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Human APOBEC3 induced mutation of human immunodeficiency virus type-1 contributes to adaptation and evolution in natural infection.

Kim EY, Lorenzo-Redondo R, Little SJ, Chung YS, Phalora PK, Maljkovic Berry I, Archer J, Penugonda S, Fischer W, Richman DD, Bhattacharya T, Malim MH, Wolinsky SM - PLoS Pathog. (2014)

Bottom Line: Human APOBEC3 proteins have additionally been proposed to induce infrequent, potentially non-lethal G-to-A mutations that make subtle contributions to sequence diversification of the viral genome and adaptation though acquisition of beneficial mutations.Viral substitutions were highest in the preferred trinucleotide contexts of the edited sites for the APOBEC3 deaminases.Consistent with the effects of immune selection, amino acid changes accumulated at the APOBEC3 editing contexts located within human leukocyte antigen (HLA)-appropriate epitopes that are known or predicted to enable peptide binding.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America.

ABSTRACT
Human APOBEC3 proteins are cytidine deaminases that contribute broadly to innate immunity through the control of exogenous retrovirus replication and endogenous retroelement retrotransposition. As an intrinsic antiretroviral defense mechanism, APOBEC3 proteins induce extensive guanosine-to-adenosine (G-to-A) mutagenesis and inhibit synthesis of nascent human immunodeficiency virus-type 1 (HIV-1) cDNA. Human APOBEC3 proteins have additionally been proposed to induce infrequent, potentially non-lethal G-to-A mutations that make subtle contributions to sequence diversification of the viral genome and adaptation though acquisition of beneficial mutations. Using single-cycle HIV-1 infections in culture and highly parallel DNA sequencing, we defined trinucleotide contexts of the edited sites for APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H. We then compared these APOBEC3 editing contexts with the patterns of G-to-A mutations in HIV-1 DNA in cells obtained sequentially from ten patients with primary HIV-1 infection. Viral substitutions were highest in the preferred trinucleotide contexts of the edited sites for the APOBEC3 deaminases. Consistent with the effects of immune selection, amino acid changes accumulated at the APOBEC3 editing contexts located within human leukocyte antigen (HLA)-appropriate epitopes that are known or predicted to enable peptide binding. Thus, APOBEC3 activity may induce mutations that influence the genetic diversity and adaptation of the HIV-1 population in natural infection.

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Site-specific editing frequencies in infected cells from single-cycle APOBEC3 titration transfection experiments.The single-cycle substitution rate for HIV-1 in the absence of human APOBEC3 was 8.6×10−4 mutations per nucleotide, whereas the mean single-cycle substitution rate for HIV-1 in the presence of human APOBEC3 ranged from about 1×10−3 to 2×10−2 per nucleotide substitution (A). The frequency of substitutions increased significantly in the region of the Gag gene of HIV-1 we sequenced in accord with increasing concentrations of the APOBEC3 proteins. The maximum single-cycle substitution rate for HIV-1 was 2×10−3 substitutions per site in the presence of APOBEC3D, 1.4×10−2 substitutions per site in the presence of APOBEC3F, 2.7×10−2 substitutions per site in the presence of APOBEC3G and 1.1×10−4 substitutions per site in the presence of APOBEC3H. The concentration of APOBEC3 at which we observed half of the estimated maximum substitution rate was 0.02 for APOBEC3D, 2.09 for APOBEC3F, 0.23 for APOBEC3G, and 0.22 µg for APOBEC3H. The single-cycle substitution rate for each mutation type (transition = Ts or transversion = Tv) of HIV-1 in the titration transfection experiments differed by 1.2 order of magnitude (B). For each of the human APOBEC3 proteins, we show the positions in the Gag gene of HIV-1 where the frequency of G-to-A mutation increased with increasing amounts of human APOBEC3 protein (Spearman rank correlation coefficient, P-value<0.05) (C). The G-to-A mutations are shown in a number of contiguous nucleotide sequence editing contexts. We used a sliding window to deduce the base frequency of G-to-A mutations (in each contiguous nucleotide context of the edited sites for each APOBEC3 protein (APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H) using the total G-to-A mutation frequency at increasing concentration. Positions with a non-significant increase in G-to-A mutations were excluded from the calculations.
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ppat-1004281-g001: Site-specific editing frequencies in infected cells from single-cycle APOBEC3 titration transfection experiments.The single-cycle substitution rate for HIV-1 in the absence of human APOBEC3 was 8.6×10−4 mutations per nucleotide, whereas the mean single-cycle substitution rate for HIV-1 in the presence of human APOBEC3 ranged from about 1×10−3 to 2×10−2 per nucleotide substitution (A). The frequency of substitutions increased significantly in the region of the Gag gene of HIV-1 we sequenced in accord with increasing concentrations of the APOBEC3 proteins. The maximum single-cycle substitution rate for HIV-1 was 2×10−3 substitutions per site in the presence of APOBEC3D, 1.4×10−2 substitutions per site in the presence of APOBEC3F, 2.7×10−2 substitutions per site in the presence of APOBEC3G and 1.1×10−4 substitutions per site in the presence of APOBEC3H. The concentration of APOBEC3 at which we observed half of the estimated maximum substitution rate was 0.02 for APOBEC3D, 2.09 for APOBEC3F, 0.23 for APOBEC3G, and 0.22 µg for APOBEC3H. The single-cycle substitution rate for each mutation type (transition = Ts or transversion = Tv) of HIV-1 in the titration transfection experiments differed by 1.2 order of magnitude (B). For each of the human APOBEC3 proteins, we show the positions in the Gag gene of HIV-1 where the frequency of G-to-A mutation increased with increasing amounts of human APOBEC3 protein (Spearman rank correlation coefficient, P-value<0.05) (C). The G-to-A mutations are shown in a number of contiguous nucleotide sequence editing contexts. We used a sliding window to deduce the base frequency of G-to-A mutations (in each contiguous nucleotide context of the edited sites for each APOBEC3 protein (APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H) using the total G-to-A mutation frequency at increasing concentration. Positions with a non-significant increase in G-to-A mutations were excluded from the calculations.

