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Characterization of two distinct early post-entry blocks to HIV-1 in common marmoset lymphocytes

View Article: PubMed Central - PubMed

ABSTRACT

In nature, primate lentiviruses infect humans and several Old World monkeys and apes. However, to date, lentiviruses infecting New World monkeys have not been described. We studied the susceptibility of common marmoset cells to HIV-1 infection and observed the presence of post-entry blocks to the early phase of HIV-1 infection in peripheral blood lymphocytes (PBLs) and a B lymphocytic cell line (B-LCL). The blocks present in these cells are dominant and phenotypically different from each other. In PBLs, the block occurs at the level of reverse transcription, reducing the accumulation of early and late transcripts, similar to the block imposed by TRIM5α. However, we have found that marmoset TRIM5α does not block HIV-1. In contrast, the restriction factor present in B-LCLs blocks HIV-1 replication at a later step, after nuclear entry, and inhibits integration. Additionally, we have identified an HIV-1 capsid mutant, N74D, that is able to escape the restriction in the marmoset B-LCLs. Our results suggest that the factors responsible for the blocks present in marmoset PBLs and B-LCLs are different. We propose the existence of at least two new restriction factors able to block HIV-1 infection in marmoset lymphocytes.

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The post-entry block(s) to HIV-1 in marmoset PBLs and B-LCLs are dominant.(A) Heterokaryons or homokaryons of human (hu) and marmoset (mar) PBLs were produced by treatment with PEG. As a control, mixtures of human and marmoset cells were treated the same way but without adding PEG. The fused and non-fused cell mixtures were challenged with HIV-1 luciferase reporter viruses pseudotyped with VSV-G. Forty-eight hours later, the infectivity of the viruses was determined by measuring the relative luciferase activity in the cells. The infectivity in the human-human homokaryons is normalized to 100%, and the relative infectivity in the other homo-heterokaryons is shown. The results of four (+PEG) and two (no PEG) independent experiments and their means are shown. (B) Human and marmoset B-LCLs were fused with PEG to generate hetero- or homokaryons. As a control, mixtures of human and marmoset cells were treated the same way but without adding PEG. The fused and non-fused cell mixtures were challenged with HIV-1 luciferase reporter viruses pseudotyped with VSV-G. Forty-eight hours later, the infectivity of the viruses was determined by measuring the relative luciferase activity in the cells. The infectivity in the human-human homokaryons is normalized to 100%, and the relative infectivity in the other homo-heterokaryons is shown. The results of three independent experiments and their means are shown. Reported p-values (2-tailed) were obtained with an unpaired t-test.
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f3: The post-entry block(s) to HIV-1 in marmoset PBLs and B-LCLs are dominant.(A) Heterokaryons or homokaryons of human (hu) and marmoset (mar) PBLs were produced by treatment with PEG. As a control, mixtures of human and marmoset cells were treated the same way but without adding PEG. The fused and non-fused cell mixtures were challenged with HIV-1 luciferase reporter viruses pseudotyped with VSV-G. Forty-eight hours later, the infectivity of the viruses was determined by measuring the relative luciferase activity in the cells. The infectivity in the human-human homokaryons is normalized to 100%, and the relative infectivity in the other homo-heterokaryons is shown. The results of four (+PEG) and two (no PEG) independent experiments and their means are shown. (B) Human and marmoset B-LCLs were fused with PEG to generate hetero- or homokaryons. As a control, mixtures of human and marmoset cells were treated the same way but without adding PEG. The fused and non-fused cell mixtures were challenged with HIV-1 luciferase reporter viruses pseudotyped with VSV-G. Forty-eight hours later, the infectivity of the viruses was determined by measuring the relative luciferase activity in the cells. The infectivity in the human-human homokaryons is normalized to 100%, and the relative infectivity in the other homo-heterokaryons is shown. The results of three independent experiments and their means are shown. Reported p-values (2-tailed) were obtained with an unpaired t-test.

