Dataset Information

Mass spectrometry analysis reveals differences in the host cell protein species found in pseudotyped lentiviral vectors.

ABSTRACT: Lentiviral vectors (LVs) have been successfully used in clinical trials showing long term therapeutic benefits. Studying the role of cellular proteins in lentivirus HIV-1 life cycle can help understand virus assembly and budding, leading to improvement of LV production for gene therapy. Lentiviral vectors were purified using size exclusion chromatography (SEC). The cellular protein composition of LVs produced by two different methods was compared: the transient transfection system pseudotyped with the VSV-G envelope, currently used in clinical trials, and a stable producer cell system using a non-toxic envelope derived from cat endogenous retrovirus RD114, RDpro. Proteins of LVs purified by size exclusion chromatography were identified by tandem mass spectrometry (MS/MS). A smaller number of cellular protein species were detected in stably produced vectors compared to transiently produced vector samples. This may be due to the presence of co-purified VSV-G vesicles in transiently produced vectors. AHNAK (Desmoyokin) was unique to RDpro-Env vectors. The potential role in LV particle production of selected proteins identified by MS analysis including AHNAK was assessed using shRNA gene knockdown technique. Down-regulation of the selected host proteins AHNAK, ALIX, and TSG101 in vector producer cells did not result in a significant difference in vector production.

SUBMITTER: Johnson S

PROVIDER: S-EPMC5910304 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Mass spectrometry analysis reveals differences in the host cell protein species found in pseudotyped lentiviral vectors.

Johnson Sabine S Wheeler Jun X JX Thorpe Robin R Collins Mary M Takeuchi Yasuhiro Y Zhao Yuan Y

Biologicals : journal of the International Association of Biological Standardization 20180201

Lentiviral vectors (LVs) have been successfully used in clinical trials showing long term therapeutic benefits. Studying the role of cellular proteins in lentivirus HIV-1 life cycle can help understand virus assembly and budding, leading to improvement of LV production for gene therapy. Lentiviral vectors were purified using size exclusion chromatography (SEC). The cellular protein composition of LVs produced by two different methods was compared: the transient transfection system pseudotyped wi ...[more]

PMID: 29361371

Similar Datasets

Project description:UnlabelledTo investigate the potential benefits which may arise from pseudotyping the HIV-1 lentiviral vector with its homologous gp41 envelope glycoprotein (GP) cytoplasmic tail (CT), we created chimeric RVG/HIV-1gp41 GPs composed of the extracellular and transmembrane sequences of RVG and either the full-length gp41 CT or C terminus gp41 truncations sequentially removing existing conserved motifs. Lentiviruses (LVs) pseudotyped with the chimeric GPs were evaluated in terms of particle release (physical titer), biological titers, infectivity, and in vivo central nervous system (CNS) transduction. We report here that LVs carrying shorter CTs expressed higher levels of envelope GP and showed a higher average infectivity than those bearing full-length GPs. Interestingly, complete removal of GP CT led to vectors with the highest transduction efficiency. Removal of all C-terminal gp41 CT conserved motifs, leaving just 17 amino acids (aa), appeared to preserve infectivity and resulted in a significantly increased physical titer. Furthermore, incorporation of these 17 aa in the RVG CT notably enhanced the physical titer. In vivo stereotaxic delivery of LV vectors exhibiting the best in vitro titers into rodent striatum facilitated efficient transduction of the CNS at the site of injection. A particular observation was the improved retrograde transduction of neurons in connected distal sites that resulted from the chimeric envelope R5 which included the "Kennedy" sequence (Ken) and lentivirus lytic peptide 2 (LLP2) conserved motifs in the CT, and although it did not exhibit a comparable high titer upon pseudotyping, it led to a significant increase in distal retrograde transduction of neurons.ImportanceIn this study, we have produced novel chimeric envelopes bearing the extracellular domain of rabies fused to the cytoplasmic tail (CT) of gp41 and pseudotyped lentiviral vectors with them. Here we report novel effects on the transduction efficiency and physical titer of these vectors, depending on CT length and context. We also managed to achieve increased neuronal transduction in vivo in the rodent CNS, thus demonstrating that the efficiency of these vectors can be enhanced following merely CT manipulation. We believe that this paper is a novel contribution to the field and opens the way for further attempts to surface engineer lentiviral vectors and make them more amenable for applications in human disease.

Dataset Information

Mass spectrometry analysis reveals differences in the host cell protein species found in pseudotyped lentiviral vectors.

Publications

Mass spectrometry analysis reveals differences in the host cell protein species found in pseudotyped lentiviral vectors.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets