Optimizing HIV-1 Viral Production via a Single Plasmid Transfection System

Optimizing HIV-1 Viral Production via a Single Plasmid Transfection System Objective Results High genetic variability of HIV-1 envelope proteins pose challenges to generating an effective vaccine. By optimizing transfection using a single plasmid, the lab will be able to generate diverse virus stocks more efficiently, accelerating HIV-1 antibody studies and spurring vaccine development. Background • The HIV-1 envelope mediates infection by binding to the CD4 receptor and coreceptors (primarily CCR5 or CXCR4) of susceptible cells. • A panel of CCR5 envelope variants (termed Global Panel or GP) effectively represents the global diversity of HIV-1. • The single hybrid plasmid would contain the proviral backbone of a lab strain of HIV-1 called NL4-3 (X4) and the envelope of a global panel virus (R5). • A coreceptor assay phenotypically confirms if the recombinant virus successfully acquired the global panel envelope that would make it an R5 virus. • Genetic sequencing further indicates the integration of the global panel envelope gene into the hybrid virus genome. Methods Gel of PCR products confirmed the presence of the envelope in hybrid plasmid Amplification of the envelope region by PCR in Figure 1 shows that the envelope sequences of the hybrid plasmids (lanes 4-13) are the same length (3kb) as the envelope sequence of the positive control, NL4-3 (lane 3). Figure 1: Hybrid plasmid global panel envelopes Restriction digest of miniprep products verified that the hybrid plasmid contained the complete HIV-1 genome Figure 2 shows that the hybrid plasmid in lane 8 had both the HIV-1 backbone of NL4-3 and the envelope of a global panel virus. The lengths of the hybrid plasmid’s proviral backbone and envelope are comparable to those of the positive control, wild-type global panel virus 398F1 (lane 4). Figure 2: Restriction digest of miniprep products Coreceptor assay displayed coreceptor tropism of control and hybrid samples Figure 3: Hybrid virus infection in DMEM Restriction digest with enzymes Xho1 and Pf1Ml/Van91I NL4-3 Global Panel Gel electrophoresis to isolate NL4-3 HIV-1 backbone (12kb) and global panel envelope (3kb) Table 1: Global panel virus names Common Name Given Name TRO11 Global Panel 1 CNE55 Global Panel 2 X2278 Global Panel 3 246F3 Global Panel 5 BJOX2000 Global Panel 7 CE1176 Global Panel 8 25710 Global Panel 10 398F1 Global Panel 11 CNE8 Global Panel 12 Figure 4: Coreceptor assay results summary Low RLU (relative light unit) values in Figure 3 indicate that the overall infection rate was low for all hybrid viruses other than Global Panel 12. X4tropic (NL4-3), R5-tropic (YU2, 246F3), and dual tropic (89.6) viruses were used as positive controls to reflect the expected phenotypes. Wells without an active inhibitor had higher RLU, as highlighted in Figure 4. DNA extraction and purification Ligation of hybrid plasmid Transform E. coli Discussion and Conclusions Pick colonies and perform miniprep PCR to amplify the envelope Transfection of HEK-293T cells Sequence comparison Coreceptor assay ATGCGTGC TACGCACG In this experiment, NL4-3 and global panel HIV-1 strains were cut correctly using restriction enzymes Xho1 and Pf1Ml/Van91I (see Methods). Successful transformation of E. coli with the hybrid plasmids demonstrated that the HIV-1 backbones of NL4-3 properly ligated to the global panel envelopes. The coreceptor assay revealed that a functioning hybrid virus containing the Global Panel 12 envelope was created (Figure 4). The remaining hybrid viruses were weak and failed to induce infection (Figure 3). This was most likely due to poor selection of colonies and lowered DNA concentrations following miniprep. Sequencing results were generally inconclusive. Various experimental strains came back >90% positive for the Global Panel 1 virus genome. Cross contamination most likely occurred during PCR. To optimize future production of single plasmid transfection systems, try increasing the strength of ampicillin on LB plates and maintaining higher plasmid DNA concentrations through miniprep. To reduce chances of contamination, consider working with one sample of global panel virus at a time.