scholarly journals Mutations within the Putative Membrane-Spanning Domain of the Simian Immunodeficiency Virus Transmembrane Glycoprotein Define the Minimal Requirements for Fusion, Incorporation, and Infectivity

2001 ◽  
Vol 75 (20) ◽  
pp. 9601-9612 ◽  
Author(s):  
John T. West ◽  
Patrick B. Johnston ◽  
Susan R. Dubay ◽  
Eric Hunter
Keyword(s):  
Amino Acids ◽  
Cell Fusion ◽  
Virus Replication ◽  
Expression Vectors ◽  
Wild Type ◽  
Short Delay ◽  
Minimum Requirement ◽  
Helical Turn ◽  
Membrane Spanning

ABSTRACT The membrane-spanning domain (MSD) of a number of retroviral transmembrane (TM) glycoproteins, including those from the human and simian immunodeficiency viruses (HIV and SIV), have been predicted to contain a charged arginine residue. The wild-type SIV TM glycoprotein is 354 amino acids long. The entire putative cytoplasmic domain of SIV (amino acids 193 to 354) is dispensable for virus replication in vitro, and such truncation-containing viruses are capable of reaching wild-type titers after a short delay. We show here that further truncation of eight additional amino acids to TM185 results in a protein that lacks fusogenicity but is, nevertheless, efficiently incorporated into budding virions. By analyzing a series of nonsense mutations between amino acids 193 and 185 in Env expression vectors and in the SIVmac239 proviral clone, a region of the SIV TM that contains the minimum requirement for glycoprotein-mediated cell-to-cell fusion and that for virus replication was identified. Virus entry and infectivity were evident in truncations to a minimum of 189 amino acids, whereas cell-cell fusion was observed for a protein of only 187 amino acids. Glycoprotein was efficiently incorporated into budding virions in truncations up to 185 amino acids, indicating that such proteins are membrane anchored and are transported to the cell surface. However, truncation of the TM to 180 amino acids resulted in a protein that displays a transport defect and may be retained in the endoplasmic reticulum. Based on our analyses of these mutants, an alternative model for the MSD of SIV is proposed. Our model suggests that membrane-imbedded charged residues can be neutralized by side-chain interactions with lipid polar head groups. As a consequence, the membrane-spanning region can be reduced by more than a helical turn. This new model accounts for the ability of truncations within the predicted MSD to remain membrane anchored and maintain biological activity.

scholarly journals The anti-inflammatory peptide Ac-SDKP is released from thymosin-β4 by renal meprin-α and prolyl oligopeptidase

2016 ◽  
Vol 310 (10) ◽  
pp. F1026-F1034 ◽  
Author(s):  
Nitin Kumar ◽  
Pablo Nakagawa ◽  
Branislava Janic ◽  
Cesar A. Romero ◽  
Morel E. Worou ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rat Kidney ◽  
Plasma Concentrations ◽  
Potential Candidate ◽  
Thymosin Β4 ◽  
Wild Type ◽  
Prolyl Oligopeptidase ◽  
Anti Inflammatory

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a natural tetrapeptide with anti-inflammatory and antifibrotic properties. Previously, we have shown that prolyl oligopeptidase (POP) is involved in the Ac-SDKP release from thymosin-β4 (Tβ4). However, POP can only hydrolyze peptides shorter than 30 amino acids, and Tβ4 is 43 amino acids long. This indicates that before POP hydrolysis takes place, Tβ4 is hydrolyzed by another peptidase that releases NH2-terminal intermediate peptide(s) with fewer than 30 amino acids. Our peptidase database search pointed out meprin-α metalloprotease as a potential candidate. Therefore, we hypothesized that, prior to POP hydrolysis, Tβ4 is hydrolyzed by meprin-α. In vitro, we found that the incubation of Tβ4 with both meprin-α and POP released Ac-SDKP, whereas no Ac-SDKP was released when Tβ4 was incubated with either meprin-α or POP alone. Incubation of Tβ4 with rat kidney homogenates significantly released Ac-SDKP, which was blocked by the meprin-α inhibitor actinonin. In addition, kidneys from meprin-α knockout (KO) mice showed significantly lower basal Ac-SDKP amount, compared with wild-type mice. Kidney homogenates from meprin-α KO mice failed to release Ac-SDKP from Tβ4. In vivo, we observed that rats treated with the ACE inhibitor captopril increased plasma concentrations of Ac-SDKP, which was inhibited by the coadministration of actinonin (vehicle, 3.1 ± 0.2 nmol/l; captopril, 15.1 ± 0.7 nmol/l; captopril + actinonin, 6.1 ± 0.3 nmol/l; P < 0.005). Similar results were obtained with urinary Ac-SDKP after actinonin treatment. We conclude that release of Ac-SDKP from Tβ4 is mediated by successive hydrolysis involving meprin-α and POP.