Mentions: We screened for mutations in HIV-1 nascent retroviral cDNA with high throughput 454 pyrosequencing using a statistical framework to improve measurement accuracy. Barcoded oligonucleotide primers with unique molecular identifiers were used to amplify a PCR pool that was then sequenced with sufficient depth of coverage to redundantly cover the chosen portion of the viral genome. By stringent filtering and correcting the raw sequencing data, the platform efficiently reduces most sequencing errors generated during pyrosequencing [31]. We separated the resulting viral sequences by sample using the index sequence. Sequence alignments made to the HIV-1 pIIIB/Δvif reference sequence were optimized to reduce alignment errors introduced by insertions-deletions (indels) associated with the pyrosequencing chemistry, correct for incomplete extension miscalls, and filter out less abundant sequencing reads. We used a statistical analytical framework to filter the data for error correction and then built the haplotypes present in the viral populations. With this approach, we found a significantly lower average mutation frequency than the typical analysis of these data (average of 8.45×10−4) (Figure 1A), consistent with the estimations of others [2]. The nucleotide substitution rate for each mutation type (transition or transversion) differed by 1.2 orders of magnitude (Figure 1B). The different rates of nucleotide substitution likely reflect viral RT and RNA polymerase II fidelity and the asymmetrical substitution bias for faster accumulation of G-to-A mutations, the expected result of APOBEC3 editing. Random PCR amplification bias did not affect the reliability of the measurements [32].


Human APOBEC3 induced mutation of human immunodeficiency virus type-1 contributes to adaptation and evolution in natural infection.

Kim EY, Lorenzo-Redondo R, Little SJ, Chung YS, Phalora PK, Maljkovic Berry I, Archer J, Penugonda S, Fischer W, Richman DD, Bhattacharya T, Malim MH, Wolinsky SM - PLoS Pathog. (2014)

Site-specific editing frequencies in infected cells from single-cycle APOBEC3 titration transfection experiments.The single-cycle substitution rate for HIV-1 in the absence of human APOBEC3 was 8.6×10−4 mutations per nucleotide, whereas the mean single-cycle substitution rate for HIV-1 in the presence of human APOBEC3 ranged from about 1×10−3 to 2×10−2 per nucleotide substitution (A). The frequency of substitutions increased significantly in the region of the Gag gene of HIV-1 we sequenced in accord with increasing concentrations of the APOBEC3 proteins. The maximum single-cycle substitution rate for HIV-1 was 2×10−3 substitutions per site in the presence of APOBEC3D, 1.4×10−2 substitutions per site in the presence of APOBEC3F, 2.7×10−2 substitutions per site in the presence of APOBEC3G and 1.1×10−4 substitutions per site in the presence of APOBEC3H. The concentration of APOBEC3 at which we observed half of the estimated maximum substitution rate was 0.02 for APOBEC3D, 2.09 for APOBEC3F, 0.23 for APOBEC3G, and 0.22 µg for APOBEC3H. The single-cycle substitution rate for each mutation type (transition = Ts or transversion = Tv) of HIV-1 in the titration transfection experiments differed by 1.2 order of magnitude (B). For each of the human APOBEC3 proteins, we show the positions in the Gag gene of HIV-1 where the frequency of G-to-A mutation increased with increasing amounts of human APOBEC3 protein (Spearman rank correlation coefficient, P-value<0.05) (C). The G-to-A mutations are shown in a number of contiguous nucleotide sequence editing contexts. We used a sliding window to deduce the base frequency of G-to-A mutations (in each contiguous nucleotide context of the edited sites for each APOBEC3 protein (APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H) using the total G-to-A mutation frequency at increasing concentration. Positions with a non-significant increase in G-to-A mutations were excluded from the calculations.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4117599&req=5