Mentions: To determine whether the block to HIV-1 infection in marmoset PBLs was due to the presence of a dominant factor and not to a deficiency in a factor needed for HIV-1 replication, we prepared heterokaryons and homokaryons of restricted and unrestricted PBLs using polyethylene glycol (PEG). As a control, we also prepared mixtures of human and marmoset PBLs that were not treated with PEG in parallel, and tested the infectivity of HIV-1 in the PEG-fused and non-fused cell mixtures. If the block were due to the presence of a dominant factor, we would expect to observe low HIV-1 infectivity in heterokaryons formed between restricted (marmoset) and unrestricted (human) cells. On the contrary, if the block were due to the absence of a factor needed for HIV-1 infection, we would expect to see HIV-1 infectivity at levels close to those seen in unrestricted cells or in the non-fused human-marmoset cell mixtures. As shown in Fig. 3A, HIV-1 infectivity was reduced in heterokaryons formed between human and marmoset PBLs by 2.6-fold compared to human-human homokaryons. We did not observe a significant reduction in infectivity in the non-fused human-marmoset cell mixtures (Fig. 3A, right). These results suggest that the early block to HIV-1 present in marmoset PBLs is most likely due to the presence of a dominant-acting factor, as is the case of the block present in rhesus cells22. The block in human-marmoset heterokaryons was not as pronounced as in marmoset-marmoset homokaryons, which showed a 12-fold lower infectivity compared to human-human homokaryons. This effect can be attributed to the formation of some human-human homokaryons and/or to incomplete cell-cell fusion. We have not been able to assess the efficiency of heterokaryon formation in our infection experiments because treatment of the cells with dyes that would allow this measurement resulted in background levels of infectivity in the PEG-treated cells. However, in pilot experiments using Vibrant DiO and Dil cell labeling solutions (Molecular Probes) to evaluate cell-cell fusion, the efficiency of heterokaryon formation ranged from 60% to 80%. Thus, it is likely that most cells have fused under the conditions of our experiment.


