Ty1 Gag comprises the capsid of virus-like particles and provides nucleic acid chaperone (NAC) functions during retrotransposition in budding yeast. A subgenomic Ty1 mRNA encodes a truncated Gag protein (p22) that is cleaved by Ty1 protease to form p18. p22/p18 strongly inhibits transposition and can be considered an element-encoded restriction factor. Here, we show that only p22 and its short derivatives restrict Ty1 mobility whereas other regions of GAG inhibit mobility weakly if at all. Mutational analyses suggest that p22/p18 is synthesized from either of two closely spaced AUG codons. Interestingly, AUG1p18 and AUG2p18 proteins display different properties, even though both contain a region crucial for RNA binding and NAC activity. AUG1p18 shows highly reduced NAC activity but specific binding to Ty1 RNA, whereas AUG2p18 shows the converse behavior. p22/p18 affects RNA encapsidation and a mutant derivative defective for RNA binding inhibits the RNA chaperone activity of the C-terminal region (CTR) of Gag-p45. Moreover, affinity pulldowns show that p18 and the CTR interact. These results support the idea that one aspect of Ty1 restriction involves inhibition of Gag-p45 NAC functions by p22/p18-Gag interactions.

The trinucleotide repeat expansion disorders (TREDs) constitute of a group of >40 hereditary neurodegenerative human diseases associated with abnormal expansion of repeated sequences, such as CAG repeats. The pathogenic factor is a transcribed RNA or protein whose function in the cell is compromised. The disorders are progressive and incurable. Consequently, many ongoing studies are oriented at developing therapies. We have analyzed crystal structures of RNA containing CAG repeats in complex with synthetic cyclic mismatch-binding ligands (CMBLs). The models show well-defined interactions between the molecules in which the CMBLs mimic nucleobases as they form pseudo-canonical base pairs with adenosine residues and engage in extensive stacking interactions with neighboring nucleotides. The binding of ligands is associated with major structural changes of the CAG repeats, which is consistent with results of biochemical studies. The results constitute an early characterization of the first lead compounds in the search for therapy against TREDs. The crystallographic data indicate how the compounds could be further refined in future biomedical studies.

The Gag polyprotein is a multifunctional regulator of retroviral replication and major structural component of immature virions. The nucleic acid chaperone (NAC) activity is considered necessary to retroviral Gag functions, but so far, NAC activity has only been confirmed for HIV-1 and RSV Gag polyproteins. The nucleocapsid (NC) domain of Gag is proposed to be crucial for interactions with nucleic acids and NAC activity. The major function of matrix (MA) domain is targeting and binding of Gag to the plasma membrane but MA can also interact with RNA and influence NAC activity of Gag. Here, we characterize RNA binding properties and NAC activity of HIV-2 MA and Gag, lacking p6 domain (GagΔp6) and discuss potential contribution of NC and MA domains to HIV-2 GagΔp6 functions and interactions with RNA. We found that HIV-2 GagΔp6 is a robust nucleic acid chaperone. HIV-2 MA protein promotes nucleic acids aggregation and tRNALys3 annealing in vitro. The NAC activity of HIV-2 NC is affected by salt which is in contrast to HIV-2 GagΔp6 and MA. At a physiological NaCl concentration the tRNALys3 annealing activity of HIV-2 GagΔp6 or MA is higher than HIV-2 NC. The HIV-2 NC and GagΔp6 show strong binding to the packaging signal (Ψ) of HIV-2 RNA and preference for the purine-rich sequences, while MA protein binds mainly to G residues without favouring Ψ RNA. Moreover, HIV-2 GagΔp6 and NC promote HIV-2 RNA dimerization while our data do not support MA domain participation in this process in vitro. We present that contrary to HIV-1 MA, HIV-2 MA displays NAC activity and we propose that MA domain may enhance the activity of HIV-2 GagΔp6. The role of the MA domain in the NAC activity of Gag may differ significantly between HIV-1 and HIV-2. The HIV-2 NC and MA interactions with RNA are not equivalent. Even though both NC and MA can facilitate tRNALys3 annealing, MA does not participate in RNA dimerization in vitro. Our data on HIV-2 indicate that the role of the MA domain in the NAC activity of Gag differs not only between, but also within, retroviral genera.