Mutational analysis of Moloney murine leukaemia virus surface protein gp70

Skov, H.; Andersen, Klaus B.

doi:10.1099/0022-1317-74-4-707

Cited by 15 publications

(21 citation statements)

References 28 publications

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“…Although the receptor binding site on the envelope protein has not been precisely defined, several charged residues in this amino-terminal region of SU have been identified (12,15). In particular, a negatively charged residue (aspartate 84) was shown to be critical for virus binding and infection (12).…”

Section: Vol 77 2003mentioning

confidence: 99%

Identification of a Critical Basic Residue on the Ecotropic Murine Leukemia Virus Receptor

Qian

Donald

Wang

et al. 2003

J Virol

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Susceptibility to ecotropic murine leukemia viruses (MLV) is restricted to mice and rats at the level of virus binding to the host cell receptor. Asparagine 232, valine 233, tyrosine 235, and glutamic acid 237 in the third extracellular domain (EL3) of the receptor are critical determinants of the host range difference between mice and humans. However, placing these residues in the human homolog confers only partial binding, indicating that other divergent sequences are involved. We sought to determine if the other sequences lie within or outside EL3. Here we report the identification of lysine 234 as another critical residue that influences virus binding and infection, as well as evidence that the unidentified sequences lie outside EL3. Each of the four basic residues in the third extracellular domain were changed to an acidic residue and initially examined in combination with a change at position 235 or position 237. Substitution of lysine 211, 215, or 222 combined with substitution of the critical tyrosine 235 or glutamic acid 237 did not affect virus infection. However, combined substitution of lysine 234, a conserved residue between mice and humans, and tyrosine 235 resulted in a marked decrease in virus infection and binding. A lysine 234 change alone reduced virus binding, contrary to previous observations that at least two of the other four residues must be changed before binding is reduced. Interestingly, there was no decrease in infection when lysine 234 was replaced in combination with glutamic acid 237. This result suggests that residue 234 may act by influencing the local structure of residues 233 to 235, whereas the presence of a glycine at position 236 may prevent this influence from extending to residue 237. With this report, the involvement of all the residues divergent between mice and humans in the third extracellular domain has been ruled out, suggesting that as yet unidentified determinants lie in other extracellular domains.The host range of the ecotropic murine leukemia virus (MLV) is restricted to mice and rats by the availability of their cell surface receptor ATRC1, an integral membrane protein with 14 membrane-spanning domains (3). In the host cell, ATRC1 functions as the principal transporter of cationic amino acids (9, 18). Other mammals, including humans, have homologous proteins that function as cationic amino acid transporters but lack ecotropic MLV receptor capability (1, 2).Homology scanning mutagenesis of the receptor and its human homolog led to the identification of residues 211 to 239 as the putative virus binding domain (2, 21) (Fig. 1A). This domain resides on the extracellular side of the plasma membrane since the two N-linked glycosylation sites (asparagines 223 and 229) within it are glycosylated in vivo (10). Replacing two or more of asparagine 232, valine 233, tyrosine 235, and glutamate 237 results in loss of virus surface protein (SU) binding and virus infection, although single substitution of any of these residues did not reduce binding or infection (2), indicatin...

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Section: Vol 77 2003mentioning

confidence: 99%

Identification of a Critical Basic Residue on the Ecotropic Murine Leukemia Virus Receptor

Qian

Donald

Wang

et al. 2003

J Virol

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“…A chimeric protein encoding only the amino-terminal 88 amino acids of the Moloney MuLV (MoMuLV) envelope can still mediate entry via the ecoR, albeit with reduced efficiency, suggesting that at least some specific interactions are mediated by these first 88 amino acids (19). Numerous insertion and site-directed mutations have been made in the binding domain of the Friend MuLV and MoMuLV envelopes (4,5,8,10,15,25). While many of these mutations prevent the propagation of the virus, none have been shown to interfere with receptor binding.…”

mentioning

confidence: 99%

Identification of a subdomain in the Moloney murine leukemia virus envelope protein involved in receptor binding

