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Adeno-associated viral (AAV) vectors are widely used for gene therapy, providing treatment for diseases caused by absent or defective genes. Despite the success of gene therapy, AAV manufacturing is still challenging, with production yields being limited. With increased patient demand, improvements in host cell productivity through various engineering strategies will be necessary. Here, we study the host cell proteome of AAV5-producing HEK293 cells using reversed phase nano-liquid chromatography and tandem mass spectrometry (RPLC-MS/MS). Relative label-free quantitation (LFQ) was performed, allowing a comparison of transfected vs. untransfected cells. Gene ontology enrichment and pathway analysis revealed differential expression of proteins involved in fundamental cellular processes such as metabolism, proliferation, and cell death. Furthermore, changes in expression of proteins involved in endocytosis and lysosomal degradation were observed. Our data provides highly valuable insights into cellular mechanisms involved during recombinant AAV production by HEK293 cells, thus potentially enabling further improvements of gene therapy product manufacturing.

bAPOBEC3 proteins are restriction factors that induce G¡A hypermutation in retroviruses during replication as a result of cytidine deamination of minus-strand DNA transcripts. However, the mechanism of APOBEC inhibition of murine leukemia viruses (MuLVs) does not appear to be G¡A hypermutation and is unclear. In this report, the incorporation of mA3 in virions resulted in a loss in virion reverse transcriptase (RT) activity and RT fidelity that correlated with the loss of virion-specific infectivity.A POBEC3G (hA3G) in humans and APOBEC3 (mA3) in mice are cytidine deaminases that act on single-stranded DNA during reverse transcription, resulting in G¡A hypermutation of newly synthesized proviral DNA (1, 2). Although exogenous murine leukemia viruses (MuLVs) are relatively insensitive to the actions of mA3 (2-7), several studies have reported partial inhibition of exogenous MuLVs after incorporation of mA3 (2,3,6,(8)(9)(10)(11). Furthermore, the finding that Rfv3, a resistance gene for Friend erythroleukemia, encodes mA3 and is responsible for a decreased infectious titer of the Friend MuLV (Fr-MuLV) (11-13) strongly suggests that mA3 inhibits the replication of exogenous MuLVs in vivo. Exogenous ecotropic MuLVs, such as the FrMuLV and Moloney MuLV (Mo-MuLV), are inhibited through mechanisms that do not appear to involve cytidine deamination (2, 9, 10). In this study, the effects of mA3 on the infectivity, reverse transcriptase (RT) activity, and frequency of mutations of the ecotropic MuLV CasFr KP were examined. Virion-associated mA3 suppresses CasFr KP MuLV infectivity. In order to examine the effects of mA3 incorporated into MuLV virions, we derived clonal cell lines infected with CasFr KP (14). The 3T3mA3 cells were derived by transfection of a plasmid encoding the full-length mA3 derived from the BALB/c mouse strain and was tagged at the C termini with hemagglutinin (HA) (5). Infected clonal cell lines were obtained from 3T3 cells as well as from 3T3 cells expressing mA3 (3T3mA3) and were designated 3T3/CasFr KP and 3T3mA3/CasFr KP , respectively. In agreement with earlier reports (2, 7, 9, 13, 15), the clonal cell line expressing mA3 (3T3mA3/CasFr KP ) released virions that had incorporated an easily detectable level of mA3 (Fig. 1).The infectivity of CasFr KP containing mA3 was compared to that of CasFr KP devoid of mA3 by a focal immunofluorescence assay (FIA) (16) and normalized for virion number using the level of p30 CA protein (Fig. 2A). The specific infectivity of virions released from cells expressing mA3 exhibited over a 90% reduction in infectivity (Fig. 2B), corroborating earlier studies of inhibition by mA3 (2,3,6,(8)(9)(10)(11).Decrease in RT activity in virions containing mA3. A recent study examined the efficiency of virion reverse transcription by monitoring the appearance of strong-stop DNA during the course of the RT reaction using virions isolated from C57BL/6 and BALB/c mice as well as those from mA3 knockout (KO) mice (17). Both mouse strains exhibited a similar decrease in RT activity...