scholarly journals Suppression of a Fusion Defect by Second Site Mutations in the Ecotropic Murine Leukemia Virus Surface Protein

1999 ◽  
Vol 73 (6) ◽  
pp. 5034-5042 ◽  
Author(s):  
Tatiana Zavorotinskaya ◽  
Lorraine M. Albritton
Keyword(s):  
Host Cell ◽  
Cell Fusion ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Surface Protein ◽  
Host Cells ◽  
Virus Surface ◽  
Fusion Defect ◽  
Murine Leukemia ◽  
Cell Cell

ABSTRACT Entry of ecotropic murine leukemia virus initiates when the envelope surface protein recognizes and binds to the virus receptor on host cells. The envelope transmembrane protein then mediates fusion of viral and host cell membranes and penetration into the cytoplasm. Using a genetic selection, we isolated an infectious retrovirus variant containing three changes in the surface protein—histidine 8 to arginine, glutamine 227 to arginine, and aspartate 243 to tyrosine. Single replacement of histidine 8 with arginine (H8R) resulted in almost complete loss of infectivity, even though the mutant envelope proteins were stable and efficiently incorporated into virions. Virions carrying H8R envelope were proficient at binding cells expressing receptor but failed to induce cell-cell fusion of XC cells, indicating that the histidine at position 8 plays an essential role in fusion during penetration of the host cell membrane. Thus, there is at least one domain in SU that is involved in fusion; the fusion functions do not reside exclusively in TM. In contrast, envelope with all three changes induced cell-cell fusion of XC cells and produced virions that were 10,000-fold more infectious than those containing only the H8R substitution, indicating that changes at positions 227 and 243 can suppress a fusion defect caused by loss of histidine 8 function. Moreover, the other two changes acted synergistically, indicating that both compensate for the loss of the same essential function of histidine 8. The ability of these changes to suppress this fusion defect might provide a means for overcoming postbinding defects found in targeted retroviral vectors for use in human gene therapy.

scholarly journals Role of the Mutation Q252R in Activating Membrane Fusion in the Murine Leukemia Virus Surface Envelope Protein

2003 ◽  
Vol 77 (20) ◽  
pp. 10841-10849 ◽  
Author(s):  
Chi-Wei Lu ◽  
Monica J. Roth
Keyword(s):  
Cell Fusion ◽  
Receptor Binding ◽  
Envelope Protein ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Host Cells ◽  
Cell Binding ◽  
Surface Envelope ◽  
Murine Leukemia ◽  
Cell Cell

ABSTRACT Entry of retroviruses into host cells requires the fusion between the viral and cellular membranes. It is unclear how receptor binding induces conformational changes within the surface envelope protein (SU) that activate the fusion machinery residing in the transmembrane envelope protein (TM). In this report, we have isolated a point mutation, Q252R, within the proline-rich region of the 4070A murine leukemia virus SU that altered the virus-cell binding characteristics and induced cell-cell fusion. Q252R displays a SU shedding-sensitive phenotype. Cell-cell fusion is receptor dependent and is observed only in the presence of MuLV Gag-Pol. Both cellular binding and fusion by Q252R are greatly enhanced in conjunction of G100R, a mutation within the SU variable region A which increases viral binding through an independent mechanism. Deletion of a conserved histidine (His36) at the SU N terminus abolished cell-cell fusion by G100R/Q252R Env without compromising virus-cell binding. Although G100R/Q252R virus has no detectable titer, replacement of the N-terminal nine 4070A SU amino acids with the equivalent ecotropic MuLV sequence restored viral infectivity. These studies provide insights into the functional cooperation between multiple elements of SU required to signal receptor binding and activate the fusion machinery.

scholarly journals Receptor-Triggered but Alkylation-Arrested Env of Murine Leukemia Virus Reveals the Transmembrane Subunit in a Prehairpin Conformation

2006 ◽  
Vol 80 (19) ◽  
pp. 9921-9925 ◽  
Author(s):  
Michael Wallin ◽  
Maria Ekström ◽  
Henrik Garoff
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Surface Protein ◽  
Intermediate Stage ◽  
Central Feature ◽  
Fusion Inhibition ◽  
Mediated Reduction ◽  
Murine Leukemia

ABSTRACT A central feature of the prevailing model for retrovirus fusion is conversion of the transmembrane (TM) subunit from a prehairpin to a hairpin-like structure. The fusion inhibition of many retroviruses, except murine leukemia virus (MLV), with peptides corresponding to interacting regions in the hairpin supports the model. MLV fusion is controlled by isomerization of the intersubunit disulfide in Env. We show here that TM peptides bind to MLV Env that has been arrested at an intermediate stage of activation by alkylation of the isomerization-active thiol in the surface subunit. This inhibits fusion rescue by dithiothreitol-mediated reduction of the surface protein-TM disulfide.

