Enhancement of antitumor activity of gammaretrovirus carrying IL-12 gene through genetic modification of envelope targeting HER2 receptor: a promising strategy for bladder cancer therapy
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Gammaretrovirus
The 10A1 murine leukemia virus (MuLV) is a recombinant type C retrovirus isolated from a mouse infected with amphotropic MuLV (A-MuLV). 10A1 and A-MuLV have 91% amino acid identity in their envelope proteins yet display different host ranges. For example, CHO-K1 cells are resistant to A-MuLV but susceptible to infection by 10A1. We have now determined that retroviral vectors bearing altered A-MuLV envelope proteins containing 10A1-derived residues at positions 71 (A71G), 74 (Q74K), and 139 (V139M) transduce CHO-K1 cells at efficiencies similar to those achieved with 10A1 enveloped vectors. A-MuLV enveloped retroviral vectors with these three 10A1 residues were also able to transduce A-MuLV-infected NIH 3T3 cells. This observation is consistent with the ability of vectors bearing this altered A-MuLV envelope protein to recognize the 10A1-specific receptor present on NIH 3T3 cells and supports the possibility that residues at positions 71, 74, and 139 of the 10A1 envelope SU protein account for the expanded host range of 10A1.
3T3 cells
Gammaretrovirus
Viral protein
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Certain glycosaminoglycans (GAGs), including heparin, inhibit infection by murine leukemia virus (MLV). We now show that this is due to inhibition of virus attachment independent of the interaction between viral envelope proteins (Env) and their cellular receptors. Heparin blocked the binding of both Env-deficient and amphotropic MLV (MLV-A) particles to NIH 3T3 fibroblasts, CHO cells which lack the amphotropic retroviral receptor Pit-2, and CHO cells transfected with Pit-2 (CHO-Pit-2). Heparin also inhibited the transduction of NIH 3T3 cells by MLV-A over a similar concentration range. This effect was observed within 15 min of exposure to retrovirus. Preloading target cells with heparin had no effect on transduction and both MLV-A and Env-deficient retrovirus bound efficiently to heparin-coated agarose beads, suggesting that heparin interacts with the virus rather than the target cell. This requires both a strong negative charge and a specific structure since GAGs with different charge and carbohydrate composition inhibited virus infection variably. The specificity of GAG-virus interaction also depends on the producer cells, since virus packaged by murine GP+EnvAM12 cells was 1,000-fold more sensitive to inhibition by chondroitin sulfate A than was virus packaged by human FLYA13 packaging cells. No evidence for an interaction between MLV and cell surface proteoglycans was found, however, since the attachment of MLV-A and envelope-defective virus to proteoglycan-deficient CHOpgsA-745 cells was similar to that seen with both wild-type and CHO-Pit-2 cells. Although the molecular mechanism is unclear, this study presents evidence that Env receptor-independent attachment is an important step in MLV infection.
Gammaretrovirus
3T3 cells
Helper virus
Viral transformation
Transduction (biophysics)
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Retroviral recombinants result from template switching between copackaged viral genomes. Here, marker reassortment between coexpressed vectors was measured during single replication cycles, and human immunodeficiency virus type 1 (HIV-1) recombination was observed six- to sevenfold more frequently than murine leukemia virus (MLV) recombination. Template switching was also assayed by using transduction-type vectors in which donor and acceptor template regions were joined covalently. In this situation, where RNA copackaging could not vary, MLV and HIV-1 template switching rates were indistinguishable. These findings argue that MLV's lower intermolecular recombination frequency does not reflect enzymological differences. Instead, these data suggest that recombination rates differ because coexpressed MLV RNAs are less accessible to the recombination machinery than are coexpressed HIV RNAs. This hypothesis provides a plausible explanation for why most gammaretrovirus recombinants, although relatively rare, display evidence of multiple nonselected crossovers. By implying that recombinogenic template switching occurs roughly four times on average during the synthesis of every MLV or HIV-1 DNA, these results suggest that virtually all products of retroviral replication are biochemical recombinants.
