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. 2021 Jul 26;95(16):e00020-21. doi: 10.1128/JVI.00020-21

FIG 5.

FIG 5

Impaired viral infectivity of HIV-1 progeny from TSLF-CD4-CCR5 cells. (A) Various tree shrew TSLF cell lines were infected at an MOI of 1.0 with replication-competent HIV-1Bal and HIV-1IIIB; at 3 dpi, the supernatant was normalized to 0.3 ng of p24 to infect TZM-bl reporter cells. After 3 days, luciferase activity was detected. (B and C) HOS-CD4-CCR5 and TSLF-CD4-CCR5 cells were infected at an MOI of 1.0 with replication-competent HIV-1Bal (B) and HIV-1IIIB (C); at 3 dpi, the supernatant normalized to 0.3 ng of p24 was used to reinfect HOS-CD4-CCR5, TSLF-CD4-CCR5, TSLF, and TZM-bl cells. At 3 dpi, p24 expression was detected by ELISA. (D) Integrated HIV-1 DNA was detected in the genome of second-round target cells by nested PCR. (E) Tree shrew TSLF and HOS-CD4-CCR5 cells were infected with replication-competent HIV-1IIIB; at 3 dpi, the supernatant was normalized to 0.8 ng of p24-equivalent virus to reinfect cells. (F) TSLF-CD4-CCR5 cells infected with HIV-1IIIB. At 24 h, after washing free virus, TSLF-CD4-CCR5 cells were cocultured with TZM-bl to mimic cell-to-cell infection, with luciferase activity detected at 3 dpi. NC, no virus; PC, HIV-1IIIB. (G) RNA from culture supernatant was extracted and amplified; progeny virus full-length genome was divided into three sections. Target sections were detected by agarose gel electrophoresis and sequenced. Fragment 1 was 2.5 kb, and fragments 2 and 3 were 3.5 kb. (H) TSLF-CD4-CCR5 cells were infected at an MOI of 1.0 with replication-competent HIV-1IIIB. At 3 dpi, viral particles were examined by electron microscopy (three images on left). Viral particles in H9 cells were used as controls (right). (I) Progeny viruses from TSLF-CD4-CCR5 and HOS-CD4-CCR5 cells were used to reinfect C8166 cells. After 4, 8, and 24 h, whole DNA was extracted, and virus products of ssDNA, lateRT, and 2LTR were determined by RT-qPCR. (J) Integrated HIV-1 DNA was detected in the genome of C8166 cells by nested PCR at 36 hpi. Supernatant from TSLF cells, which could not be infected by HIV-1IIIB, is marked as “mock.”