KHNYN is essential for the zinc finger antiviral protein (ZAP) to restrict HIV-1 containing clustered CpG dinucleotides
- King’s College London, United Kingdom
- University College London, United Kingdom
Figures
Figure 1
KHNYN is a ZAP-interacting factor identified by yeast two-hybrid screening.
A yeast two-hybrid screen for ZAP-S and ZAP-L interacting factors identified a region in KHNYN-1 and KHNYN-2. The selected interaction domain (SID) is the amino acid sequence shared by all prey …
Figure 2 with 1 supplement
KHNYN interacts with ZAP and selectively inhibits HIV-1 containing clustered CpG dinucleotides.
(A) Lysates of HEK293T cells transfected either with pGFP-FLAG, pKHNYN-1-FLAG or pKHNYN-2-FLAG were immunoprecipitated with an anti-ZAP antibody. Post-nuclear supernatants and immunoprecipitation …
Figure 2—figure supplement 1
HIV-1EnvCpG86-561 contains 36 introduced CpG dinucleotides.
MacVector ClustalW alignment of nucleotides 1–600 of env from wild-type HIV-1 and HIV-1EnvCpG86-561. CpG dinucleotides introduced through synonymous mutations are highlighted in red.
Figure 3
ZAP is required for KHNYN to inhibit infectious virion production for HIV-1 with clustered CpG dinucleotides.
(A) ZAP, TRIM25 and Actin expression in HeLa cells, HeLa Control CRISPR cells (expressing a guide RNA targeting the firefly luciferase gene), HeLa ZAP CRISPR guide 1 (ZAP-G1) cells and HeLa TRIM25 …
Figure 4
ZAP is required for KHNYN to inhibit genomic RNA abundance for HIV-1 with clustered CpG dinucleotides.
(A–C) HeLa Control CRISPR cells or ZAP-G1 CRISPR cells were transfected with 500 ng pHIV-1 or pHIV-1EnvCpG86-561 and 500 ng of pGFP-FLAG or 62.5 ng pKHNYN-1/2-FLAG plus 437.5 ng of pGFP-FLAG. …
Figure 5
TRIM25 is required for KHNYN to inhibit HIV-1 with clustered CpG dinucleotides.
(A–B) HeLa Control CRISPR cells or TRIM25-G1 CRISPR cells were transfected with 500 ng pHIV-1 or pHIV-1EnvCpG86-561 and 500 ng of pGFP-FLAG or 62.5 ng pKHNYN-1-FLAG or pKHNYN-2-FLAG plus 437.5 ng of …
Figure 6 with 2 supplements
The KH and NYN domains are necessary for KHNYN antiviral activity.
(A) Schematic of KHNYN-1 and KHNYN-2 domains and mutations in the NYN domain. (B–C) HeLa cells were transfected with 500 ng pHIV-1 or pHIV-1EnvCpG86-561 and either 500 ng of pGFP-FLAG or 62.5 ng …
Figure 6—figure supplement 1
KHNYN mutant proteins localize to the cytoplasm.
Panels show representative fields for the localization of KHNYN-1/2-FLAG wild type or mutant proteins in 293T Control CRISPR cells. Cells were stained with an anti-FLAG antibody (red) and DAPI …
Figure 6—figure supplement 2
NYN domain alignment.
(A) MacVector ClustalW alignment of the NYN domains from KHNYN and ZC3H12A. The amino acid identity and similarity is shown. The four aspartic acid residues that coordinate the magnesium ion in the …
Figure 7 with 3 supplements
KHNYN depletion increases infectious virus production for HIV-1 with clustered CpG dinucleotides and MLV Gag expression.
(A) 100 ng pKHNYN-1-FLAG, pKHNYN-2-FLAG, pKHNYN-1-FLAG with a mutation in the PAM that prevents it from being targeted by KHNYN guide 1 (pKHNYN-1-CRG1-FLAG) or pKHNYN-2-CRG1-FLAG plus 400 ng of pGFP …
Figure 7—figure supplement 1
KHNYN is necessary for CpG dinucleotides to inhibit HIV-1 infectious virus production.
(A) 100 ng pKHNYN-1-FLAG, pKHNYN-2-FLAG, pKHNYN-1-FLAG with a mutation in the PAM that prevents it from being targeted by KHNYN guide 2 (pKHNYN-1-CRG2-FLAG) or pKHNYN-2-CRG2-FLAG plus 400 ng pGFP …
Figure 7—figure supplement 2
CRISPR-Cas9-induced mutations in KHNYN.
(A–B) The nucleotide sequence surrounding the guide RNA sequence for KHNYN guide 1 (A) and KHNYN guide 2 (B). The sequence in the parental cells not transduced with lentiCRISPRv2 (untransduced) as …
Figure 7—figure supplement 3
KHNYN depletion does not substantially increase Sindbis virus replication.
(A–B) HeLa Control CRISPR cells, ZAP-G1 CRISPR cells, TRIM25 CRISPR cells and KHNYN-G1 CRISPR clone B were infected with Sindbis virus and the amount of infectious virus was measured in the cell …
Figure 8
KHNYN is necessary for CpG dinucleotides to inhibit HIV-1 genomic RNA expression.
(A–C) HeLa Control CRISPR cells, ZAP-G1 CRISPR cells, KHNYN-G1 CRISPR bulk cells or two independent clones were transfected with 500 ng pHIV-1 or pHIV-1EnvCpG86-561 and 500 ng pGFP-FLAG. RNA was …
Figure 9
Deletion of the KH-like domain or mutations in the NYN domain in KHNYN prevent reconstitution of CpG-dependent inhibition of HIV-1 infectious virus production in KHNYN CRISPR cells.
(A–B) HeLa KHNYN-G1 CRISPR clone B cells were transfected with 500 ng pHIV-1 or pHIV-1EnvCpG86-561 and 500 ng of pGFP-FLAG or 31.25 ng, 62.5 ng, 125 ng, 250 ng or 500 ng of pKHNYN-1-CRG1-FLAG or …
Additional files
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Supplementary file 1
Key resources table.
- https://doi.org/10.7554/eLife.46767.018
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Transparent reporting form
- https://doi.org/10.7554/eLife.46767.019