Gammaretrovirus
Transduction (biophysics)
Helper virus
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Gammaretrovirus
Matrix (chemical analysis)
Group-specific antigen
Bovine leukemia virus
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ABSTRACT Immature retroviral particles are assembled by self-association of the structural polyprotein precursor Gag. During maturation the Gag polyprotein is proteolytically cleaved, yielding mature structural proteins, matrix (MA), capsid (CA), and nucleocapsid (NC), that reassemble into a mature viral particle. Proteolytic cleavage causes the N terminus of CA to fold back to form a β-hairpin, anchored by an internal salt bridge between the N-terminal proline and the inner aspartate. Using an in vitro assembly system of capsid-nucleocapsid protein (CANC), we studied the formation of virus-like particles (VLP) of a gammaretrovirus, the xenotropic murine leukemia virus (MLV)-related virus (XMRV). We show here that, unlike other retroviruses, XMRV CA and CANC do not assemble tubular particles characteristic of mature assembly. The prevention of β-hairpin formation by the deletion of either the N-terminal proline or 10 initial amino acids enabled the assembly of ΔProCANC or Δ10CANC into immature-like spherical particles. Detailed three-dimensional (3D) structural analysis of these particles revealed that below a disordered N-terminal CA layer, the C terminus of CA assembles a typical immature lattice, which is linked by rod-like densities with the RNP.
Gammaretrovirus
Virus-like particle
Cleavage (geology)
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Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus found in association with human prostate cancer and chronic fatigue syndrome, although these associations are controversial. XMRV shows at most 94% identity to known mouse retroviruses. Here we used XMRV-specific PCR to search for a more closely related source of XMRV in mice. While we could not find a complete copy, we did find a 3,600-bp region of XMRV in an endogenous retrovirus present in NIH/3T3 cells. These results show that XMRV has clear ancestors in mice and highlight another possible source of contamination in PCR assays for XMRV.
Gammaretrovirus
Endogenous retrovirus
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ABSTRACT A potentially powerful approach for in vivo gene delivery is to target retrovirus to specific cells through interactions between cell surface receptors and appropriately modified viral envelope proteins. Previously, relatively large (>100 residues) protein ligands to cell surface receptors have been inserted at or near the N terminus of retroviral envelope proteins. Although viral tropism could be altered, the chimeric envelope proteins lacked full activity, and coexpression of wild-type envelope was required for production of transducing virus. Here we analyze more than 40 derivatives of ecotropic Moloney murine leukemia virus (MLV) envelope, containing insertions of short RGD-containing peptides, which are ligands for integrin receptors. In many cases pseudotyped viruses containing only the chimeric envelope protein could transduce human cells. The precise location, size, and flanking sequences of the ligand affected transduction specificity and efficiency. We conclude that retroviral tropism can be rationally reengineered by insertion of short peptide ligands and without the need to coexpress wild-type envelope.
Transduction (biophysics)
Gammaretrovirus
Tissue tropism
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Gammaretrovirus
Cell fusion
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Gammaretrovirus
Rous sarcoma virus
Endogenous retrovirus
Infectivity
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Immature retrovirus particles are assembled from the multidomain Gag protein. In these particles, the Gag proteins are arranged radially as elongated rods. We have previously characterized the properties of HIV-1 Gag in solution. In the absence of nucleic acid, HIV-1 Gag displays moderately weak interprotein interactions, existing in monomer-dimer equilibrium. Neutron scattering and hydrodynamic studies suggest that the protein is compact, and biochemical studies indicate that the two ends can approach close in three-dimensional space, implying the need for a significant conformational change during assembly. We now describe the properties of the Gag protein of Moloney murine leukemia virus (MLV), a gammaretrovirus. We found that this protein is very different from HIV-1 Gag: it has much weaker protein-protein interaction and is predominantly monomeric in solution. This has allowed us to study the protein by small-angle X-ray scattering and to build a low-resolution molecular envelope for the protein. We found that MLV Gag is extended in solution, with an axial ratio of ∼7, comparable to its dimensions in immature particles. Mutational analysis suggests that runs of prolines in its matrix and p12 domains and the highly charged stretch at the C terminus of its capsid domain all contribute to this extended conformation. These differences between MLV Gag and HIV-1 Gag and their implications for retroviral assembly are discussed.
Group-specific antigen
Gammaretrovirus
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