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HIV-1 and HIV-2 exhibit similar mutation frequencies and spectra in the absence of G-to-A hypermutation.

Rawson JM, Landman SR, Reilly CS, Mansky LM - Retrovirology (2015)

Bottom Line: To address this hypothesis, we performed Illumina sequencing of multiple amplicons prepared from cells infected with HIV-1 or HIV-2, resulting in ~4.7 million read pairs and the identification of ~200,000 mutations after data processing.However, the observed differences were primarily due to reduced levels of G-to-A hypermutation for HIV-2.Overall, these data imply that differences in replication fidelity are likely not a major contributing factor to the unique clinical features of HIV-2 infection.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Virology, University of Minnesota, Minneapolis, MN, USA. rawso018@umn.edu.

ABSTRACT

Background: Human immunodeficiency virus type 2 (HIV-2) is often distinguished clinically by lower viral loads, reduced transmissibility, and longer asymptomatic periods than for human immunodeficiency virus type 1 (HIV-1). Differences in the mutation frequencies of HIV-1 and HIV-2 have been hypothesized to contribute to the attenuated progression of HIV-2 observed clinically.

Results: To address this hypothesis, we performed Illumina sequencing of multiple amplicons prepared from cells infected with HIV-1 or HIV-2, resulting in ~4.7 million read pairs and the identification of ~200,000 mutations after data processing. We observed that: (1) HIV-2 displayed significantly lower total mutation, substitution, and transition mutation frequencies than that of HIV-1, along with a mutation spectrum markedly less biased toward G-to-A transitions, (2) G-to-A hypermutation consistent with the activity of APOBEC3 proteins was observed for both HIV-1 and HIV-2 despite the presence of Vif, (3) G-to-A hypermutation was significantly higher for HIV-1 than for HIV-2, and (4) HIV-1 and HIV-2 total mutation frequencies were not significantly different in the absence of G-to-A hypermutants.

Conclusions: Taken together, these data demonstrate that HIV-2 exhibits a distinct mutational spectrum and a lower mutation frequency relative to HIV-1. However, the observed differences were primarily due to reduced levels of G-to-A hypermutation for HIV-2. These findings suggest that HIV-2 may be less susceptible than HIV-1 to APOBEC3-mediated hypermutation, but that the fidelities of other mutational sources (such as reverse transcriptase) are relatively similar for HIV-1 and HIV-2. Overall, these data imply that differences in replication fidelity are likely not a major contributing factor to the unique clinical features of HIV-2 infection.

No MeSH data available.


Related in: MedlinePlus

Experimental strategy for investigating HIV-1 and HIV-2 mutagenesis by Illumina DNA sequencing. Vector virus stocks were produced by co-transfecting 293T cells with HIV-1 or HIV-2 Env-deficient vectors and HIV-1 or HIV-2 CXCR4-tropic Env expression constructs. Virus stocks were concentrated, DNase I-treated to reduce plasmid carryover, and titered in U373-MAGI cells. To prepare samples for Illumina sequencing, 1 × 106 U373-MAGI cells were infected at an MOI of 1.0, generating approximately 1 × 106 proviruses per experimental replicate. This assay represents a single round of viral replication, as producer cells and target cells cannot be re-infected, due to a lack of receptor or Env expression, respectively. Polymerase chain reaction (PCR) of five amplicons (Gag, Vif, HSA, EGFP-1, and EGFP-2) was performed from the proviral DNA. Amplicons from the HIV-1 and HIV-2 proviral DNAs were either identical (HSA, EGFP-1 and 2) or homologous (Gag and Vif) in sequence. The EGFP-1 and EGFP-2 amplicons represent non-overlapping segments of the egfp gene. Sequencing libraries were prepared from the amplicons, pooled in an equimolar fashion to normalize coverage, and subjected to 2 ×150 bp sequencing on the Illumina MiSeq, generating approximately 4.7 million read pairs after data processing.
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Fig1: Experimental strategy for investigating HIV-1 and HIV-2 mutagenesis by Illumina DNA sequencing. Vector virus stocks were produced by co-transfecting 293T cells with HIV-1 or HIV-2 Env-deficient vectors and HIV-1 or HIV-2 CXCR4-tropic Env expression constructs. Virus stocks were concentrated, DNase I-treated to reduce plasmid carryover, and titered in U373-MAGI cells. To prepare samples for Illumina sequencing, 1 × 106 U373-MAGI cells were infected at an MOI of 1.0, generating approximately 1 × 106 proviruses per experimental replicate. This assay represents a single round of viral replication, as producer cells and target cells cannot be re-infected, due to a lack of receptor or Env expression, respectively. Polymerase chain reaction (PCR) of five amplicons (Gag, Vif, HSA, EGFP-1, and EGFP-2) was performed from the proviral DNA. Amplicons from the HIV-1 and HIV-2 proviral DNAs were either identical (HSA, EGFP-1 and 2) or homologous (Gag and Vif) in sequence. The EGFP-1 and EGFP-2 amplicons represent non-overlapping segments of the egfp gene. Sequencing libraries were prepared from the amplicons, pooled in an equimolar fashion to normalize coverage, and subjected to 2 ×150 bp sequencing on the Illumina MiSeq, generating approximately 4.7 million read pairs after data processing.

