Human T-cell leukemia virus type 1 Gag domains have distinct RNA-binding specificities with implications for RNA packaging and dimerization

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) is the first retrovirus that has conclusively been shown to cause human diseases. In HIV-1, specific interactions between the nucleocapsid (NC) domain of the Gag protein and genomic RNA (gRNA) mediate gRNA dimerization and selective packaging; however, the mechanism for gRNA packaging in HTLV-1, a deltaretrovirus, is unclear. In other deltaretroviruses, the matrix (MA) and NC domains of Gag are both involved in gRNA packaging, but MA binds nucleic acids with higher affinity and has more robust chaperone activity, suggesting that this domain may play a primary role. Here, we show that the MA domain of HTLV-1, but not the NC domain, binds short hairpin RNAs derived from the putative gRNA packaging signal. RNA probing of the HTLV-1 5' leader and cross-linking studies revealed that the primer-binding site and a region within the putative packaging signal form stable hairpins that interact with MA. In addition to a previously identified palindromic dimerization initiation site (DIS), we identified a new DIS in HTLV-1 gRNA and found that both palindromic sequences bind specifically the NC domain. Surprisingly, a mutant partially defective in dimer formation in vitro exhibited a significant increase in RNA packaging into HTLV-1-like particles, suggesting that efficient RNA dimerization may not be strictly required for RNA packaging in HTLV-1. Moreover, the lifecycle of HTLV-1 and other deltaretroviruses may be characterized by NC and MA functions that are distinct from those of the corresponding HIV-1 proteins, but together provide the functions required for viral replication.

Keywords: Gag; HTLV-1; RNA binding protein; RNA dimerization; RNA folding; RNA structure; matrix domain; nucleocapsid; retrovirus; viral replication.

Figure 1.

HTLV-1 gRNA-derived 5′ leader constructs (HTLV-1_CH isolate) used in this study. A, Mfold-predicted secondary structures (101) of short gRNA constructs SL(450–470), SL(474–508), and SL(447–512) derived from the putative HTLV-1 packaging signal within the gag coding region. B, schematic representation of HTLV-1 5′ leader and three constructs encompassing the 5′-UTR (R and U5) and the 5′ end of the gag coding region (5′ gag). The locations of SL(450–470) and SL(474–508) as well as the SD and PBS are indicated.

Figure 2.

Binding assays with HTLV-1 MA and NC. A, results of FA binding assays wherein 20 nm fluorescently labeled ssDNA20 or RNA was titrated with increasing concentrations of HTLV-1 MA or NC proteins. The averages of three experiments with standard deviations are shown. B and C, results of EMSA binding studies investigating HTLV-1 MA (B) and NC (C) binding interactions with the 5′ leader. The percentage of bound RNA is plotted as a function of protein concentration with curves fit to the Hill equation (96). Error bars represent standard deviations from two independent experiments.

Figure 3.

SHAPE analysis of HTLV-1 5′ leader RNA. The secondary structure model was predicted by incorporating the averaged NMIA reactivities for each nt from three to seven independent experiments as pseudo free-energy constraints in RNAstructure (99, 100). Nucleotides are colored according to SHAPE reactivity as indicated in the key. The PBS region, a previously identified DIS (DIS-2) (40, 47), a newly identified DIS (DIS-1), SL(450–470), SL1, and the U5:AUG interaction are labeled and indicated by black boxes or lines. The AUG start codon is highlighted using a red box. Gray nt indicate regions where SHAPE data could not be accurately analyzed due to primer binding and signal distortion near the RNA ends.

Figure 4.

Dimerization study of the HTLV-1 5′ leader. A, mutations introduced to disrupt the palindromic loops at nt positions 348–350 (M348), 382–386 (M382), and 498–500 (M498) of the full-length 5′ leader RNA. In each case, stable GAGA tetraloops were substituted for WT sequences individually or simultaneously (3M) (highlighted in red). B, schematic showing tetraloop mutations introduced into the loop regions of the full 5′ leader RNA. The predicted intermolecular contacts formed by each palindromic sequence are shown next to each putative DIS. C, native agarose gel showing the results of dimerization assays of WT and mutated HTLV-1 5′ leader. RNAs (0.5 μm) were either folded in low-salt buffer (50 mm Hepes, pH 7.5, and 1 mm MgCl₂) (lanes 2, 4, 6, 8, and 10) or folded in low-salt buffer followed by incubation in dimerization buffer (50 mm Hepes, pH 7.5, 150 mm NaCl, and 1 mm MgCl₂) at 37 °C for 1 h (lanes 3, 5, 7, 9, and 11). Lane 1 is a 100-bp DNA ladder. Markers for monomeric (M) and dimeric (D) RNAs are shown. The identity of the slowest migrating band is unknown but may represent a trimer. The native gel is representative of three independent experiments.

Figure 5.

Results of XL-SHAPE analysis on HTLV-1 MA (A) and NC (B) binding to the HTLV-1 5′ leader RNA. Arrows indicate sites with decreased (red) and increased (blue) NMIA reactivity upon protein binding, and violet stars indicate protein cross-linking sites. All identified sites correspond to positions with calculated reactivity changes of ≥0.3 and p < 0.05 based on an unpaired two-tail Student's t test. Experiments were performed in triplicate. Potential intermolecular dimerization of DIS-1 and DIS-2 sequences is shown in B (upper left, boxed) with SHAPE reactivity changes upon NC binding indicated (nt color scheme within the boxes is the same as in Fig. 3).

Figure 6.

HTLV-1 RNA packaging efficiency of the WT and mutant HT-UTR-MA RNA. A, serial dilution of the HTLV-1–like particles for protein quantification. Top, immunoblot displaying a serial dilution (1–20 μl of lysate per loading) of HTLV-1–like particles. Bottom, protein band intensities (in arbitrary units) (y axis) were plotted against the volume of protein loaded (x axis) to evaluate the linearity and accuracy of protein quantification. The linear equation and an R² value are indicated. B, immunoblots of both VLPs and cell lysates were probed with anti-HA antibodies, and cell lysate blots were normalized for expression based upon glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The amount of protein was determined based on the linear standard shown in A. Lane 1, WT HT-UTR-MA; lane 2, M348 mutant; lane 3, M382 mutant; lane 4, 2M mutant. C, relative expression level of WT or mutant HT-UTR-MA RNAs in VLPs as detected by a two-step quantitative RT-PCR assay. The expression level of the M348, M382, and 2M mutants was normalized to that from WT HT-UTR-MA. The results of four independent experiments are shown with the mean value indicated by a horizontal bar. D, relative -fold change of RNA packaging efficiency. The RNA packaging efficiency (y axis) was calculated as indicated under “Experimental procedures.” The RNA packaging efficiency of the M348, M382, and 2M mutants was normalized to that from WT HT-UTR-MA. The results of four independent experiments are shown with the mean value indicated by a horizontal bar. n.s., no significant difference.