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Adeno-associated virus (AAV) producer cell lines represent an effective method for large-scale production of AAV vectors. We set out to evaluate and characterize the use of an abbreviated protocol to generate "masterwells" (MWs; a nonclonal cell population) as a platform for research and preclinical vector production. In this system, a single plasmid containing three components, the vector sequence, the AAV rep, and cap genes, and a selectable marker gene is stably transfected into HeLaS3 cells. Producer cell lines generating an AAV2 vector expressing a secreted form of human placental alkaline phosphatase (SEAP) have been created. Several MWs showed vector yields in the 5×10(4) to 2×10(5) DNase-resistant particles/cell range, and the productivity was stable over >60 population doublings. Integrated plasmid copy number in three high-producing MWs ranged from approximately 12 to 50; copies were arranged in a head-to-tail configuration. Upon infection with adenovirus, rep/cap copy number was amplified approximately 100-fold and high yield appeared to be dependent on the extent of amplification. Rep/cap gene expression and vector packaging both reached a peak at 48 hr postinfection. AAV2-SEAP vector was produced in 1-liter shaker culture and purified for assessment of vector quality and potency. The data showed that the majority of the capsids from the MWs contained vector DNA (≥70%) and that purified vector was free of replication-competent AAV. In vitro and in vivo analyses demonstrated that potency of the producer cell-derived vector was comparable to vector generated via the standard transfection method.

The advent of modern molecular biology has allowed for the discovery of several mechanisms by which oncoviruses promote carcinogenesis. Remarkably, nearly all human oncogenic viruses increase levels of the transcription factor hypoxia-inducible factor 1 (HIF-1). In this review, we highlight HIF-1׳s significance in viral oncogenesis, while providing an in-depth analysis of its activation mechanisms by the following oncoviruses: human papillomaviruses (HPVs), hepatitis B/C viruses (HBV/HCVs), Epstein-Barr virus (EBV), Kaposi׳s sarcoma-associated herpes virus (KSHV), and human T-cell lymphotropic virus (HTLV-1). We discuss virus-induced HIF-1׳s role in transcriptional upregulation of metabolic, angiogenic, and microenvironmental factors that are integral for oncogenesis. Admittedly, conclusive evidence is lacking as to whether activation of HIF-1 target genes is necessary for malignant transformation or merely a result thereof. In addition, a complete understanding of host-virus interactions, the effect of viral genomic variation, and the clinical (and potential therapeutic) relevance of HIF-1 in viral oncogenesis warrant further investigation.

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