PRJNA170908
Human cells infected with HIV Genome Sequencing
TNPO3, a karyopherin specific for serine/arginine (SR)-rich splicing factors, increases the efficiency of transduction by HIV-1 and most lentiviruses, but not by MLV. Chimeric vectors generated by exchanging HIV-1 and MLV components and functional assessment of HIV-1 capsid mutant viruses showed capsid to be the viral determinant of TNPO3-dependence. CPSF6-358 is a truncated SR-protein lacking the putative nuclear localization signal that inhibits HIV-1 replication in a capsid-dependent manner. 27 HIV-1 capsid substitution mutants were tested for sensitivity to inhibition by CPSF6-358. The effect of the capsid mutants on sensitivity to inhibition by CPSF6-358 correlates with sensitivity to TNPO3 knockdown of the same panel of viruses. To determine if HIV-1 inhibition by TNPO3 knockdown (KD) results from CPSF6 accumulation in the cytoplasm, CPSF6-358 and full-length CPSF6 were expressed as fusions with the SV40 large T antigen nuclear localization signal (NLS) or with the protein kinase inhibitor nuclear export signal (NES). CPSF6-358-NLS did not inhibit HIV-1 transduction whereas CPSF6-NES did. When CPSF6 was depleted, TNPO3 KD no longer inhibited HIV-1 transduction. Rescue of the CPSF6 KD with non-targetable CPSF6-NLS did not restore the inhibitory effect of TNPO3 KD. Biochemical experiments indicated that the accumulation of CPSF6 in the cytoplasm, whether by TNPO3 KD or by the CPSF6-358 truncation, resulted in abnormally stable capsids. Our results demonstrate that the effect of TNPO3 KD on HIV-1 replication results from effects on the subcellular localization of capsid-binding protein CPSF6.