scholarly journals Development of a Novel High-Throughput Surrogate Assay to Measure HIV Envelope/CCR5/CD4-Mediated Viral/Cell Fusion Using BacMam Baculovirus Technology

2003 ◽  
Vol 8 (4) ◽  
pp. 463-470 ◽  
Author(s):  
Stephen Jenkinson ◽  
David C. Mc Coy ◽  
Sandy A. Kerner ◽  
Robert G. Ferris ◽  
Wendell K. Lawrence ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Fusion ◽  
High Throughput ◽  
Viral Envelope ◽  
Host Cells ◽  
Luciferase Reporter ◽  
Viral Fusion ◽  
Fusion Event ◽  
Surrogate Assay ◽  
Cell Cell

The initial event by which M-tropic HIV strains gain access to cells is via interaction of the viral envelope protein gp120 with the host cell CCR5 coreceptor and CD4. Inhibition of this event reduces viral fusion and entry into cells in vitro. The authors have employed BacMam baculovirus-mediated gene transduction to develop a cell/cell fusion assay that mimics the HIV viral/cell fusion process and allows high-throughput quantification of this fusion event. The assay design uses human osteosarcoma (HOS) cells stably transfected with cDNAs expressing CCR5, CD4, and long terminal repeat (LTR)-luciferase as the recipient host cell. An HEK-293 cell line transduced with BacMam viral constructs to express the viral proteins gp120, gp41, tat, and rev represents the virus. Interaction of gp120 with CCR5/CD4 results in the fusion of the 2 cells and transfer of tat to the HOS cell cytosol; tat, in turn, binds to the LTR region on the luciferase reporter and activates transcription, resulting in an increase in cellular luciferase activity. In conclusion, the cell/cell fusion assay developed has been demonstrated to be a robust and reproducible high-throughput surrogate assay that can be used to assess the effects of compounds on gp120/CCR5/CD4-mediated viral fusion into host cells.

scholarly journals Dynein Regulators Are Important for Ecotropic Murine Leukemia Virus Infection

2016 ◽  
Vol 90 (15) ◽  
pp. 6896-6905 ◽  
Author(s):  
Roger Valle-Tenney ◽  
Tatiana Opazo ◽  
Jorge Cancino ◽  
Stephen P. Goff ◽  
Gloria Arriagada
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Host Cells ◽  
Cellular Proteins ◽  
Preintegration Complex ◽  
Microtubule Network ◽  
Flag Epitope ◽  
Murine Leukemia

ABSTRACTDuring the early steps of infection, retroviruses must direct the movement of the viral genome into the nucleus to complete their replication cycle. This process is mediated by cellular proteins that interact first with the reverse transcription complex and later with the preintegration complex (PIC), allowing it to reach and enter the nucleus. For simple retroviruses, such as murine leukemia virus (MLV), the identities of the cellular proteins involved in trafficking of the PIC in infection are unknown. To identify cellular proteins that interact with the MLV PIC, we developed a replication-competent MLV in which the integrase protein was tagged with a FLAG epitope. Using a combination of immunoprecipitation and mass spectrometry, we established that the microtubule motor dynein regulator DCTN2/p50/dynamitin interacts with the MLV preintegration complex early in infection, suggesting a direct interaction between the incoming viral particles and the dynein complex regulators. Further experiments showed that RNA interference (RNAi)-mediated silencing of either DCTN2/p50/dynamitin or another dynein regulator, NudEL, profoundly reduced the efficiency of infection by ecotropic, but not amphotropic, MLV reporters. We propose that the cytoplasmic dynein regulators are a critical component of the host machinery needed for infection by the retroviruses entering the cell via the ecotropic envelope pathway.IMPORTANCERetroviruses must access the chromatin of host cells to integrate the viral DNA, but before this crucial event, they must reach the nucleus. The movement through the cytoplasm—a crowded environment where diffusion is slow—is thought to utilize retrograde transport along the microtubule network by the dynein complex. Different viruses use different components of this multisubunit complex. We found that the preintegration complex of murine leukemia virus (MLV) interacts with the dynein complex and that regulators of this complex are essential for infection. Our study provides the first insight into the requirements for retrograde transport of the MLV preintegration complex.

scholarly journals The Human TRIM5α Restriction Factor Mediates Accelerated Uncoating of the N-Tropic Murine Leukemia Virus Capsid

2006 ◽  
Vol 81 (5) ◽  
pp. 2138-2148 ◽  
Author(s):  
Michel J. Perron ◽  
Matthew Stremlau ◽  
Mark Lee ◽  
Hassan Javanbakht ◽  
Byeongwoon Song ◽  
...  
Keyword(s):  
Host Cell ◽  
Reverse Transcription ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Restriction Factor ◽  
Viral Capsid ◽  
Wild Type ◽  
Host Cell Factors ◽  
Infected Cells ◽  
Murine Leukemia

