Human RPL7 and DDX21 interact with HTLV-1 Gag and enhance tRNAPro primer annealing to genomic RNA

Abstract

Human T-cell leukemia virus type 1 (HTLV-1), an oncogenic retrovirus, uses human tRNA^Pro to prime reverse transcription (RT). However, how tRNA^Pro is annealed to the primer binding site (PBS), which is embedded in a highly structured hairpin in the genomic RNA (gRNA), remains unclear. We hypothesize that HTLV-1 Gag may have more robust chaperone activity than mature HTLV-1 nucleocapsid (NC), which in contrast to HIV-1 NC, displays relatively weak chaperone function, and that a cellular co-factor may be required to facilitate primer tRNA annealing. Recombinant HTLV-1 Gag was successfully purified for the first time and used to perform primer-annealing assays. Relative to mature NC and matrix (MA) domains, HTLV-1 Gag is only slightly more effective at chaperoning the annealing of tRNA^Pro to the PBS. To identify potential HTLV-1 Gag interacting co-chaperones of tRNA annealing in cells, we performed affinity tagging/purification-mass spectrometry (AP-MS). Two significant AP-MS hits, RPL7 and DDX21, were further validated by reciprocal co-IP studies in both HEK293T and chronically HTLV-1-infected MT-2 cells. Domain mapping studies revealed that HTLV-1 Gag interacts with RPL7 and DDX21 through the zinc fingers in the NC domain independent of the presence of RNA. In addition, we showed that both RPL7 and DDX21 are packaged into virions. RPL7 or DDX21 alone was more effective than HTLV-1 Gag at annealing tRNA^Pro to the PBS. Synergistic effects of the Gag/RPL7/DDX21 combination in facilitating tRNA^Pro annealing to the PBS were found. Taken together, the mechanistic insights gained from these studies could be exploited for the development of new therapeutic strategies aimed at targeting HTLV-1 RT.

Keywords: DDX21; Gag; HTLV-1; RPL7; reverse transcription; tRNAPro primer.

Figure 1.. Schematic representation of RNAs and proteins used in this study.

(A) Sequences and secondary structures of tRNA^Pro (75 nt, top left) and a portion of HTLV-1 5' UTR containing the primer binding site (PBS) (98 nt, WT PBS, top right) used in this work. The sequence in the HTLV-1 PBS complementary to the 3' 18-nt of tRNA^Pro is indicated by red letters. The bottom of this panel shows the tRNA^Pro-PBS annealed complex. The structure of the PBS region is based on the structure-probing results reported in Wu et al (17). (B) Sequence and secondary structure of HTLV-1 PBS region before (WT PBS, top panel) and after deleting nt 425-434 (88 nt, Δ452-434 PBS mutant, bottom of this panel). (C) Domain architecture of HTLV-1 Gag bacterial expression construct consisting of matrix (MA, p19), capsid (CA, p24), and nucleocapsid (NC, p17) domains. The Tobacco Etch Virus (TEV) protease cleavage site and a 6xHis-tag at the C-terminus are indicated. The sequence of amino acids in the NC domain is shown. (D) Domain organization of HTLV-1 Gag mammalian expression construct used in AP-MS pull-down study; the human rhinovirus (HRV) 3C protease cleavage site, green fluorescent protein (GFP), and twin-strep tag at the C-terminus are indicated. (E) Domain organization of human RPL7 bacterial expression construct with a 6xHis-tag at the C-terminus, an N-terminal basic leucine zipper (bZIP), and a C-terminal domain (CTD). (F) Domain organization of human RPL7 mammalian expression construct with HA tag at the N-terminus. (G) Domain organization of human DDX21 bacterial expression construct with a 6xHis-tag at the C-terminus, a disordered N-terminal domain (NTD), a helicase core (the helicase N-terminal and C-terminal domains), and a CTD. (H) Human DDX21 mammalian expression construct with mCherry at the N-terminus and V5 and 6xHis tags at the C-terminus.

Figure 2.. Concentration-dependence and time-course annealing assays show that HTLV-1 ΔC29 Gag chaperones tRNA^Pro annealing to the stable HTLV-1 WT PBS and the less structured HTLV-1 Δ425-434 PBS more effectively than HTLV-1 WT Gag.