Mentions: In order to compare mutation frequencies and spectra between HIV-1 and HIV-2, single cycle infections with HIV-1 or HIV-2 were performed at a high MOI (1 million U373-MAGI-X4 cells per replicate infected at an MOI of 1.0, see “Methods”; Figure 1). In this assay, producer cells cannot be re-infected due to a lack of the appropriate receptor and co-receptor, and target cells likewise cannot be re-infected due to disruption of the env genes in the HIV-1 and HIV-2 vectors. Genomic DNA was purified from infected cells and first subjected to quantitative PCR (qPCR) in order to determine the level of plasmid carryover from transfections. Plasmid carryover was quantified either by: (1) determining the plasmid backbone copy number (by measuring the ampicillin resistance gene) and dividing by the proviral copy number, or (2) determining the proviral copy number from heat-inactivated viral infections and dividing by the proviral copy number from un-treated infections (see “Methods”). We found that the level of plasmid carryover for HIV-1 was 0.2% when measured by either method, while the level of carryover was 2.8 or 1.4% for HIV-2, depending on the approach used (Additional file 1: Table S1). The significantly higher level of plasmid carryover for HIV-2 likely reflects the reduced infectivity of HIV-2 viral stocks, which resulted in larger volumes of viral stocks being used during infection. These results are comparable to those obtained in another study [5] and are too low to significantly impact measured mutation frequencies. Next, amplicons were prepared from proviral DNA for Illumina sequencing. In total, 12 samples were analyzed—three experimental replicates each of HIV-1, HIV-2, and HIV-1 and HIV-2 plasmid amplifications as controls to determine levels of background errors. Further, for each sample, five amplicons were prepared (Gag, Vif, HSA, EGFP-1, and EGFP-2), representing a mixture of viral (Gag, Vif) and marker (HSA, EGFP-1, EGFP-2) gene targets. Libraries were prepared individually from samples in order to prevent inter-sample recombination during library construction. Following this, all libraries were pooled and subjected to 2 × 150 paired-end sequencing on the Illumina MiSeq, resulting in ~4.7 million total read pairs after processing, or an average of ~79,000 read pairs/amplicon/sample (Additional file 2: Table S2). After stringent filtering of Illumina data, the mutation frequencies (expressed as mutations per base pair, or m/bp) were determined for all samples, both in terms of total mutations and every possible subdivision (i.e. substitutions, transitions, transversions, etc.). Mutation counts, frequencies, and relative percentages are listed in Additional file 3: Dataset S1, both combined across all five amplicons and separated by amplicon.Figure 1


HIV-1 and HIV-2 exhibit similar mutation frequencies and spectra in the absence of G-to-A hypermutation.

Rawson JM, Landman SR, Reilly CS, Mansky LM - Retrovirology (2015)

Experimental strategy for investigating HIV-1 and HIV-2 mutagenesis by Illumina DNA sequencing. Vector virus stocks were produced by co-transfecting 293T cells with HIV-1 or HIV-2 Env-deficient vectors and HIV-1 or HIV-2 CXCR4-tropic Env expression constructs. Virus stocks were concentrated, DNase I-treated to reduce plasmid carryover, and titered in U373-MAGI cells. To prepare samples for Illumina sequencing, 1 × 106 U373-MAGI cells were infected at an MOI of 1.0, generating approximately 1 × 106 proviruses per experimental replicate. This assay represents a single round of viral replication, as producer cells and target cells cannot be re-infected, due to a lack of receptor or Env expression, respectively. Polymerase chain reaction (PCR) of five amplicons (Gag, Vif, HSA, EGFP-1, and EGFP-2) was performed from the proviral DNA. Amplicons from the HIV-1 and HIV-2 proviral DNAs were either identical (HSA, EGFP-1 and 2) or homologous (Gag and Vif) in sequence. The EGFP-1 and EGFP-2 amplicons represent non-overlapping segments of the egfp gene. Sequencing libraries were prepared from the amplicons, pooled in an equimolar fashion to normalize coverage, and subjected to 2 ×150 bp sequencing on the Illumina MiSeq, generating approximately 4.7 million read pairs after data processing.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4496919&req=5

Fig1: Experimental strategy for investigating HIV-1 and HIV-2 mutagenesis by Illumina DNA sequencing. Vector virus stocks were produced by co-transfecting 293T cells with HIV-1 or HIV-2 Env-deficient vectors and HIV-1 or HIV-2 CXCR4-tropic Env expression constructs. Virus stocks were concentrated, DNase I-treated to reduce plasmid carryover, and titered in U373-MAGI cells. To prepare samples for Illumina sequencing, 1 × 106 U373-MAGI cells were infected at an MOI of 1.0, generating approximately 1 × 106 proviruses per experimental replicate. This assay represents a single round of viral replication, as producer cells and target cells cannot be re-infected, due to a lack of receptor or Env expression, respectively. Polymerase chain reaction (PCR) of five amplicons (Gag, Vif, HSA, EGFP-1, and EGFP-2) was performed from the proviral DNA. Amplicons from the HIV-1 and HIV-2 proviral DNAs were either identical (HSA, EGFP-1 and 2) or homologous (Gag and Vif) in sequence. The EGFP-1 and EGFP-2 amplicons represent non-overlapping segments of the egfp gene. Sequencing libraries were prepared from the amplicons, pooled in an equimolar fashion to normalize coverage, and subjected to 2 ×150 bp sequencing on the Illumina MiSeq, generating approximately 4.7 million read pairs after data processing.
Mentions: In order to compare mutation frequencies and spectra between HIV-1 and HIV-2, single cycle infections with HIV-1 or HIV-2 were performed at a high MOI (1 million U373-MAGI-X4 cells per replicate infected at an MOI of 1.0, see “Methods”; Figure 1). In this assay, producer cells cannot be re-infected due to a lack of the appropriate receptor and co-receptor, and target cells likewise cannot be re-infected due to disruption of the env genes in the HIV-1 and HIV-2 vectors. Genomic DNA was purified from infected cells and first subjected to quantitative PCR (qPCR) in order to determine the level of plasmid carryover from transfections. Plasmid carryover was quantified either by: (1) determining the plasmid backbone copy number (by measuring the ampicillin resistance gene) and dividing by the proviral copy number, or (2) determining the proviral copy number from heat-inactivated viral infections and dividing by the proviral copy number from un-treated infections (see “Methods”). We found that the level of plasmid carryover for HIV-1 was 0.2% when measured by either method, while the level of carryover was 2.8 or 1.4% for HIV-2, depending on the approach used (Additional file 1: Table S1). The significantly higher level of plasmid carryover for HIV-2 likely reflects the reduced infectivity of HIV-2 viral stocks, which resulted in larger volumes of viral stocks being used during infection. These results are comparable to those obtained in another study [5] and are too low to significantly impact measured mutation frequencies. Next, amplicons were prepared from proviral DNA for Illumina sequencing. In total, 12 samples were analyzed—three experimental replicates each of HIV-1, HIV-2, and HIV-1 and HIV-2 plasmid amplifications as controls to determine levels of background errors. Further, for each sample, five amplicons were prepared (Gag, Vif, HSA, EGFP-1, and EGFP-2), representing a mixture of viral (Gag, Vif) and marker (HSA, EGFP-1, EGFP-2) gene targets. Libraries were prepared individually from samples in order to prevent inter-sample recombination during library construction. Following this, all libraries were pooled and subjected to 2 × 150 paired-end sequencing on the Illumina MiSeq, resulting in ~4.7 million total read pairs after processing, or an average of ~79,000 read pairs/amplicon/sample (Additional file 2: Table S2). After stringent filtering of Illumina data, the mutation frequencies (expressed as mutations per base pair, or m/bp) were determined for all samples, both in terms of total mutations and every possible subdivision (i.e. substitutions, transitions, transversions, etc.). Mutation counts, frequencies, and relative percentages are listed in Additional file 3: Dataset S1, both combined across all five amplicons and separated by amplicon.Figure 1

Bottom Line: To address this hypothesis, we performed Illumina sequencing of multiple amplicons prepared from cells infected with HIV-1 or HIV-2, resulting in ~4.7 million read pairs and the identification of ~200,000 mutations after data processing.However, the observed differences were primarily due to reduced levels of G-to-A hypermutation for HIV-2.Overall, these data imply that differences in replication fidelity are likely not a major contributing factor to the unique clinical features of HIV-2 infection.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Virology, University of Minnesota, Minneapolis, MN, USA. rawso018@umn.edu.

ABSTRACT

Background: Human immunodeficiency virus type 2 (HIV-2) is often distinguished clinically by lower viral loads, reduced transmissibility, and longer asymptomatic periods than for human immunodeficiency virus type 1 (HIV-1). Differences in the mutation frequencies of HIV-1 and HIV-2 have been hypothesized to contribute to the attenuated progression of HIV-2 observed clinically.

Results: To address this hypothesis, we performed Illumina sequencing of multiple amplicons prepared from cells infected with HIV-1 or HIV-2, resulting in ~4.7 million read pairs and the identification of ~200,000 mutations after data processing. We observed that: (1) HIV-2 displayed significantly lower total mutation, substitution, and transition mutation frequencies than that of HIV-1, along with a mutation spectrum markedly less biased toward G-to-A transitions, (2) G-to-A hypermutation consistent with the activity of APOBEC3 proteins was observed for both HIV-1 and HIV-2 despite the presence of Vif, (3) G-to-A hypermutation was significantly higher for HIV-1 than for HIV-2, and (4) HIV-1 and HIV-2 total mutation frequencies were not significantly different in the absence of G-to-A hypermutants.

Conclusions: Taken together, these data demonstrate that HIV-2 exhibits a distinct mutational spectrum and a lower mutation frequency relative to HIV-1. However, the observed differences were primarily due to reduced levels of G-to-A hypermutation for HIV-2. These findings suggest that HIV-2 may be less susceptible than HIV-1 to APOBEC3-mediated hypermutation, but that the fidelities of other mutational sources (such as reverse transcriptase) are relatively similar for HIV-1 and HIV-2. Overall, these data imply that differences in replication fidelity are likely not a major contributing factor to the unique clinical features of HIV-2 infection.

No MeSH data available.


Related in: MedlinePlus