ppat-1004281-g001: Site-specific editing frequencies in infected cells from single-cycle APOBEC3 titration transfection experiments.The single-cycle substitution rate for HIV-1 in the absence of human APOBEC3 was 8.6×10−4 mutations per nucleotide, whereas the mean single-cycle substitution rate for HIV-1 in the presence of human APOBEC3 ranged from about 1×10−3 to 2×10−2 per nucleotide substitution (A). The frequency of substitutions increased significantly in the region of the Gag gene of HIV-1 we sequenced in accord with increasing concentrations of the APOBEC3 proteins. The maximum single-cycle substitution rate for HIV-1 was 2×10−3 substitutions per site in the presence of APOBEC3D, 1.4×10−2 substitutions per site in the presence of APOBEC3F, 2.7×10−2 substitutions per site in the presence of APOBEC3G and 1.1×10−4 substitutions per site in the presence of APOBEC3H. The concentration of APOBEC3 at which we observed half of the estimated maximum substitution rate was 0.02 for APOBEC3D, 2.09 for APOBEC3F, 0.23 for APOBEC3G, and 0.22 µg for APOBEC3H. The single-cycle substitution rate for each mutation type (transition = Ts or transversion = Tv) of HIV-1 in the titration transfection experiments differed by 1.2 order of magnitude (B). For each of the human APOBEC3 proteins, we show the positions in the Gag gene of HIV-1 where the frequency of G-to-A mutation increased with increasing amounts of human APOBEC3 protein (Spearman rank correlation coefficient, P-value<0.05) (C). The G-to-A mutations are shown in a number of contiguous nucleotide sequence editing contexts. We used a sliding window to deduce the base frequency of G-to-A mutations (in each contiguous nucleotide context of the edited sites for each APOBEC3 protein (APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H) using the total G-to-A mutation frequency at increasing concentration. Positions with a non-significant increase in G-to-A mutations were excluded from the calculations.
Mentions: We screened for mutations in HIV-1 nascent retroviral cDNA with high throughput 454 pyrosequencing using a statistical framework to improve measurement accuracy. Barcoded oligonucleotide primers with unique molecular identifiers were used to amplify a PCR pool that was then sequenced with sufficient depth of coverage to redundantly cover the chosen portion of the viral genome. By stringent filtering and correcting the raw sequencing data, the platform efficiently reduces most sequencing errors generated during pyrosequencing [31]. We separated the resulting viral sequences by sample using the index sequence. Sequence alignments made to the HIV-1 pIIIB/Δvif reference sequence were optimized to reduce alignment errors introduced by insertions-deletions (indels) associated with the pyrosequencing chemistry, correct for incomplete extension miscalls, and filter out less abundant sequencing reads. We used a statistical analytical framework to filter the data for error correction and then built the haplotypes present in the viral populations. With this approach, we found a significantly lower average mutation frequency than the typical analysis of these data (average of 8.45×10−4) (Figure 1A), consistent with the estimations of others [2]. The nucleotide substitution rate for each mutation type (transition or transversion) differed by 1.2 orders of magnitude (Figure 1B). The different rates of nucleotide substitution likely reflect viral RT and RNA polymerase II fidelity and the asymmetrical substitution bias for faster accumulation of G-to-A mutations, the expected result of APOBEC3 editing. Random PCR amplification bias did not affect the reliability of the measurements [32].

Bottom Line: Human APOBEC3 proteins have additionally been proposed to induce infrequent, potentially non-lethal G-to-A mutations that make subtle contributions to sequence diversification of the viral genome and adaptation though acquisition of beneficial mutations.Viral substitutions were highest in the preferred trinucleotide contexts of the edited sites for the APOBEC3 deaminases.Consistent with the effects of immune selection, amino acid changes accumulated at the APOBEC3 editing contexts located within human leukocyte antigen (HLA)-appropriate epitopes that are known or predicted to enable peptide binding.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America.

ABSTRACT
Human APOBEC3 proteins are cytidine deaminases that contribute broadly to innate immunity through the control of exogenous retrovirus replication and endogenous retroelement retrotransposition. As an intrinsic antiretroviral defense mechanism, APOBEC3 proteins induce extensive guanosine-to-adenosine (G-to-A) mutagenesis and inhibit synthesis of nascent human immunodeficiency virus-type 1 (HIV-1) cDNA. Human APOBEC3 proteins have additionally been proposed to induce infrequent, potentially non-lethal G-to-A mutations that make subtle contributions to sequence diversification of the viral genome and adaptation though acquisition of beneficial mutations. Using single-cycle HIV-1 infections in culture and highly parallel DNA sequencing, we defined trinucleotide contexts of the edited sites for APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H. We then compared these APOBEC3 editing contexts with the patterns of G-to-A mutations in HIV-1 DNA in cells obtained sequentially from ten patients with primary HIV-1 infection. Viral substitutions were highest in the preferred trinucleotide contexts of the edited sites for the APOBEC3 deaminases. Consistent with the effects of immune selection, amino acid changes accumulated at the APOBEC3 editing contexts located within human leukocyte antigen (HLA)-appropriate epitopes that are known or predicted to enable peptide binding. Thus, APOBEC3 activity may induce mutations that influence the genetic diversity and adaptation of the HIV-1 population in natural infection.

Show MeSH
Related in: MedlinePlus