(A and B): Concentration-dependence annealing assays using 20 nM 5' ³²P-labeled tRNA^Pro and 200 nM HTLV-1 WT PBS (A) or Δ425-434 PBS (B) in the presence of varying concentrations of HTLV-1 WT or ΔC29 Gag at 37°C for 1 h. F indicates free tRNA^Pro and B1 and B2 indicate different conformations of tRNA^Pro-PBS binary complexes. Heat indicates heat annealing, the positive control. (C and D): Graphs showing percentages of tRNA^Pro annealed to WT (C) or Δ425-434 PBS (D) in the presence of varying concentrations of HTLV-1 WT or ΔC29 Gag. (E-H): Time-course annealing assays using 20 nM 5' ³²P-labeled tRNA^Pro and 200 nM HTLV-1 WT PBS (E and G) or Δ425-434 PBS (F and H) in the presence of 2 μM HTLV-1 WT Gag (E and F) or ΔC29 Gag (G and H) at 37°C. (I and J): Graphs showing percentages of tRNA^Pro annealed to HTLV-1 WT PBS (I) or Δ425-434 PBS (J) at different time points in the presence of 2 μM HTLV-1 WT or ΔC29 Gag. Lines represent exponential fits of the data with the standard deviation between trials indicated.

Figure 3.. HTLV-1 Gag interacts with RPL7 and DDX21 in an RNA-independent manner as confirmed by pull-down and reciprocal co-IP.

(A) Western blot analysis of HEK293T cell lysates overexpressing GFP or HTLV-1 Gag-GFP with or without RNase treatment. Proteins were pulled down by streptactin and immunoblotted using anti-HTLV-1 p19 (MA), anti-DDX21 and anti-RPL7 antibodies. (B) FLAG or HTLV-1 Gag-FLAG expressed in HEK293T cell lysate was IP’d using an anti-FLAG antibody. Co-IP’d proteins were immunoblotted using anti-DDX21 and anti-RPL7 antibodies. (C) Gag, CA-NC, and CA in MT-2 cell lysate were IP’d using an anti-HTLV-1 p24 (CA) antibody. Co-IP’d proteins were immunoblotted using anti-DDX21 and anti-RPL7 antibodies. (D and E): RPL7-HA was IP’d using an anti-HA antibody in lysate from HEK293T cells with (D) or without (E) HTLV-1 Gag-GFP co-overexpression. Co-pulled-down proteins were immunoblotted using anti-DDX21 and anti-GFP antibodies. (F) DDX21 was IP’d by an anti-DDX21 antibody in lysate from HEK293T cells overexpressing HTLV-1 Gag-GFP. Co-immunoprecipitated proteins were immunoblotted using anti-GFP and anti-RPL7 antibodies.

Figure 4.. Zinc finger (ZF) structures in the nucleocapsid (NC) domain of HTLV-1 Gag are essential for the interactions with DDX21 and RPL7.

(A) Schematic representation of HTLV-1 Gag and NC subdomain truncation constructs used in the pull-down study. (++), (+), and (−) indicate the relative ability of each construct to co-pull-down DDX21 and RPL7. (B) GFP, WT Gag-GFP, ΔMA, ΔCA, and ΔNC Gag-GFP in HEK293T cell lysate were pulled down by streptactin and immunoblotted using an anti-GFP antibody 48 h post-transfection. Co-pulled-down proteins were immunoblotted using anti-DDX21 and anti-RPL7 antibodies. (C) GFP, WT Gag-GFP, ΔZF1, ΔZF2, ΔZF1-2, ΔNC CTD, and ΔNC Gag-GFP in HEK293T cell lysate were pulled down by streptactin and immunoblotted using an anti-HTLV-1 p19 antibody 48 h post-transfection. Co-pulled-down proteins were immunoblotted using anti-DDX21 and anti-RPL7 antibodies. (D) Cell lysate from HEK293T cells overexpressing HTLV-1 Gag-GFP 48 h post-transfection was treated with or without 10 mM EDTA. GFP or HTLV-1 Gag-GFP was pulled down by streptactin and immunoblotted using an anti-HTLV-1 p19 antibody. Co-pulled-down proteins were immunoblotted using anti-DDX21 and anti-RPL7 antibodies. β-actin was used as a loading control in panels (B - D).

Figure 5.. RPL7 interacts with HTLV-1 Gag and DDX21 through the N-terminal basic leucine zipper (b-ZIP) domain and C-terminal domain (CTD).