scholarly journals The ORF49 Protein of Murine Gammaherpesvirus 68 Cooperates with RTA in Regulating Virus Replication

2007 ◽  
Vol 81 (18) ◽  
pp. 9870-9877 ◽  
Author(s):  
Sangmi Lee ◽  
Hye-Jeong Cho ◽  
Jung-Jin Park ◽  
Yong-Sun Kim ◽  
Seungmin Hwang ◽  
...  
Keyword(s):  
Virus Replication ◽  
Wild Type Virus ◽  
Wild Type ◽  
Mapping Study ◽  
Murine Gammaherpesvirus ◽  
Downstream Target ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68 ◽  
Target Promoters

ABSTRACT Our functional mapping study of murine gammaherpesvirus 68 (MHV-68, or γHV-68) revealed that a mutant harboring a transposon at the ORF49 locus (ORF49null) evidenced a highly attenuated in vitro growth. ORF49 resides adjacent to and in an opposite direction from RTA, the primary switch of the gammaherpesvirus life cycle. A FLAG-tagged ORF49 protein was able to transcomplement ORF49null, and a revertant of ORF49null restored its attenuated growth to a level comparable to that of the wild type. The FLAG-tagged ORF49 protein promoted the ability of RTA to activate downstream target promoters and enhanced virus replication from the ORF50null virus in the presence of RTA. Furthermore, ORF49 enhanced wild-type virus replication by increasing the RTA transcript levels. Our data indicate that ORF49 may perform an important function in MHV-68 replication in cooperation with RTA.

scholarly journals Contribution of Endocytic Motifs in the Cytoplasmic Tail of Herpes Simplex Virus Type 1 Glycoprotein B to Virus Replication and Cell-Cell Fusion

2007 ◽  
Vol 81 (24) ◽  
pp. 13889-13903 ◽  
Author(s):  
Igor Beitia Ortiz de Zarate ◽  
Lilia Cantero-Aguilar ◽  
Magalie Longo ◽  
Clarisse Berlioz-Torrent ◽  
Flore Rozenberg
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Surface ◽  
Cell Fusion ◽  
Virus Replication ◽  
Herpes Simplex Virus Type ◽  
Wild Type ◽  
Simplex Virus ◽  
Cell Cell

ABSTRACT The use of endocytic pathways by viral glycoproteins is thought to play various functions during viral infection. We previously showed in transfection assays that herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is transported from the cell surface back to the trans-Golgi network (TGN) and that two motifs of gB cytoplasmic tail, YTQV and LL, function distinctly in this process. To investigate the role of each of these gB trafficking signals in HSV-1 infection, we constructed recombinant viruses in which each motif was rendered nonfunctional by alanine mutagenesis. In infected cells, wild-type gB was internalized from the cell surface and concentrated in the TGN. Disruption of YTQV abolished internalization of gB during infection, whereas disruption of LL induced accumulation of internalized gB in early recycling endosomes and impaired its return to the TGN. The growth of both recombinants was moderately diminished. Moreover, the fusion phenotype of cells infected with the gB recombinants differed from that of cells infected with the wild-type virus. Cells infected with the YTQV-mutated virus displayed reduced cell-cell fusion, whereas giant syncytia were observed in cells infected with the LL-mutated virus. Furthermore, blocking gB internalization or impairing gB recycling to the cell surface, using drugs or a transdominant negative form of Rab11, significantly reduced cell-cell fusion. These results favor a role for endocytosis in virus replication and suggest that gB intracellular trafficking is involved in the regulation of cell-cell fusion.

scholarly journals Pore mutants of HERG and KvLQT1 downregulate the reciprocal currents in stable cell lines

2010 ◽  
Vol 299 (5) ◽  
pp. H1525-H1534 ◽  
Author(s):  
Xiao-Qin Ren ◽  
Gong Xin Liu ◽  
Louise E. Organ-Darling ◽  
Renjian Zheng ◽  
Karim Roder ◽  
...  
Keyword(s):  
Cell Lines ◽  
Rabbit Model ◽  
Chinese Hamster ◽  
Expression Vectors ◽  
Delayed Rectifier ◽  
Wild Type ◽  
Cho Cell ◽  
Surface Plasmon Resonance Analysis ◽  
Transgenic Rabbit

