested for their incorporation into VLPs of either species. They were found to assemble only into viral particles of the species from which their carboxy-terminal domain originated. Thus, the 64-terminal-residue sequence suffices to draw the 1308 (MHV)- or 1433 (FIPV)-amino-acid-long mature S protein into VLPs. Both chimeric S proteins appeared to cause cell fusion when expressed individually, suggesting that they were biologically fully active. This was indeed confirmed by incorporating one of the proteins into virions which thereby acquired a new host cell tropism, as will be reported elsewhere.

scholarly journals Vaccinia Virus A26 and A27 Proteins Form a Stable Complex Tethered to Mature Virions by Association with the A17 Transmembrane Protein

2008 ◽  
Vol 82 (24) ◽  
pp. 12384-12391 ◽  
Author(s):  
Amanda R. Howard ◽  
Tatiana G. Senkevich ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Matrix Protein ◽  
Transmembrane Domain ◽  
Noncovalent Complex ◽  
Transmembrane Protein ◽  
Amino Acid Identity ◽  
Noncovalent Interaction ◽  
Terminal Segment ◽  
Stable Complex ◽  
Cowpox Virus

ABSTRACT During vaccinia virus replication, mature virions (MVs) are wrapped with cellular membranes, transported to the periphery, and exported as extracellular virions (EVs) that mediate spread. The A26 protein is unusual in that it is present in MVs but not EVs. This distribution led to a proposal that A26 negatively regulates wrapping. A26 also has roles in the attachment of MVs to the cell surface and incorporation of MVs into proteinaceous A-type inclusions in some orthopoxvirus species. However, A26 lacks a transmembrane domain, and nothing is known regarding how it associates with the MV, regulates incorporation of the MV into inclusions, and possibly prevents EV formation. Here, we provide evidence that A26 forms a disulfide-bonded complex with A27 that is anchored to the MV through a noncovalent interaction with the A17 transmembrane protein. In the absence of A27, A26 was unstable, and only small amounts were detected. The interaction of A26 with A27 depended on a C-terminal segment of A26 with 45% amino acid identity to A27. Deletion of A26 failed to enhance EV formation by vaccinia virus, as had been predicted. Nevertheless, the interaction of A26 and A27 may have functional significance, since each is thought to mediate binding to cells through interaction with laminin and heparan sulfate, respectively. We also found that A26 formed a noncovalent complex with A25, a truncated form of the cowpox virus A-type inclusion matrix protein. The latter association suggests a mechanism for incorporation of virions into A-type inclusions in other orthopoxvirus strains.

scholarly journals Canine Influenza Virus is Mildly Restricted by Canine Tetherin Protein

Viruses ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 565
Author(s):  
Yun Zheng ◽  
Xiangqi Hao ◽  
Qingxu Zheng ◽  
Xi Lin ◽  
Xin Zhang ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Cellular Damage ◽  
Interferon Response ◽  
Type I ◽  
Canine Influenza Virus ◽  
Immune Factor ◽  
Canine Influenza ◽  
The Impact

Tetherin (BST2/CD317/HM1.24) has emerged as a key host-cell ·defence molecule that acts by inhibiting the release and spread of diverse enveloped virions from infected cells. We analysed the biological features of canine tetherin and found it to be an unstable hydrophilic type I transmembrane protein with one transmembrane domain, no signal peptide, and multiple glycosylation and phosphorylation sites. Furthermore, the tissue expression profile of canine tetherin revealed that it was particularly abundant in immune organs. The canine tetherin gene contains an interferon response element sequence that can be regulated and expressed by canine IFN-α. A CCK-8 assay showed that canine tetherin was effective in helping mitigate cellular damage caused by canine influenza virus (CIV) infection. Additionally, we found that the overexpression of canine tetherin inhibited replication of the CIV and that interference with the canine tetherin gene enhanced CIV replication in cells. The impact of canine tetherin on CIV replication was mild. However, these results elucidate the role of the innate immune factor, canine tetherin, during CIV infection for the first time.

scholarly journals Emergence of a new SARS-CoV-2 variant from GR clade with a novel S glycoprotein mutation V1230L in West Bengal, India

2021 ◽  
Author(s):  
Rakesh Sarkar ◽  
Ritubrita Saha ◽  
Pratik Mallick ◽  
Ranjana Sharma ◽  
Amandeep Kaur ◽  
...  
Keyword(s):  
South African ◽  
West Bengal ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Viral Envelope ◽  
Protein Coding ◽  
S Protein ◽  
Genomic Epidemiology ◽  
Clade B ◽  
New Variant

