Author: admin     Date: May 23, 2022

Lentivirus is a gene vector developed from HIV-1(human immunodeficiency type I virus,80-120 nm), which are mainly composed of lipid membrane, capsid and internal RNA genome. Lentivirus can effectively integrate into the genomes of the host cells, thus enabling the stable expression of its genes.

Currently, the most frequently used methods for measuring lentivirus titer are:

1) transducing units (TU) which is based on infection activity;

2) copy number determination which is based on real-time quantitative PCR (qPCR);

3) analysis of viral p24 concentration which is based on enzyme-linked immunosorbent assay (ELISA).

Transducing units (TU) is a common method of titer determination for lentivirus, indicating the number of bioactive virus within a suspension per milliliter and reflecting the infection ability of lentivirus. qPCR has the advantages of fast speed and good reproducibility, calculating copy number by expanding the viral genome. However, a common concern is that the free RNA would result in overestimation of the viral concentration. p24 is the main structural protein forming the lentivirus capsid (each lentivirus has around 2,000 p24 molecules). By using ELISA, lentivirus particle concentration can be obtained. However, free p24 would also result in inaccuracy of data. These 3 common methods each demonstrate separate aspects of a lentivirus sample, requiring 2-3 techniques to determine the purity of lentivirus samples, which can be time-consuming. Whether lentivirus could transmit genes into host cells has two critical considerations:

1) the expression of VSV-G protein (a vesicular stomatitis virus G glycoprotein), which determines the specific binding to the receptors on the host cells ——"recognition";

2) the virus was successfully loaded with the target gene——"cargo".

Therefore, the expression of surface-specific membrane proteins and the loading of internal nucleic acids are also key points when optimizing the production process of viral vectors.