Reduced APOBEC3H variant anti-viral activities are associated with altered RNA binding activities - PubMed (original) (raw)
Reduced APOBEC3H variant anti-viral activities are associated with altered RNA binding activities
Anjie Zhen et al. PLoS One. 2012.
Abstract
APOBEC3H (A3H) is a member of the APOBEC3 family of proteins with varying activities against retroviruses and retrotransposons. The A3H gene contains several single nucleotide polymorphisms and up to seven haplotypes have been detected in humans. Although variations in anti-viral function among A3H haplotypes are not fully understood, only 15N105R-containing A3H variants are known to have potent activities against Vif-deficient HIV-1. Unique motif RLYY(F/Y)W of APOBEC3G (A3G) and APOBEC3F (A3F) required for 7SL RNA binding and HIV-1 incorporation is also conserved in all A3H variants. Like A3G, A3H HapII also demonstrated high binding affinity to host small RNAs such as 7SL and Y RNAs. Mutation of a critical amino acid, W115A resulted in reduced expression level, decreased affinity for 7SL RNA, impairment of virion packaging and reduced anti-viral activity. By comparison, A3H HapI had lower binding affinities to host small RNAs and reduced efficiency of virion incorporation, resulting in significantly reduced anti-viral activity. The SNP ΔN15 commonly found in A3H HapIII and HapIV abolished their abilities to associate with RNAs, and A3H HapIIΔ15N failed to package into HIV-1 virions or exhibited any anti-viral activity. Finally, we showed that A3H variants had distinct cellular localization patterns, which correlated with their different RNA binding affinities. Thus, Pol-III RNA such as 7SL RNA binding is a conserved feature of potent anti-HIV human APOBEC3 cytidine deaminases.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Conservation of the unique RNA binding motif RLYY(F/Y/H)W in A3H variants.
Amino acid sequences from various human AID/APOBEC proteins were aligned using ClustalW2. A unique motif RLYY(F/Y/H)W on the N-terminus of A3G and A3F required for their binding of 7SL RNA and virion incorporation is conserved on all A3H variants.
Figure 2. RNA binding activities of A3G and A3H HapII.
To compare the RNA binding affinities of A3H HapII and A3G, 293T cells were transfected with empty vector, HA-tagged A3H HapII or A3G. Forty-eight hours post-transfection, the cell lysates were collected and immunoprecipitated using anti-HA affinity matrix. The immunoprecipitated RNAs were purified and reverse transcribed. The relative amounts of 7SL, Y RNAs and GAPDH RNA associated with A3G and A3H HapII in comparison to the control were measured by qRT-PCR.
Figure 3. Single point mutation on 115W impairs A3H HapII RNA binding activity and viral packaging.
A) A3H HapII115A showed no anti-viral activity against HIV-1 NL4-3ΔVif compared to A3H HapII. B) A3H HapII115A had reduced protein expression level compared to A3H HapII (compare cell lysates in lanes 2 and 5), but even when expressed at similar levels (compare cell lysates in lanes 3 and 5), HapII 115A lost its ability to be packaged into the virus (viral lysate lane 5). C) A3H HapII115A had impaired ability to bind to 7SL, Y RNAs as well as GAPDH RNA.
Figure 4. A3H HapI showed an impaired ability to package into HIV-1 virions.
To compare anti-viral activity and packaging of A3H HapI and HapII, pNL4-3Δvif was transfected with decreasing doses of A3H HapII and HapI. A) A3H HapI showed significantly reduced anti-viral activity compared to A3H HapII. B) Even when A3H HapI was expressed at a similar level as A3H HapII (compare cell lysates in lanes 3 and 5), it had a reduced ability to be packaged into the virion (compare viral lysates in lanes 3 and 5).
Figure 5. A3H HapI had a reduced ability to associate with 7SL and Y RNAs.
Single mutation G105R restored its RNA binding activity. A) To compare RNA binding activities between A3H HapII and HapI, 293T cells were transfected with HA-tagged A3H HapII and HapI, and the cell lysates were immunoprecipitated using anti-HA affinity matrix agarose beads. Immunoprecipitated RNA were purified and reverse transcribed, and the relative amounts of 7SL, Y RNAs, GAPDH, β-actin and 5s RNA were measured using qRT-PCR. B) Relative RNA binding activities of A3H HapI and HapI105R compared to HapII.
Figure 6. The Δ15N deletion abolished A3H HapII anti-viral activity and virion incorporation.
To compare anti-viral activity and packaging of A3H HapII and HapIIΔ15N, pNL4–3Δvif was transfected with decreasing doses of A3H HapII and HapIIΔ15N. A) A3H HapIIΔ15N had no anti-viral activity compared to A3H HapII. B) Even when expressed at similar levels as A3H HapII (cell lysates in lane 3 and 5), A3H HapIIΔ15N completely lost its ability to be packaged into HIV-1.
Figure 7. A3H HapIIΔ15N has little RNA binding affinity.
A) To compare the RNA binding activity between A3H HapII and HapI, 293T cells were transfected with HA-tagged A3H HapII and HapI, and the cell lystes were immunoprecipitated using anti-HA affinity matrix. Immunoprecipitated RNA were purified and reverse transcribed, and the relative amounts of 7SL, Y RNAs and GAPDH were measured. B) Homology modeling of A3H HapII showing that 15N lies close to the putative RLYYHW RNA binding motif.
Figure 8. Cellular localization of A3H variants.
Hela cells were transfected with various A3H variant constructs, fixed, permeabilized and stained with a mouse anti-HA antibody followed by a secondary Texas Red conjugated anti-mouse antibody. The nucleus was stained with DAPI. The cells were examined by deconvolution microscopy as described in Methods. A series of Z-stack images were acquired with z–step of 0.2 µm and raw image sequences were processed with velocity imaging system and deconvolved. Various cellular localization patterns of the A3H variants were observed. The numbers of cells observed in each location were counted and summarized as percentages of total cells in the table.
References
- Malim MH (2006) Natural resistance to HIV infection: The Vif-APOBEC interaction. C R Biol 329: 871–875. - PubMed
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