India is currently facing the devastating second wave of COVID-19 pandemic resulting in approximately 4000 deaths per day. To control this pandemic continuous mutational surveillance and genomic epidemiology of circulating strains is very important. In this study, we performed mutational analysis of the protein coding genes of SARS-CoV-2 strains (n=2000) collected during January 2021 to March 2021. Our data revealed the emergence of a new variant in West Bengal, India, which is characterized by the presence of 11 co-existing mutations including D614G, P681H and V1230L in S-glycoprotein. This new variant was identified in 70 out of 412 sequences submitted from West Bengal. Interestingly, among these 70 sequences, 16 sequences also harbored E484K in the S glycoprotein. Phylogenetic analysis revealed strains of this new variant emerged from GR clade (B.1.1) and formed a new cluster. We propose to name this variant as GRL or lineage B.1.1/S:V1230L due to the presence of V1230L in S glycoprotein along with GR clade specific mutations. Co-occurrence of P681H, previously observed in UK variant, and E484K, previously observed in South African variant and California variant, demonstrates the convergent evolution of SARS-CoV-2 mutation. V1230L, present within the transmembrane domain of S2 subunit of S glycoprotein, has not yet been reported from any country. Substitution of valine with more hydrophobic amino acid leucine at position 1230 of the transmembrane domain, having role in S protein binding to the viral envelope, could strengthen the interaction of S protein with the viral envelope and also increase the deposition of S protein to the viral envelope, and thus positively regulate virus infection. P618H and E484K mutation have already been demonstrated in favor of increased infectivity and immune invasion respectively. Therefore, the new variant having G614G, P618H, P1230L and E484K is expected to have better infectivity, transmissibility and immune invasion characteristics, which may pose additional threat along with B.1.617 in the ongoing COVID-19 pandemic in India.

scholarly journals High Physiological Levels of LMP1 Result in Phosphorylation of eIF2α in Epstein-Barr Virus-Infected Cells

2004 ◽  
Vol 78 (4) ◽  
pp. 1657-1664 ◽  
Author(s):  
Ngan Lam ◽  
Mark L. Sandberg ◽  
Bill Sugden
Keyword(s):  
Gene Expression ◽  
Epstein Barr Virus ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Barr Virus ◽  
Signaling Domain ◽  
Infected Cells ◽  
Epstein Barr ◽  
Green Fluorescent ◽  
Carboxy Terminal

ABSTRACT LMP1 is an Epstein-Barr virus (EBV)-encoded membrane protein essential for the proliferation of EBV-infected lymphoblasts (E. Kilger, A. Kieser, M. Baumann, and W. Hammerschmidt, EMBO J. 17:1700-1709, 1998). LMP1 also inhibits gene expression and induces cytostasis in transfected cells when it is expressed at levels as little as twofold higher than the average for EBV-positive lymphoblasts (M. Sandberg, A. Kaykas, and B. Sugden, J. Virol. 74:9755-9761, 2000; A. Kaykas and B. Sugden, Oncogene 19:1400-1410, 2000). We have found that in three different clones of EBV-infected lymphoblasts the levels of expression of LMP1 in individual cells in each clone ranged over 100-fold. This difference is due to a difference in levels of the LMP1 transcript. In these clones, cells expressing high levels of LMP1 incorporated less BrdU. We also found that induction of expression of LMP1 or of a derivative of LMP1 with its transmembrane domain fused to green fluorescent protein instead of its carboxy-terminal signaling domain resulted in phosphorylation of eIF2α in EBV-negative Burkitt's lymphoma cells. This induction of phosphorylation of eIF2α was also detected in EBV-infected lymphoblasts, in which high levels of LMP1 correlated with high levels of phosphorylation of eIF2α. Our results indicate that inhibition of gene expression and of cell proliferation by LMP1 occurs normally in EBV-infected cells.

scholarly journals Upregulation of BST-2 by Type I Interferons Reduces the Capacity of Vpu To Protect HIV-1-Infected Cells from NK Cell Responses

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Jérémie Prévost ◽  
Suzanne Pickering ◽  
Mitchell J. Mumby ◽  
Halima Medjahed ◽  
Gabrielle Gendron-Lepage ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Nk Cell ◽  
Restriction Factor ◽  
Type I ◽  
Cell Responses ◽  
Viral Release ◽  
Type I Ifns ◽  
Infected Cells ◽  
Ifn Treatment ◽  
Hiv 1

