Comparative host protein interactions with HTLV-1 p30 and HTLV-2 p28: insights into difference in pathobiology of human retroviruses

Abstract

Background: Human T lymphotropic virus type-1 (HTLV-1) and type 2 (HTLV-2) are closely related human retroviruses, but have unique disease associations. HTLV-1 is the causative agent of an aggressive T-cell leukemia known as adult T-cell leukemia (ATL), HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), and other inflammatory diseases. HTLV-2 infection has not been clearly associated with any disease condition. Although both viruses can transform T cells in vitro, the HTLV-1 provirus is mainly detected in CD4+ T cells whereas HTLV-2 is mainly detected in CD8+ T cells of infected individuals. HTLV-1 and HTLV-2 encode accessory proteins p30 and p28, respectively, which share partial amino acid homology and are required for viral persistence in vivo. The goal of this study was to identify host proteins interacting with p30 and p28 in order to understand their role in pathogenesis.

Results: Affinity-tag purification coupled with mass spectrometric (MS) analyses revealed 42 and 22 potential interacting cellular partners of p30 and p28, respectively. Of these, only three cellular proteins, protein arginine methyltransferase 5 (PRMT5), hnRNP K and 60 S ribosomal protein L8 were detected in both p30 and p28 fractions. To validate the proteomic results, four interacting proteins were selected for further analyses using immunoblot assays. In full agreement with the MS analysis two cellular proteins REGγ and NEAF-interacting protein 30 (NIP30) selectively interacted with p30 and not with p28; heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1) bound to p28 and not to p30; and PRMT5 interacted with both p30 and p28. Further studies demonstrated that reduced levels of PRMT5 resulted in decreased HTLV-2 viral gene expression whereas the viral gene expression of HTLV-1 was unchanged.

Conclusion: The comparisons of p30 and p28 host protein interaction proteome showed striking differences with some degree of overlap. PRMT5, one of the host proteins that interacted with both p30 and p28 differentially affected HTLV-1 and HTLV-2 viral gene expression suggesting that PRMT5 is involved at different stages of HTLV-1 and HTLV-2 biology. These findings suggest that distinct host protein interaction profiles of p30 and p28 could, in part, be responsible for differences in HTLV-1 and HTLV-2 pathobiology. This study provides new avenues of investigation into mechanisms of viral infection, tropism and persistence.

Figure 1

Validation of p30 interaction with REGγ and NIP30.A) S-tag affinity purification with Mock, S-GFP, S-p30-HA and S-p28-HA transfected 293T cell lysates. The purified product was analyzed by immunoblotting using indicated antibodies. The expression of individual protein and S-tag purification was confirmed using the indicated antibodies. B) 293T cells were transfected with S-p30-HA and S-p28-HA and immunoprecipitated with non-specific IgG (NSIgG) or anti-REGγ antibody and probed with anti-HA antibody. Immunoblotting of input using anti-REGγ and anti-HA antibodies confirmed the expression of the interacting proteins. C) 293T cells lysates expressing S-p30-HA and S-p28-HA were immunoprecipitated with non-specific IgG (NSIgG) or anti-NIP30 antibody and probed with anti-HA antibody. The expression of p30, p28 and NIP30 was confirmed with indicated antibodies.

Figure 2

Validation of p28 interaction with hnRNP H1.A) Ectopically expressed Mock, S-GFP, S-p30-HA, S-p28-HA in 293T cells were S-tag affinity purified and probed with anti-hnRNP H1 antibodies. Endogenous expression of hnRNP H1 was confirmed by immunoblotting with anti-hnRNP H1 antibody. B) The immunoprecipitation assay was performed on cell lysates transfected with S-p30-HA and S-p28-HA. Non-specific IgG (NSIgG) or anti-hnRNP H1 antibodies were used for immunoprecipitation and subsequently probed with anti-HA antibody. Immunoblotting using anti-HA and anti-hnRNP H1 was used to validate the expression of all proteins.

Figure 3

Validation of p30 and p28 interaction with PRMT5. Mock, S-GFP, S-p30-HA and S-p28-HA expressed in 293T cells were purified by S-tag affinity purification and immunoblotting with anti-PRMT5 antibody. The expression of PRMT5 was confirmed by immunoblotting the cell lysates with anti-PRMT5 antibody. B) 293T cells co- transfected with p30 or p28 or Flag PRMT5 as indicated in the figure. Immunoprecipitation was performed using anti-Flag antibodies and subsequently probed for p30 and p28 using anti-HA antibodies. The expression of Flag-PRMT5, p30 and p28 was validated by immunoblotting as shown in the figure.

Figure 4

PRMT5 knockdown and HTLV gene expression.A) 293T cells expressing normal and knockdown levels of PRMT5 were transfected with HTLV-1 molecular clone. Immunoblotting was performed using anti-PRMT5 antibodies to confirm the knockdown and with anti-HTLV p24 antibodies to monitor p24 levels. The amount of p19 Gag production was analyzed by p19 ELISA. B) 293T cells transduced with scrambled shRNA or shRNA against PRMT5 were transfected with HTLV-2 molecular clone. Knockdown of PRMT5 was confirmed by immunoblotting with anti-PRMT5 antibodies. The intracellular p24 and extracellular p19 Gag production was analyzed by immunoblotting with anti-HTLV p24 antibodies and with p19 ELISA respectively. Student t-test resulted in a p value of 0.011 (_*) indicating that decrease in p19 production with lower levels of PRMT5 is significant. Immunoblotting with anti-Actin antibodies was used to validate equal loading.

Figure 5

Analysis of p30 interacting host proteins.A) Functional distribution of HTLV-1 p30-interacting proteins summarized in the pie chart graph as percentages. B), C) and D) Ingenuity pathways analysis results in three network pathways with broad cellular functions with the involvement of p30-interacting proteins. The proteins that were identified in this study to interact with p30 are indicated in bold, and previously identified p30-interacting proteins are indicated in bold italics. The interaction between two proteins is indicated with a straight line; arrows indicate action upon in the direction of the arrow; and dashed lines indicate indirect interactions.

Figure 6

Analysis of p28 interacting host proteins.A) Functional distribution of HTLV-2 p28-interacting proteins summarized in the pie chart graph as percentages. B) and C) Ingenuity pathways analysis results in two network pathways with broad cellular functions with the involvement of p28-interacting proteins. The proteins that were identified in this study to interact with p28 are indicated in bold. The interaction between two proteins is indicated with a straight line; arrows indicate action upon in the direction of the arrow; and dashed lines indicate indirect interactions.