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Enrichment of intersubtype HIV-1 recombinants in a dual infection system using HIV-1 strain-specific siRNAs.

Gao Y, Abreha M, Nelson KN, Baird H, Dudley DM, Abraha A, Arts EJ - Retrovirology (2011)

Bottom Line: Using this system, a strong bias is selected for recombination breakpoints in the C2 region, whereas other HIV-1 env regions, most notably the hypervariable regions, were nearly devoid of intersubtype recombination breakpoints.Sequence conservation plays an important role in selecting for recombination breakpoints, but the lack of breakpoints in many conserved env regions suggest that other mechanisms are at play.These findings show that siRNAs can be used as an efficient in vitro tool for enriching recombinants, to facilitate further study on mechanisms of intersubytpe HIV-1 recombination, and to generate replication-competent intersubtype recombinant proteins with a breadth in HIV-1 diversity for future vaccine studies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, 10900 Euclid Ave, Cleveland, Ohio 44106, USA. yxg18@case.edu

ABSTRACT

Background: Intersubtype HIV-1 recombinants in the form of unique or stable circulating recombinants forms (CRFs) are responsible for over 20% of infections in the worldwide epidemic. Mechanisms controlling the generation, selection, and transmission of these intersubtype HIV-1 recombinants still require further investigation. All intersubtype HIV-1 recombinants are generated and evolve from initial dual infections, but are difficult to identify in the human population. In vitro studies provide the most practical system to study mechanisms, but the recombination rates are usually very low in dual infections with primary HIV-1 isolates. This study describes the use of HIV-1 isolate-specific siRNAs to enrich intersubtype HIV-1 recombinants and inhibit the parental HIV-1 isolates from a dual infection.

Results: Following a dual infection with subtype A and D primary HIV-1 isolates and two rounds of siRNA treatment, nearly 100% of replicative virus was resistant to a siRNA specific for an upstream target sequence in the subtype A envelope (env) gene as well as a siRNA specific for a downstream target sequence in the subtype D env gene. Only 20% (10/50) of the replicating virus had nucleotide substitutions in the siRNA-target sequence whereas the remaining 78% (39/50) harbored a recombination breakpoint that removed both siRNA target sequences, and rendered the intersubtype D/A recombinant virus resistant to the dual siRNA treatment. Since siRNAs target the newly transcribed HIV-1 mRNA, the siRNAs only enrich intersubtype env recombinants and do not influence the recombination process during reverse transcription. Using this system, a strong bias is selected for recombination breakpoints in the C2 region, whereas other HIV-1 env regions, most notably the hypervariable regions, were nearly devoid of intersubtype recombination breakpoints. Sequence conservation plays an important role in selecting for recombination breakpoints, but the lack of breakpoints in many conserved env regions suggest that other mechanisms are at play.

Conclusion: These findings show that siRNAs can be used as an efficient in vitro tool for enriching recombinants, to facilitate further study on mechanisms of intersubytpe HIV-1 recombination, and to generate replication-competent intersubtype recombinant proteins with a breadth in HIV-1 diversity for future vaccine studies.

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Schematic illustration on how siRNAs may enrich for intersubtype HIV-1 recombinants. HIV-1 v120-A and v126-D were used for mono- or dual-infection of U87.CD4.CXCR4 cells with equal or different MOI in this study. This figure illustrates how two siRNAs specific for the 5' end of v120-A env and 3' end of v126-D env might enrich for v126-D/v120-A recombinants after an initial dual infection and then propagation with siRNAs. During the initial dual infection (panel A), v120-A and v126-D are produced from monoinfected cells while a co-infected cell can produce a heterodiploid virus particle containing an RNA genome from each virus. If the heterodiploid virus infects and replicates in the initial dually infected cultures, a v120-A/v126-D or v126-D/v120-A recombinant virus can then be produced in the next round of infection but it is likely in lower abundance than the parental viruses. The five general types of virus, produced from initial dual infection, are then used to infect fresh cells treated with siRNAs (Panel B). In this round, siRNA120a primarily inhibits HIV-1 v120-A whereas siRNA126a would inhibit v126-D. In addition, both siRNAs in a single cell would block infection by a heterodiploid virus as well as v120-A/v126-D recombinant because these two types of viruses would be sensitive to one or both siRNAs. Because siRNA120a targets the 5'end of v120-A env gene and siRNA126a targets the 3'end of v126-D gene, a virus resistant to both siRNA would harbor chimeric env genome with 5' end/upstream region from v126-D and a 3' end/downstream region from v120-A. In addition the breakpoint would have to be between the two siRNA target sequences in the env gene.
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Figure 1: Schematic illustration on how siRNAs may enrich for intersubtype HIV-1 recombinants. HIV-1 v120-A and v126-D were used for mono- or dual-infection of U87.CD4.CXCR4 cells with equal or different MOI in this study. This figure illustrates how two siRNAs specific for the 5' end of v120-A env and 3' end of v126-D env might enrich for v126-D/v120-A recombinants after an initial dual infection and then propagation with siRNAs. During the initial dual infection (panel A), v120-A and v126-D are produced from monoinfected cells while a co-infected cell can produce a heterodiploid virus particle containing an RNA genome from each virus. If the heterodiploid virus infects and replicates in the initial dually infected cultures, a v120-A/v126-D or v126-D/v120-A recombinant virus can then be produced in the next round of infection but it is likely in lower abundance than the parental viruses. The five general types of virus, produced from initial dual infection, are then used to infect fresh cells treated with siRNAs (Panel B). In this round, siRNA120a primarily inhibits HIV-1 v120-A whereas siRNA126a would inhibit v126-D. In addition, both siRNAs in a single cell would block infection by a heterodiploid virus as well as v120-A/v126-D recombinant because these two types of viruses would be sensitive to one or both siRNAs. Because siRNA120a targets the 5'end of v120-A env gene and siRNA126a targets the 3'end of v126-D gene, a virus resistant to both siRNA would harbor chimeric env genome with 5' end/upstream region from v126-D and a 3' end/downstream region from v120-A. In addition the breakpoint would have to be between the two siRNA target sequences in the env gene.

Mentions: This study examined the possible influence of siRNA inhibition on HIV-1 replication in a dual infection and how siRNA may be used as a tool to enrich for HIV-1 recombination in specific regions (e.g. env) of the HIV-1 genome. Several specific siRNAs were designed and tested. They include a siRNA120a specifically targeting upstream (C1) of env in virus v120-A, a primary CXCR4-tropic HIV-1 isolate from a subtype A infected Ugandan, and a siRNA126a specifically targeting downstream (gp41) of virus v126-D, a primary CXCR4-tropic HIV-1 isolate from a subtype D infected Ugandan [23]. Theoretically, any RNA containing either target region will be degraded by siRNA120a or siRNA126a; and only recombinants not containing env region upstream of v120-A and env region downstream of v126-D can survive, be propagated, and be enriched (Figure 1).


Enrichment of intersubtype HIV-1 recombinants in a dual infection system using HIV-1 strain-specific siRNAs.

Gao Y, Abreha M, Nelson KN, Baird H, Dudley DM, Abraha A, Arts EJ - Retrovirology (2011)

Schematic illustration on how siRNAs may enrich for intersubtype HIV-1 recombinants. HIV-1 v120-A and v126-D were used for mono- or dual-infection of U87.CD4.CXCR4 cells with equal or different MOI in this study. This figure illustrates how two siRNAs specific for the 5' end of v120-A env and 3' end of v126-D env might enrich for v126-D/v120-A recombinants after an initial dual infection and then propagation with siRNAs. During the initial dual infection (panel A), v120-A and v126-D are produced from monoinfected cells while a co-infected cell can produce a heterodiploid virus particle containing an RNA genome from each virus. If the heterodiploid virus infects and replicates in the initial dually infected cultures, a v120-A/v126-D or v126-D/v120-A recombinant virus can then be produced in the next round of infection but it is likely in lower abundance than the parental viruses. The five general types of virus, produced from initial dual infection, are then used to infect fresh cells treated with siRNAs (Panel B). In this round, siRNA120a primarily inhibits HIV-1 v120-A whereas siRNA126a would inhibit v126-D. In addition, both siRNAs in a single cell would block infection by a heterodiploid virus as well as v120-A/v126-D recombinant because these two types of viruses would be sensitive to one or both siRNAs. Because siRNA120a targets the 5'end of v120-A env gene and siRNA126a targets the 3'end of v126-D gene, a virus resistant to both siRNA would harbor chimeric env genome with 5' end/upstream region from v126-D and a 3' end/downstream region from v120-A. In addition the breakpoint would have to be between the two siRNA target sequences in the env gene.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3025951&req=5

Figure 1: Schematic illustration on how siRNAs may enrich for intersubtype HIV-1 recombinants. HIV-1 v120-A and v126-D were used for mono- or dual-infection of U87.CD4.CXCR4 cells with equal or different MOI in this study. This figure illustrates how two siRNAs specific for the 5' end of v120-A env and 3' end of v126-D env might enrich for v126-D/v120-A recombinants after an initial dual infection and then propagation with siRNAs. During the initial dual infection (panel A), v120-A and v126-D are produced from monoinfected cells while a co-infected cell can produce a heterodiploid virus particle containing an RNA genome from each virus. If the heterodiploid virus infects and replicates in the initial dually infected cultures, a v120-A/v126-D or v126-D/v120-A recombinant virus can then be produced in the next round of infection but it is likely in lower abundance than the parental viruses. The five general types of virus, produced from initial dual infection, are then used to infect fresh cells treated with siRNAs (Panel B). In this round, siRNA120a primarily inhibits HIV-1 v120-A whereas siRNA126a would inhibit v126-D. In addition, both siRNAs in a single cell would block infection by a heterodiploid virus as well as v120-A/v126-D recombinant because these two types of viruses would be sensitive to one or both siRNAs. Because siRNA120a targets the 5'end of v120-A env gene and siRNA126a targets the 3'end of v126-D gene, a virus resistant to both siRNA would harbor chimeric env genome with 5' end/upstream region from v126-D and a 3' end/downstream region from v120-A. In addition the breakpoint would have to be between the two siRNA target sequences in the env gene.
Mentions: This study examined the possible influence of siRNA inhibition on HIV-1 replication in a dual infection and how siRNA may be used as a tool to enrich for HIV-1 recombination in specific regions (e.g. env) of the HIV-1 genome. Several specific siRNAs were designed and tested. They include a siRNA120a specifically targeting upstream (C1) of env in virus v120-A, a primary CXCR4-tropic HIV-1 isolate from a subtype A infected Ugandan, and a siRNA126a specifically targeting downstream (gp41) of virus v126-D, a primary CXCR4-tropic HIV-1 isolate from a subtype D infected Ugandan [23]. Theoretically, any RNA containing either target region will be degraded by siRNA120a or siRNA126a; and only recombinants not containing env region upstream of v120-A and env region downstream of v126-D can survive, be propagated, and be enriched (Figure 1).

Bottom Line: Using this system, a strong bias is selected for recombination breakpoints in the C2 region, whereas other HIV-1 env regions, most notably the hypervariable regions, were nearly devoid of intersubtype recombination breakpoints.Sequence conservation plays an important role in selecting for recombination breakpoints, but the lack of breakpoints in many conserved env regions suggest that other mechanisms are at play.These findings show that siRNAs can be used as an efficient in vitro tool for enriching recombinants, to facilitate further study on mechanisms of intersubytpe HIV-1 recombination, and to generate replication-competent intersubtype recombinant proteins with a breadth in HIV-1 diversity for future vaccine studies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, 10900 Euclid Ave, Cleveland, Ohio 44106, USA. yxg18@case.edu

ABSTRACT

Background: Intersubtype HIV-1 recombinants in the form of unique or stable circulating recombinants forms (CRFs) are responsible for over 20% of infections in the worldwide epidemic. Mechanisms controlling the generation, selection, and transmission of these intersubtype HIV-1 recombinants still require further investigation. All intersubtype HIV-1 recombinants are generated and evolve from initial dual infections, but are difficult to identify in the human population. In vitro studies provide the most practical system to study mechanisms, but the recombination rates are usually very low in dual infections with primary HIV-1 isolates. This study describes the use of HIV-1 isolate-specific siRNAs to enrich intersubtype HIV-1 recombinants and inhibit the parental HIV-1 isolates from a dual infection.

Results: Following a dual infection with subtype A and D primary HIV-1 isolates and two rounds of siRNA treatment, nearly 100% of replicative virus was resistant to a siRNA specific for an upstream target sequence in the subtype A envelope (env) gene as well as a siRNA specific for a downstream target sequence in the subtype D env gene. Only 20% (10/50) of the replicating virus had nucleotide substitutions in the siRNA-target sequence whereas the remaining 78% (39/50) harbored a recombination breakpoint that removed both siRNA target sequences, and rendered the intersubtype D/A recombinant virus resistant to the dual siRNA treatment. Since siRNAs target the newly transcribed HIV-1 mRNA, the siRNAs only enrich intersubtype env recombinants and do not influence the recombination process during reverse transcription. Using this system, a strong bias is selected for recombination breakpoints in the C2 region, whereas other HIV-1 env regions, most notably the hypervariable regions, were nearly devoid of intersubtype recombination breakpoints. Sequence conservation plays an important role in selecting for recombination breakpoints, but the lack of breakpoints in many conserved env regions suggest that other mechanisms are at play.

Conclusion: These findings show that siRNAs can be used as an efficient in vitro tool for enriching recombinants, to facilitate further study on mechanisms of intersubytpe HIV-1 recombination, and to generate replication-competent intersubtype recombinant proteins with a breadth in HIV-1 diversity for future vaccine studies.

Show MeSH
Related in: MedlinePlus