The Dengue virus (DENV) genome contains multiple cis -acting elements required for translation and replication. Previous studies indicated that a 719-nt subgenomic minigenome (DENV-MINI) is an efficient template for translation and (−) strand RNA synthesis in vitro . We performed a detailed structural analysis of DENV-MINI RNA, combining chemical acylation techniques, Pb ²⁺ ion-induced hydrolysis and site-directed mutagenesis. Our results highlight protein-independent 5′–3′ terminal interactions involving hybridization between recognized cis -acting motifs. Probing analyses identified tandem dumbbell structures (DBs) within the 3′ terminus spaced by single-stranded regions, internal loops and hairpins with embedded GNRA-like motifs. Analysis of conserved motifs and top loops (TLs) of these dumbbells, and their proposed interactions with downstream pseudoknot (PK) regions, predicted an H-type pseudoknot involving TL1 of the 5′ DB and the complementary region, PK2. As disrupting the TL1/PK2 interaction, via ‘flipping’ mutations of PK2, previously attenuated DENV replication, this pseudoknot may participate in regulation of RNA synthesis. Computer modeling implied that this motif might function as autonomous structural/regulatory element. In addition, our studies targeting elements of the 3′ DB and its complementary region PK1 indicated that communication between 5′–3′ terminal regions strongly depends on structure and sequence composition of the 5′ cyclization region.

HIV-1 Rev is an ~13 kD accessory protein expressed during the early stage of virus replication. After translation, Rev enters the nucleus and binds the Rev response element (RRE), a ~350 nucleotide, highly structured element embedded in the env gene in unspliced and singly spliced viral RNA transcripts. Rev-RNA assemblies subsequently recruit Crm1 and other cellular proteins to form larger complexes that are exported from the nucleus. Once in the cytoplasm, the complexes dissociate and unspliced and singly-spliced viral RNAs are packaged into nascent virions or translated into viral structural proteins and enzymes, respectively. Rev binding to the RRE is a complex process, as multiple copies of the protein assemble on the RNA in a coordinated fashion via a series of Rev-Rev and Rev-RNA interactions. Our understanding of the nature of these interactions has been greatly advanced by recent studies using X-ray crystallography, small angle X-ray scattering (SAXS) and single particle electron microscopy as well as biochemical and genetic methodologies. These advances are discussed in detail in this review, along with perspectives on development of antiviral therapies targeting the HIV-1 RRE.

Synthesis of integration-competent, double-stranded DNA from the (+)-RNA strand genome of retroviruses and long terminal repeat-containing retrotransposons reflects a multistep process catalyzed by the virus-encoded reverse transcriptase (RT). In conjunction with RNA- and DNA-templated DNA synthesis, a hydrolytic activity of the same enzyme (RNase H) is required to remove genomic RNA of the RNA/DNA replication intermediate. Together, these combined synthetic and degradative functions ensure correct selection, extension, and removal of the RNA primers of (−)- and (+)-strand DNA synthesis (tRNA and the polypurine tract, respectively). For HIV-1 RT, a quarter century of research has not only illuminated the biochemical properties, structure, and conformational dynamics of this highly versatile enzyme but has also witnessed drug discovery advances from the first Food and Drug Administration-approved anti-RT drug to recent use of RT inhibitors as potential colorectal microbicides. Salient features of HIV-1 RT and extension of these findings into programs of drug discovery are reviewed here.

Despite the wealth of information available for the reverse transcriptase (RT)-associated ribonuclease H (RNaseH) domain of lentiviruses, gammaretroviruses and long terminal repeat containing retro...