5'-Cap sequestration is an essential determinant of HIV-1 genome packaging.

HIV-1 selectively packages two copies of its 5'-capped RNA genome (gRNA) during virus assembly, a process mediated by the nucleocapsid (NC) domain of the viral Gag polyprotein and encapsidation signals located within the dimeric 5' leader of the viral RNA. Although residues within the leader that promote packaging have been identified, the determinants of authentic packaging fidelity and efficiency remain unknown. Here, we show that a previously characterized 159-nt region of the leader that possesses all elements required for RNA dimerization, high-affinity NC binding, and packaging in a noncompetitive RNA packaging assay (ΨCES) is unexpectedly poorly packaged when assayed in competition with the intact 5' leader. ΨCES lacks a 5'-tandem hairpin element that sequesters the 5' cap, suggesting that cap sequestration may be important for packaging. Consistent with this hypothesis, mutations within the intact leader that expose the cap without disrupting RNA structure or NC binding abrogated RNA packaging, and genetic addition of a 5' ribozyme to ΨCES to enable cotranscriptional shedding of the 5' cap promoted ΨCES-mediated RNA packaging to wild-type levels. Additional mutations that either block dimerization or eliminate subsets of NC binding sites substantially attenuated competitive packaging. Our studies indicate that packaging is achieved by a bipartite mechanism that requires both sequestration of the 5' cap and exposure of NC binding sites that reside fully within the ΨCES region of the dimeric leader. We speculate that cap sequestration prevents irreversible capture by the cellular RNA processing and translation machinery, a mechanism likely employed by other viruses that package 5'-capped RNA genomes.

Identification of the initial nucleocapsid recognition element in the HIV-1 RNA packaging signal.

Selective packaging of the HIV-1 genome during virus assembly is mediated by interactions between the dimeric 5'-leader of the unspliced viral RNA and the nucleocapsid (NC) domains of a small number of assembling viral Gag polyproteins. Here, we show that the dimeric 5'-leader contains more than two dozen NC binding sites with affinities ranging from 40 nM to 1.4 µM, and that all high-affinity sites (Kd ≲ 400 nM) reside within a ∼150-nt region of the leader sufficient to promote RNA packaging (core encapsidation signal, ΨCES). The four initial binding sites with highest affinity reside near two symmetrically equivalent three-way junction structures. Unlike the other high-affinity sites, which bind NC with exothermic energetics, binding to these sites occurs endothermically due to concomitant unwinding of a weakly base-paired [UUUU]:[GGAG] helical element. Mutations that stabilize base pairing within this element eliminate NC binding to this site and severely impair RNA packaging into virus-like particles. NMR studies reveal that a recently discovered small-molecule inhibitor of HIV-1 RNA packaging that appears to function by stabilizing the structure of the leader binds directly to the [UUUU]:[GGAG] helix. Our findings suggest a sequential NC binding mechanism for Gag-genome assembly and identify a potential RNA Achilles' heel to which HIV therapeutics may be targeted.

A Novel Genus of Actinobacterial Tectiviridae.

Streptomyces phages WheeHeim and Forthebois are two novel members of the Tectiviridae family. These phages were isolated on cultures of the plant pathogen Streptomyces scabiei, known for its worldwide economic impact on potato crops. Transmission electron microscopy showed viral particles with double-layered icosahedral capsids, and frequent instances of protruding nanotubes harboring a collar-like structure. Mass-spectrometry confirmed the presence of lipids in the virion, and serial purification of colonies from turbid plaques and immunity testing revealed that both phages are temperate. Streptomycesphages WheeHeim and Forthebois have linear dsDNA chromosomes (18,266 bp and 18,251 bp long, respectively) with the characteristic two-segment architecture of the Tectiviridae. Both genomes encode homologs of the canonical tectiviral proteins (major capsid protein, packaging ATPase and DNA polymerase), as well as PRD1-type virion-associated transglycosylase and membrane DNA delivery proteins. Comparative genomics and phylogenetic analyses firmly establish that these two phages, together with Rhodococcusphage Toil, form a new genus within the Tectiviridae, which we have tentatively named Deltatectivirus. The identification of a cohesive clade of Actinobacteria-infecting tectiviruses with conserved genome structure but with scant sequence similarity to members of other tectiviral genera confirms that the Tectiviridae are an ancient lineage infecting a broad range of bacterial hosts.