(A) Schematic representation of human RPL7 WT and domain truncation constructs used in this study. On the right side, letters indicate the construct used in each lane in (B) and (C). Relative binding of RPL7 variants to HTLV-1 Gag and DDX21 are shown as (++), (+), or (−). (B) GFP or Gag-GFP was pulled down by streptactin and immunoblotted using an anti-HTLV-1 p19 antibody in cell lysate from HEK293T cells co-transfected with HTLV-1 Gag-GFP and WT or truncation mutant constructs of HA-RPL7 48 h post-transfection. Co-pulled-down HA-RPL7 was immunoblotted using an anti-HA antibody. (C) RPL7-HA WT or truncation mutants were IP’d and immunoblotted using anti-HA antibody 48 h post-transfection. Co-IP’d DDX21 was immunoblotted using anti-DDX21 antibody. (B and C): β-actin was used as a loading control.

Figure 6.. DDX21 interacts with HTLV-1 Gag through the helicase core and the C-terminal domain.

(A) Schematic representation of human DDX21 WT and domain truncation constructs used in the pull-down study. Letters on the right represent the construct used in each lane in (B). Relative binding of DDX21 variants to HTLV-1 Gag are reported as (++), (+), or (−). (B) Gag-GFP was pulled down by streptactin and immunoblotted using an anti-HTLV-1 p19 antibody in cell lysate from HEK293T cells co-transfected with HTLV-1 Gag-GFP and WT or truncation mutant constructs of mCherry-DDX21-V5 48 h post-transfection. Co-pulled-down DDX21-V5 was immunoblotted using an anti-V5 antibody. β-actin was used as a loading control.

Figure 7.. RPL7 and DDX21 are packaged into HTLV-1 virions.

(A) Schematic diagram of purification of HTLV-1 virus from MT-2 cell culture medium through a sucrose cushion and further fractionation of the viral pellet through an OptiPrep density gradient. (B) Fractionated viral lysate was immunoblotted using anti-HTLV-1 p24, anti-DDX21, and anti-RPL7 antibodies.

Figure 8.. Concentration-dependence and time-course annealing assays show that human DDX21 and RPL7 facilitate tRNA^Pro annealing to the HTLV-1 PBS more efficiently than HTLV-1 WT and ΔC29 Gag.

(A and B): Concentration-dependence annealing assays using 20 nM 5' ³²P-labeled tRNA^Pro and 200 nM HTLV-1 WT PBS or Δ425-434 PBS in the presence of varying concentrations of RPL7 (A) or DDX21 (B) at 37°C for 1 h. F indicates free tRNA^Pro and B1 and B2 indicate different tRNA^Pro-PBS binary complexes. Heat indicates heat annealing, the positive control. (C and D): Graphs showing percentages of tRNA^Pro annealed to WT (C) or Δ425-434 PBS (D) in the presence of varying concentrations of HTLV-1 WT Gag, ΔC29 Gag, RPL7, or DDX21. (E-H): Time-course annealing assays using 20 nM 5' ³²P-labeled tRNA^Pro and 200 nM HTLV-1 WT PBS (E and G) or Δ425-434 PBS (F and H) in the presence of 0.8 μM RPL7 (E and F) or DDX21 (G and H) at 37°C. (I and J): Graphs showing percentages of tRNA^Pro annealed to HTLV-1 WT PBS (I) or Δ425-434 PBS (J) at different time points in the presence of 2 μM HTLV-1 WT Gag, 2 μM ΔC29 Gag, 0.8 μM RPL7, or 0.8 μM DDX21. Lines represent exponential fits of the data with the standard deviation between trials indicated.

Figure 9.. HTLV-1 Gag, RPL7, and DDX21 act synergistically to chaperone tRNA^Pro annealing to the HTLV-1 WT PBS.

(A) Time-course annealing assays using 20 nM 5' ³²P-labeled tRNA^Pro and 200 nM HTLV-1 WT PBS in the presence of 2 μM HTLV-1 WT Gag, 0.8 μM RPL7, and 0.8 μM DDX21 at 37°C for varying time. F indicates free tRNA^Pro and B1 and B2 indicate different tRNA^Pro-PBS binary complexes. Heat indicates heat annealing, the positive control. (B) Graph for percentages of tRNA^Pro annealed to HTLV-1 WT PBS at different time points in the presence of 2 μM HTLV-1 WT Gag, 0.8 μM RPL7, and 0.8 μM DDX21. Lines represent exponential fits of the data with the standard deviation between trials indicated.

Figure 10.. Model for HTLV-1 Gag, human RPL7, and human DDX21 synergistically facilitating tRNA^Pro annealing to the HTLV-1 PBS in two steps.

In step 1, DDX21 unwinds the stable hairpin in the HTLV-1 PBS region to make the PBS less structured. In step 2, HTLV-1 Gag, RPL7, and DDX21 synergistically facilitate tRNA^Pro annealing to the less structured PBS.