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Sliva, Katja; Erlwein, Otto; Bittner, Alexandra; Schnierle, Barbara S.

doi:10.1186/1743-422x-1-14

Cited by 24 publications

(11 citation statements)

References 15 publications

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“…We have not tested whether the higher expression of the env ORF translates into an accumulation of the ENV protein, which has been linked to the generation of cytopathic effects in some MLV 42 . That said, we have not observed any cytopathic effect, in agreement with the fact that most MLV do not generate such effects 43 .…”

Section: Resultssupporting

confidence: 90%

Characterization of a new case of XMLV (Bxv1) contamination in the human cell line Hep2 (clone 2B)

Loiseau

Cordaux

Giraud

et al. 2020

Sci Rep

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The use of misidentified cell lines contaminated by other cell lines and/or microorganisms has generated much confusion in the scientific literature. Detailed characterization of such contaminations is therefore crucial to avoid misinterpretation and ensure robustness and reproducibility of research. Here we use DNA-seq data produced in our lab to first confirm that the Hep2 (clone 2B) cell line (Sigma-Aldrich catalog number: 85011412-1VL) is indistinguishable from the HeLa cell line by mapping integrations of the human papillomavirus 18 (HPV18) at their expected loci on chromosome 8. We then show that the cell line is also contaminated by a xenotropic murine leukemia virus (XMLV) that is nearly identical to the mouse Bxv1 provirus and we characterize one Bxv1 provirus, located in the second intron of the pseudouridylate synthase 1 (PUS1) gene. Using an RNA-seq dataset, we confirm the high expression of the E6 and E7 HPV18 oncogenes, show that the entire Bxv1 genome is moderately expressed, and retrieve a Bxv1 splicing event favouring expression of the env gene. Hep2 (clone 2B) is the fourth human cell line so far known to be contaminated by the Bxv1 XMLV. This contamination has to be taken into account when using the cell line in future experiments.

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Section: Resultssupporting

confidence: 90%

Characterization of a new case of XMLV (Bxv1) contamination in the human cell line Hep2 (clone 2B)

Loiseau

Cordaux

Giraud

et al. 2020

Sci Rep

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“…MLVeGFP: Replicative Moloney-MLV, referred to as MLVeGFP throughout this study, was produced from the pMOV-eGFP expression plasmid 70 – 72 . The eGFP reporter is inserted in frame within the proline-rich region of the viral envelope glycoprotein, is expressed on the exterior surface of the virus, and does not alter infectivity nor ecotropic receptor specificity 70 .…”

Section: Methodsmentioning

confidence: 99%

Single-Particle Discrimination of Retroviruses from Extracellular Vesicles by Nanoscale Flow Cytometry

Tang

Renner

Fritzsche

et al. 2017

Sci Rep

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Retroviruses and small EVs overlap in size, buoyant densities, refractive indices and share many cellderived surface markers making them virtually indistinguishable by standard biochemical methods. This poses a significant challenge when purifying retroviruses for downstream analyses or for phenotypic characterization studies of markers on individual virions given that EVs are a major contaminant of retroviral preparations. Nanoscale flow cytometry (NFC), also called flow virometry, is an adaptation of flow cytometry technology for the analysis of individual nanoparticles such as extracellular vesicles (EVs) and retroviruses. In this study we systematically optimized NFC parameters for the detection of retroviral particles in the range of 115-130 nm, including viral production, sample labeling, laser power and voltage settings. By using the retroviral envelope glycoprotein as a selection marker, and evaluating a number of fluorescent dyes and labeling methods, we demonstrate that it is possible to confidently distinguish retroviruses from small EVs by NFC. Our findings make it now possible to individually phenotype genetically modified retroviral particles that express a fluorescent envelope glycoprotein without removing EV contaminants from the sample.Retroviruses, such as the human immunodeficiency virus (HIV), are enveloped RNA viruses that range between 90-150 nm in diameter, depending on the species 1-3 . When nascent virions egress from infected cells, they bear contents of the cytosol (e.g., proteins, mRNAs, miRNAs), as well as a portion of the cell membrane embedded with surface receptors to form the viral envelope [4][5][6] . The phenotypic analysis of host-derived markers on the surface of individual viruses is of considerable interest, as it can provide information on the identity of the specific cell types that are infected in a host. However, a major hurdle in purifying retroviruses for single-particle characterization studies is the removal of EVs that are concomitantly released by the cells 7-11 . EV is a broad term that describes all particles with a membrane bilayer released from cells; these can include exosomes, microvesicles, and apoptotic vesicles [12][13][14][15][16] . Small EVs, that are in the size range of retroviruses constitute a major contaminant of virus preparations as they are biochemically and biophysically similar to retroviruses in terms of their refractive indices, buoyant densities, and surface markers 5,7,[17][18][19] . Additionally, EVs can also package retroviral proteins and RNAs that further complicate discrimination [20][21][22][23][24] .Nanoscale flow cytometry (NFC), also called flow virometry, is a new and powerful tool in the field of virology that enables the phenotypic analysis of the markers at the surface of individual virions 11,[25][26][27][28][29][30][31][32] . Virus populations can now be profiled and sorted in multi-parameter analyses, much in the same way as cells 25,27,30,32,33 . However, NFC analysis with current instrumentation can be challenging due to ...

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“…The pMOV-eGFP expression vector encodes a replicative Moloney murine leukemia virus (MoMLV) with the eGFP reporter gene inserted in the proline-rich region of the ecotropic env gene (33). The single-cycle HIV[p8.9] pseudovirus is generated by a multi-plasmid expression system using a packaging vector for HIV-1 (pCSGW), an expression vector for Gag-Pol-Rev (p8.9) and the pMDG plasmid encoding the envelope glycoprotein of the vesicular stomatitis virus (34,35).…”

Section: Methodsmentioning

confidence: 99%

Binding of RNA by APOBEC3G controls deamination-independent restriction of retroviruses

Bélanger¹,

Savoie²,

Gerpe³

et al. 2013

Nucleic Acids Research

116

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APOBEC3G (A3G) is a host-encoded protein that potently restricts the infectivity of a broad range of retroviruses. This can occur by mechanisms dependent on catalytic activity, resulting in the mutagenic deamination of nascent viral cDNA, and/or by other means that are independent of its catalytic activity. It is not yet known to what extent deamination-independent processes contribute to the overall restriction, how they exactly work or how they are regulated. Here, we show that alanine substitution of either tryptophan 94 (W94A) or 127 (W127A) in the non-catalytic N-terminal domain of A3G severely impedes RNA binding and alleviates deamination-independent restriction while still maintaining DNA mutator activity. Substitution of both tryptophans (W94A/W127A) produces a more severe phenotype in which RNA binding and RNA-dependent protein oligomerization are completely abrogated. We further demonstrate that RNA binding is specifically required for crippling late reverse transcript accumulation, preventing proviral DNA integration and, consequently, restricting viral particle release. We did not find that deaminase activity made a significant contribution to the restriction of any of these processes. In summary, this work reveals that there is a direct correlation between A3G’s capacity to bind RNA and its ability to inhibit retroviral infectivity in a deamination-independent manner.

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Cited by 24 publications

References 15 publications

Characterization of a new case of XMLV (Bxv1) contamination in the human cell line Hep2 (clone 2B)

Characterization of a new case of XMLV (Bxv1) contamination in the human cell line Hep2 (clone 2B)

Single-Particle Discrimination of Retroviruses from Extracellular Vesicles by Nanoscale Flow Cytometry

Binding of RNA by APOBEC3G controls deamination-independent restriction of retroviruses

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