Characterization of two distinct early post-entry blocks to HIV-1 in common marmoset lymphocytes
The post-entry block(s) to HIV-1 in marmoset PBLs and B-LCLs are dominant.(A) Heterokaryons or homokaryons of human (hu) and marmoset (mar) PBLs were produced by treatment with PEG. As a control, mixtures of human and marmoset cells were treated the same way but without adding PEG. The fused and non-fused cell mixtures were challenged with HIV-1 luciferase reporter viruses pseudotyped with VSV-G. Forty-eight hours later, the infectivity of the viruses was determined by measuring the relative luciferase activity in the cells. The infectivity in the human-human homokaryons is normalized to 100%, and the relative infectivity in the other homo-heterokaryons is shown. The results of four (+PEG) and two (no PEG) independent experiments and their means are shown. (B) Human and marmoset B-LCLs were fused with PEG to generate hetero- or homokaryons. As a control, mixtures of human and marmoset cells were treated the same way but without adding PEG. The fused and non-fused cell mixtures were challenged with HIV-1 luciferase reporter viruses pseudotyped with VSV-G. Forty-eight hours later, the infectivity of the viruses was determined by measuring the relative luciferase activity in the cells. The infectivity in the human-human homokaryons is normalized to 100%, and the relative infectivity in the other homo-heterokaryons is shown. The results of three independent experiments and their means are shown. Reported p-values (2-tailed) were obtained with an unpaired t-test.
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f3: The post-entry block(s) to HIV-1 in marmoset PBLs and B-LCLs are dominant.(A) Heterokaryons or homokaryons of human (hu) and marmoset (mar) PBLs were produced by treatment with PEG. As a control, mixtures of human and marmoset cells were treated the same way but without adding PEG. The fused and non-fused cell mixtures were challenged with HIV-1 luciferase reporter viruses pseudotyped with VSV-G. Forty-eight hours later, the infectivity of the viruses was determined by measuring the relative luciferase activity in the cells. The infectivity in the human-human homokaryons is normalized to 100%, and the relative infectivity in the other homo-heterokaryons is shown. The results of four (+PEG) and two (no PEG) independent experiments and their means are shown. (B) Human and marmoset B-LCLs were fused with PEG to generate hetero- or homokaryons. As a control, mixtures of human and marmoset cells were treated the same way but without adding PEG. The fused and non-fused cell mixtures were challenged with HIV-1 luciferase reporter viruses pseudotyped with VSV-G. Forty-eight hours later, the infectivity of the viruses was determined by measuring the relative luciferase activity in the cells. The infectivity in the human-human homokaryons is normalized to 100%, and the relative infectivity in the other homo-heterokaryons is shown. The results of three independent experiments and their means are shown. Reported p-values (2-tailed) were obtained with an unpaired t-test.
Mentions: To determine whether the block to HIV-1 infection in marmoset PBLs was due to the presence of a dominant factor and not to a deficiency in a factor needed for HIV-1 replication, we prepared heterokaryons and homokaryons of restricted and unrestricted PBLs using polyethylene glycol (PEG). As a control, we also prepared mixtures of human and marmoset PBLs that were not treated with PEG in parallel, and tested the infectivity of HIV-1 in the PEG-fused and non-fused cell mixtures. If the block were due to the presence of a dominant factor, we would expect to observe low HIV-1 infectivity in heterokaryons formed between restricted (marmoset) and unrestricted (human) cells. On the contrary, if the block were due to the absence of a factor needed for HIV-1 infection, we would expect to see HIV-1 infectivity at levels close to those seen in unrestricted cells or in the non-fused human-marmoset cell mixtures. As shown in Fig. 3A, HIV-1 infectivity was reduced in heterokaryons formed between human and marmoset PBLs by 2.6-fold compared to human-human homokaryons. We did not observe a significant reduction in infectivity in the non-fused human-marmoset cell mixtures (Fig. 3A, right). These results suggest that the early block to HIV-1 present in marmoset PBLs is most likely due to the presence of a dominant-acting factor, as is the case of the block present in rhesus cells22. The block in human-marmoset heterokaryons was not as pronounced as in marmoset-marmoset homokaryons, which showed a 12-fold lower infectivity compared to human-human homokaryons. This effect can be attributed to the formation of some human-human homokaryons and/or to incomplete cell-cell fusion. We have not been able to assess the efficiency of heterokaryon formation in our infection experiments because treatment of the cells with dyes that would allow this measurement resulted in background levels of infectivity in the PEG-treated cells. However, in pilot experiments using Vibrant DiO and Dil cell labeling solutions (Molecular Probes) to evaluate cell-cell fusion, the efficiency of heterokaryon formation ranged from 60% to 80%. Thus, it is likely that most cells have fused under the conditions of our experiment.

View Article: PubMed Central - PubMed

ABSTRACT

In nature, primate lentiviruses infect humans and several Old World monkeys and apes. However, to date, lentiviruses infecting New World monkeys have not been described. We studied the susceptibility of common marmoset cells to HIV-1 infection and observed the presence of post-entry blocks to the early phase of HIV-1 infection in peripheral blood lymphocytes (PBLs) and a B lymphocytic cell line (B-LCL). The blocks present in these cells are dominant and phenotypically different from each other. In PBLs, the block occurs at the level of reverse transcription, reducing the accumulation of early and late transcripts, similar to the block imposed by TRIM5α. However, we have found that marmoset TRIM5α does not block HIV-1. In contrast, the restriction factor present in B-LCLs blocks HIV-1 replication at a later step, after nuclear entry, and inhibits integration. Additionally, we have identified an HIV-1 capsid mutant, N74D, that is able to escape the restriction in the marmoset B-LCLs. Our results suggest that the factors responsible for the blocks present in marmoset PBLs and B-LCLs are different. We propose the existence of at least two new restriction factors able to block HIV-1 infection in marmoset lymphocytes.

No MeSH data available.


Related in: MedlinePlus