We previously reported a transgenic rabbit model of long QT syndrome based on overexpression of pore mutants of repolarizing K+ channels KvLQT1 (LQT1) and HERG (LQT2).The transgenes in these rabbits eliminated the slow and fast components of the delayed rectifier K+ current ( IKs and IKr, respectively), as expected. Interestingly, the expressed pore mutants of HERG and KvLQT1 downregulated the remaining reciprocal repolarizing currents, IKs and IKr, without affecting the steady-state levels of the native polypeptides. Here, we sought to further explore the functional interactions between HERG and KvLQT1 in heterologous expression systems. Stable Chinese hamster ovary (CHO) cell lines expressing KvLQT1-minK or HERG were transiently transfected with expression vectors coding for mutant or wild-type HERG or KvLQT1. Transiently expressed pore mutant or wild-type KvLQT1 downregulated IKr in HERG stable CHO cell lines by 70% and 44%, respectively. Immunostaining revealed a severalfold lower surface expression of HERG, which could account for the reduction in IKr upon KvLQT1 expression. Deletion of the KvLQT1 NH2-terminus did not abolish the downregulation, suggesting that the interactions between the two channels are mediated through their COOH-termini. Similarly, transiently expressed HERG reduced IKs in KvLQT1-minK stable cells. Coimmunoprecipitations indicated a direct interaction between HERG and KvLQT1, and surface plasmon resonance analysis demonstrated a specific, physical association between the COOH-termini of KvLQT1 and HERG. Here, we present an in vitro model system consistent with the in vivo reciprocal downregulation of repolarizing currents seen in transgenic rabbit models, illustrating the importance of the transfection method when studying heterologous ion channel expression and trafficking. Moreover, our data suggest that interactions between KvLQT1 and HERG are mediated through COOH-termini.

scholarly journals A C-terminal Hydrophobic Region is Required for Homo-Oligomerization of the Hepatitis E Virus Capsid (ORF2) Protein

2001 ◽  
Vol 1 (3) ◽  
pp. 122-128 ◽  
Author(s):  
Li Xiaofang ◽  
Mohammad Zafrullah ◽  
Faizan Ahmad ◽  
Shahid Jameel
Keyword(s):  
Amino Acids ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Wild Type ◽  
Hydrophobic Region ◽  
Reading Frame ◽  
Orf2 Protein ◽  
Acute Form ◽  
Open Reading Frame 2

Hepatitis E virus (HEV) is the causative agent of hepatitis E, an acute form of viral hepatitis. The open reading frame 2 (ORF2) of HEV encodes the viral capsid protein, which can self-oligomerize into virus-like particles. To understand the domains within this protein important for capsid biogenesis, we have carried out in vitro analyses of association and folding patterns of wild type and mutant ORF2 proteins. When expressedin vitroor in transfected cells, the ORF2 protein assembled as dimers, trimers and higher order forms. While N-terminal deletions upto 111 amino acids had no effect, the deletion of amino acids 585–610 led to reduced homo-oligomerization. This deletion also resulted in aberrant folding of the protein, as determined by its sensitivity to trypsin. This study suggests that a C-terminal hydrophobic region encompassing amino acids 585–610 of the ORF2 protein might be critical for capsid biogenesis.

scholarly journals The Slow Wallerian Degeneration Protein, WldS, Binds Directly to VCP/p97 and Partially Redistributes It within the Nucleus

2006 ◽  
Vol 17 (3) ◽  
pp. 1075-1084 ◽  
Author(s):  
Heike Laser ◽  
Laura Conforti ◽  
Giacomo Morreale ◽  
Till G.M. Mack ◽  
Molly Heyer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Wallerian Degeneration ◽  
Nuclear Protein ◽  
Ubiquitin Proteasome System ◽  
Wild Type ◽  
Ubiquitin Proteasome ◽  
Direct Binding ◽  
Synthesis Activity ◽  
Additional Role

Slow Wallerian degeneration (WldS) mutant mice express a chimeric nuclear protein that protects sick or injured axons from degeneration. The C-terminal region, derived from NAD+ synthesizing enzyme Nmnat1, is reported to confer neuroprotection in vitro. However, an additional role for the N-terminal 70 amino acids (N70), derived from multiubiquitination factor Ube4b, has not been excluded. In wild-type Ube4b, N70 is part of a sequence essential for ubiquitination activity but its role is not understood. We report direct binding of N70 to valosin-containing protein (VCP; p97/Cdc48), a protein with diverse cellular roles including a pivotal role in the ubiquitin proteasome system. Interaction with WldS targets VCP to discrete intranuclear foci where ubiquitin epitopes can also accumulate. WldS lacking its N-terminal 16 amino acids (N16) neither binds nor redistributes VCP, but continues to accumulate in intranuclear foci, targeting its intrinsic NAD+ synthesis activity to these same foci. Wild-type Ube4b also requires N16 to bind VCP, despite a more C-terminal binding site in invertebrate orthologues. We conclude that N-terminal sequences of WldS protein influence the intranuclear location of both ubiquitin proteasome and NAD+ synthesis machinery and that an evolutionary recent sequence mediates binding of mammalian Ube4b to VCP.

scholarly journals Cell surface expression of v-fms-coded glycoproteins is required for transformation.

