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 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.

A purine loop and the primer binding site are critical for the selective encapsidation of mouse mammary tumor virus genomic RNA by Pr77Gag

“…Within the ssPurines, the GGAG at the 5′ end and the AG at the 3′ end, respectively, are crucial for packaging (Figure 9 ). Interestingly, a sequence similar to the MMTV 5′ GGAG was found to be involved in the Pr55 Gag binding in HIV-1 (in the form of an asymmetrical internal loop 5′ G/AGG 3′ ( 20 ) and HTLV-1 NC and MA binding (5′GAG 3′; 70 ). Similarly, in HIV-2, a 5′ GGRG 3′ motif located upstream of DIS was found to be important for RNA packaging and has been suggested as Gag binding site ( 71 ).…”

Rous Sarcoma Virus Genomic RNA Dimerization Capability In Vitro Is Not a Prerequisite for Viral Infectivity

“…RSV 5 -leader RNA (636 nt) was probed by SHAPE using N-methylisatoic anhydride (NMIA) as described previously with minor alterations [51]. Briefly, 2.3 µM RNA was folded in 50 mM HEPES, pH 7.5 by heating to 80 • C for 2 min, then 60 • C for 2 min.…”

“…Primer extension reactions were incubated at 55 • C for 1 h and then the enzyme was inactivated at 70 • C for 15 min. The RNAs were hydrolyzed using NaOH, as described [51]. Dideoxy sequencing reactions were performed on the template plasmid used for in vitro transcription, and all the reactions (XL-SHAPE, dideoxy sequencing) were analyzed by capillary electrophoresis, as described [51].…”

“…The RNAs were hydrolyzed using NaOH, as described [51]. Dideoxy sequencing reactions were performed on the template plasmid used for in vitro transcription, and all the reactions (XL-SHAPE, dideoxy sequencing) were analyzed by capillary electrophoresis, as described [51]. The sequences of the primers used in the primer extension reactions were as follows: 5 -AGC CGC CTT CAA TGC CC-3 (Primer 1, complementary to nt 615-631 of RSV 5 -leader RNA), 5 -GGT TTT ACA CGC GGA CGA AAT CAC C-3 (Primer 2, complementary to nt 397-421) and 5 -CCT CTA CTA GGG TCA TCG TCC GC-3 (Primer 3, complementary to nt 190-212).…”

Portrait of the Intrinsically Disordered Side of the HTLV-1 Proteome

“…However, the HTLV-1 NC nonspecifically binds to genomic RNA and exhibits very poor chaperone activity. 48 It has been reported that zinc-finger binding domains can interact with each other, while NTD can interact with the CTD via electrostatic interactions when NC is not bound to RNA. Such an intramolecular interaction is postulated to control the binding of this protein to DNA and impairing chaperone properties of this protein.…”