ABSTRACTThe HIV-1 accessory protein Vpu enhances viral release by counteracting the restriction factor BST-2. Furthermore, Vpu promotes NK cell evasion by downmodulating cell surface NTB-A and PVR, known ligands of the NK cell receptors NTB-A and DNAM-1, respectively. While it has been established that Vpu’s transmembrane domain (TMD) is required for the interaction and intracellular sequestration of BST-2, NTB-A, and PVR, it remains unclear how Vpu manages to target these proteins simultaneously. In this study, we show that upon upregulation, BST-2 is preferentially downregulated by Vpu over its other TMD substrates. We found that type I interferon (IFN)-mediated BST-2 upregulation greatly impairs the ability of Vpu to downregulate NTB-A and PVR. Our results suggest that occupation of Vpu by BST-2 affects its ability to downregulate other TMD substrates. Accordingly, knockdown of BST-2 increases Vpu’s potency to downmodulate NTB-A and PVR in the presence of type I IFN treatment. Moreover, we show that expression of human BST-2, but not that of the macaque orthologue, decreases Vpu’s capacity to downregulate NTB-A. Importantly, we show that type I IFNs efficiently sensitize HIV-1-infected cells to NTB-A- and DNAM-1-mediated direct and antibody-dependent NK cell responses. Altogether, our results reveal that type I IFNs decrease Vpu’s polyfunctionality, thus reducing its capacity to protect HIV-1-infected cells from NK cell responses.IMPORTANCEThe restriction factor BST-2 and the NK cell ligands NTB-A and PVR are among a growing list of membrane proteins found to be downregulated by HIV-1 Vpu. BST-2 antagonism enhances viral release, while NTB-A and PVR downmodulation contributes to NK cell evasion. However, it remains unclear how Vpu can target multiple cellular factors simultaneously. Here we provide evidence that under physiological conditions, BST-2 is preferentially targeted by Vpu over NTB-A and PVR. Specifically, we show that type I IFNs decrease Vpu’s polyfunctionality by upregulating BST-2, thus reducing its capacity to protect HIV-1-infected cells from NK cell responses. This indicates that there is a hierarchy of Vpu substrates upon IFN treatment, revealing that for the virus, targeting BST-2 as part of its resistance to IFN takes precedence over evading NK cell responses. This reveals a potential weakness in HIV-1’s immunoevasion mechanisms that may be exploited therapeutically to harness NK cell responses against HIV-1.

scholarly journals AMIGO, a transmembrane protein implicated in axon tract development, defines a novel protein family with leucine-rich repeats

2003 ◽  
Vol 160 (6) ◽  
pp. 963-973 ◽  
Author(s):  
Juha Kuja-Panula ◽  
Marjaana Kiiltomäki ◽  
Takashi Yamashiro ◽  
Ari Rouhiainen ◽  
Heikki Rauvala
Keyword(s):  
Hippocampal Neurons ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Transmembrane Protein ◽  
Protein Family ◽  
Type I ◽  
Fiber Tracts ◽  
Neuronal Tissues ◽  
Immunoglobulin Domain ◽  
Leucine Rich Repeats

Ordered differential display identified a novel sequence induced in neurons by the neurite-promoting protein amphoterin. We named this gene amphoterin-induced gene and ORF (AMIGO), and also cloned two other novel genes homologous to AMIGO (AMIGO2 and AMIGO3). Together, these three AMIGOs form a novel family of genes coding for type I transmembrane proteins which contain a signal sequence for secretion and a transmembrane domain. The deduced extracellular parts of the AMIGOs contain six leucine-rich repeats (LRRs) flanked by cysteine-rich LRR NH2- and COOH-terminal domains and by one immunoglobulin domain close to the transmembrane region. A substrate-bound form of the recombinant AMIGO ectodomain promoted prominent neurite extension in hippocampal neurons, and in solution, the same AMIGO ectodomain inhibited fasciculation of neurites. A homophilic and heterophilic binding mechanism is shown between the members of the AMIGO family. Our results suggest that the members of the AMIGO protein family are novel cell adhesion molecules among which AMIGO is specifically expressed on fiber tracts of neuronal tissues and participates in their formation.

Sign in / Sign up

Export Citation Format

Share Document