1984 ◽  
Vol 4 (10) ◽  
pp. 1999-2009 ◽  
Author(s):  
M F Roussel ◽  
C W Rettenmier ◽  
A T Look ◽  
C J Sherr
Keyword(s):  
Amino Acids ◽  
Protein Kinase ◽  
Cell Surface ◽  
Gene Product ◽  
Kinase Activity ◽  
Specific Protein ◽  
Protein Kinase Activity ◽  
Wild Type ◽  
Specific Protein Kinase

The viral oncogene v-fms encodes a transforming glycoprotein with in vitro tyrosine-specific protein kinase activity. Although most v-fms-coded molecules remain internally sequestered in transformed cells, a minor population of molecules is transported to the cell surface. An engineered deletion mutant lacking 348 base pairs of the 3.0-kilobase-pair v-fms gene encoded a polypeptide that was 15 kilodaltons smaller than the wild-type v-fms gene product. The in-frame deletion of 116 amino acids was adjacent to the transmembrane anchor peptide located near the middle of the predicted protein sequence and 432 amino acids from the carboxyl terminus. The mutant polypeptide acquired N-linked oligosaccharide chains, was proteolytically processed in a manner similar to the wild-type glycoprotein, and exhibited an associated tyrosine-specific protein kinase activity in vitro. However, the N-linked oligosaccharides of the mutant glycoprotein were not processed to complex carbohydrate chains, and the glycoprotein was not detected at the cell surface. Cells expressing high levels of the mutant glycoprotein did not undergo morphological transformation and did not form colonies in semisolid medium. The transforming activity of the v-fms gene product therefore appears to be mediated through target molecules on the plasma membrane.

scholarly journals A Second-Site Mutation That Restores Replication of a Tat-Defective Human Immunodeficiency Virus

1999 ◽  
Vol 73 (4) ◽  
pp. 2781-2789 ◽  
Author(s):  
Koen Verhoef ◽  
Ben Berkhout
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Virus Replication ◽  
Suppressor Mutation ◽  
Large Set ◽  
Wild Type ◽  
Suppressor Mutations ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We previously constructed a large set of mutants of the human immunodeficiency virus type 1 (HIV-1) regulatory protein Tat with conservative amino acid substitutions in the activation domain. These Tat variants were analyzed in the context of the infectious virus, and several mutants were found to be defective for replication. In an attempt to obtain second-site suppressor mutations that could provide information on the Tat protein structure, some of the replication-impaired viruses were used as a parent for the isolation of revertant viruses with improved replication capacity. Sequence analysis of revertant viruses frequently revealed changes within thetat gene, most often first-site reversions either to the wild-type amino acid or to related amino acids that restore, at least partially, the Tat function and virus replication. Of 30 revertant cultures, we identified only one second-site suppressor mutation. The inactive Y26A mutant yielded the second-site suppressor mutation Y47N that partially restored trans-activation activity and virus replication. Surprisingly, when the suppressor mutation was introduced in the wild-type Tat background, it also improved thetrans-activation function of this protein about twofold. We conclude that the gain of function measured for the Y47N change is not specific for the Y26A mutant, arguing against a direct interaction of Tat amino acids 26 and 47 in the three-dimensional fold of this protein. Other revertant viruses did not contain any additional Tat changes, and some viruses revealed putative second-site Tat mutations that did not significantly improve Tat function and virus replication. We reason that these mutations were introduced by chance through founder effects or by linkage to suppressor mutations elsewhere in the virus genome. In conclusion, the forced evolution of mutant HIV-1 genomes, which is an efficient approach for the analysis of RNA regulatory motifs, seems less suited for the analysis of the structure of this small transcription factor, although protein variants with interesting properties can be generated.

scholarly journals The NB Protein of Influenza B Virus Is Not Necessary for Virus Replication In Vitro

2003 ◽  
Vol 77 (10) ◽  
pp. 6050-6054 ◽  
Author(s):  
Masato Hatta ◽  
Yoshihiro Kawaoka
Keyword(s):  
Cell Culture ◽  
Virus Replication ◽  
Wild Type Virus ◽  
Influenza B Virus ◽  
Influenza B ◽  
Type Virus ◽  
Wild Type ◽  
B Virus

ABSTRACT The NB protein of influenza B virus is thought to function as an ion channel and therefore would be expected to have an essential function in viral replication. Because direct evidence for its absolute requirement in the viral life cycle is lacking, we generated NB knockout viruses by reverse genetics and tested their growth properties both in vitro and in vivo. Mutants not expressing NB replicated as efficiently as the wild-type virus in cell culture, whereas in mice they showed restricted growth compared with findings for the wild-type virus. Thus, the NB protein is not essential for influenza B virus replication in cell culture but promotes efficient growth in mice.

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