ABSTRACT The host cell factors TRIM5αhu and Fv-1 restrict N-tropic murine leukemia virus (N-MLV) infection at an early postentry step before or after reverse transcription, respectively. Interestingly, the identity of residue 110 of the MLV capsid determines susceptibility to both TRIM5αhu and Fv-1. In this study, we investigate the fate of the MLV capsid in cells expressing either the TRIM5αhu or Fv-1 restriction factor. The expression of TRIM5αhu, but not Fv-1, specifically promoted the premature conversion of particulate N-MLV capsids within infected cells to soluble capsid proteins. The TRIM5αhu-mediated disassembly of particulate N-MLV capsids was dependent upon residue 110 of the viral capsid. Furthermore, the deletion or disruption of TRIM5αhu domains necessary for potent N-MLV restriction completely abrogated the disappearance of particulate N-MLV capsids observed with wild-type TRIM5αhu. These results suggest that premature disassembly of the viral capsid contributes to the restriction of N-MLV infection by TRIM5αhu, but not by Fv-1.

scholarly journals G541R within the 4070A TM Protein Regulates Fusion in Murine Leukemia Viruses

2003 ◽  
Vol 77 (22) ◽  
pp. 12011-12021 ◽  
Author(s):  
Lucille O'Reilly ◽  
Monica J. Roth
Keyword(s):  
Cell Fusion ◽  
Viral Vector ◽  
Leukemia Virus ◽  
Critical Role ◽  
Viral Envelope ◽  
Vector System ◽  
Wild Type ◽  
C Terminus ◽  
Murine Leukemia ◽  
Cell Cell

ABSTRACT The mutation G541R within the ectodomain of TM was isolated in three independent chimeric enveloped murine leukemia virus (MuLV) viral populations originally impaired in viral passage and in wild-type 4070A. Isolation of G541R in multiple populations suggested it played a critical role in viral envelope function. Using a viral vector system, the observed effects of the G541R mutation within MuLV envelope proteins were pleiotropic and included effects on the regulation of SU-TM interactions and membrane fusion. G541R suppresses enhanced cell-cell fusion events attributable to the absence of the R-peptide yet does not adversely affect virus titers. The ability to suppress cell-cell fusion is dependent on the presence of the C terminus of the amphotropic 4070A SU protein. Within the wild-type 4070A envelope background, the mutation results in a decreased level of Env at the cell surface that is mirrored in the virion. The TM mutation alters recognition of the SU C terminus by a monoclonal antibody, suggestive of an altered conformation. The presence of G541R allowed the virus to achieve a balance between cytopathogenicity and replication and restored productive viral entry.

scholarly journals A Unique Heparin-Binding Domain in the Envelope Protein of the Neuropathogenic PVC-211 Murine Leukemia Virus May Contribute to Its Brain Capillary Endothelial Cell Tropism

2001 ◽  
Vol 75 (24) ◽  
pp. 12439-12445 ◽  
Author(s):  
Atsushi Jinno-Oue ◽  
Miho Oue ◽  
Sandra K. Ruscetti
Keyword(s):  
Amino Acid ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Surface Protein ◽  
Binding Domain ◽  
Heparin Binding ◽  
Brain Capillary ◽  
Envelope Surface ◽  
Heparin Binding Domain ◽  
Murine Leukemia

ABSTRACT Previous studies from our laboratory demonstrated that PVC-211 murine leukemia virus (MuLV), a neuropathogenic variant of Friend MuLV (F-MuLV), had undergone genetic changes which allowed it to efficiently infect rat brain capillary endothelial cells (BCEC) in vivo and in vitro. Two amino acid changes from F-MuLV in the putative receptor binding domain (RBD) of the envelope surface protein of PVC-211 MuLV (Glu-116 to Gly and Glu-129 to Lys) were shown to be sufficient for conferring BCEC tropism on PVC-211 MuLV. Recent examination of the unique RBD of PVC-211 MuLV revealed that the substitution of Lys for Glu at position 129 created a new heparin-binding domain that overlapped a heparin-binding domain common to ecotropic MuLVs. In this study we used heparin-Sepharose columns to demonstrate that PVC-211 MuLV, but not F-MuLV, can bind efficiently to heparin and that one or both of the amino acids in the RBD of PVC-211 MuLV that are associated with BCEC tropism are responsible. We further showed that heparin can enhance or inhibit MuLV infection and that the mode of action is dependent on heparin concentration, sulfation of heparin, and the affinity of the virus for heparin. Our results suggest that the amino acid changes that occurred in the envelope surface protein of PVC-211 MuLV may allow the virus to bind strongly to the surface of BCEC via heparin-like molecules, increasing the probability that the virus will bind to its cell surface receptor and efficiently infect these cells.

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