MacKrell

Soong

Curtis

et al. 1996

J Virol

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We have mutated amino acids within the receptor-binding domain of Moloney murine leukemia virus envelope in order to identify residues involved in receptor binding. Analysis of mutations in the region of amino acids 81 to 88 indicates that this region is important for specific envelope-receptor interactions. None of the aspartate 84 (D-84) mutants studied bind measurably, although they are efficiently incorporated into particles. D-84 mutants have titers that correspond to the severity of the substitution. This observation suggests that D-84 may provide a direct receptor contact. Mutations in the other charged amino acids in this domain (R-83, E-86, and E-87) yield titers similar to those of wild-type envelope, but the affinity of the mutant envelope in the binding assay is decreased by nonconservative substitutions in parallel to the severity of the change. These other amino acids may either provide secondary receptor contacts or assist in maintaining a structure in the domain that favors efficient binding. We also studied other regions of high hydrophilicity. Our initial characterization indicates that amino acids 106 to 111 and 170 to 188 do not play a major role in receptor binding. Measurements of relative binding affinity and titer indicate that most mutations in the region of amino acids 120 to 131 did not significantly affect receptor binding. However, SU encoded by mutants H123V, R124L, and C131A as well as C81A could not be detected in particles and therefore did not bind measurably. Therefore, the region encompassed by amino acids 81 to 88 appears to be directly involved in receptor binding.

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“…The plasmid pKA1558 (encompassing a molecular clone of Moloney murine leukaemia virus (MoMuLV); Skov & Andersen, 1993) was used for molecular cloning of hybrid viruses. First, a 30 nt linker containing an SfiI site was introduced at nt 5893 (all positions according to GenBank accession no.…”

Section: Methodsmentioning

confidence: 99%

“…The infectious molecular clone of ecotropic MLV strain MoMuLV pKA1558 (Skov & Andersen, 1993) served as the source of MLV genome components. After modification of SfiI restriction sites to facilitate exchange of env genes, the resulting clone pKA1558LDS was still able to generate replication-competent MLV after transfection of 293T cells (data not shown).…”

Section: Generation Of Mlv/hiv-1 and Mlv/sivagm Hybrid Virusesmentioning

confidence: 99%

Genetic engineering of onco/lentivirus hybrids results in formation of infectious but not of replication-competent viruses

Steidl

Schüle

Mühlebach

et al. 2004

Journal of General Virology

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To achieve specific gene transfer into human CD4 + cells, murine leukaemia virus (MLV)-based pseudotype vector particles were generated employing Env variants derived from human or simian immunodeficiency virus (HIV-1 or SIVagm). Here, we describe the generation of full-length onco/lentivirus hybrid genomes comprising components of MLV and HIV-1 or SIVagm, respectively, to assess the possibility of replication-competent hybrid virus formation. The env reading frame of an infectious molecular clone of MLV was replaced with the analogous coding regions of HIV-1 or SIVagm encompassing the env gene and accessory genes. Resulting MLV/HIV-1 or MLV/SIVagm hybrid genomes were transfected into 293T cells. Expression of viral proteins and budding of retroviral particles was shown by specific immunostaining and electron microscopy. The viral particles mediated CD4-and co-receptor-specific infection of human cells as demonstrated by PCR and immunostaining in the respective target cells. However, no productive infection resulting in the generation of infectious virus was detected in these cells. Thus, these onco/lentivirus hybrids, although able to initiate single-round infection, were not replication competent. Thus, MLV-based pseudotype vectors carrying Env variants of HIV-1 or SIVagm are not prone to form replication-competent retroviruses, suggesting a favourable safety profile for MLV-based CD4-specific pseudotype vectors.

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Mutational analysis of Moloney murine leukaemia virus surface protein gp70

Cited by 15 publications

References 28 publications

Identification of a Critical Basic Residue on the Ecotropic Murine Leukemia Virus Receptor

Identification of a Critical Basic Residue on the Ecotropic Murine Leukemia Virus Receptor

Identification of a subdomain in the Moloney murine leukemia virus envelope protein involved in receptor binding

Genetic engineering of onco/lentivirus hybrids results in formation of infectious but not of replication-competent viruses

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