Identification of a putative Gag binding site critical for feline immunodeficiency virus genomic RNA packaging.

The retroviral Gag precursor plays a central role in the selection and packaging of viral genomic RNA (gRNA) by binding to virus-specific packaging signal(s) (psi or ψ). Previously, we mapped the feline immunodeficiency virus (FIV) ψ to two discontinuous regions within the 5' end of the gRNA that assumes a higher order structure harboring several structural motifs. To better define the region and structural elements important for gRNA packaging, we methodically investigated these FIV ψ sequences using genetic, biochemical, and structure-function relationship approaches. Our mutational analysis revealed that the unpaired U85CUG88 stretch within FIV ψ is crucial for gRNA encapsidation into nascent virions. High-throughput selective 2' hydroxyl acylation analyzed by primer extension (hSHAPE) performed on wild type (WT) and mutant FIV ψ sequences, with substitutions in the U85CUG88 stretch, revealed that these mutations had limited structural impact and maintained nucleotides 80-92 unpaired, as in the WT structure. Since these mutations dramatically affected packaging, our data suggest that the single-stranded U85CUG88 sequence is important during FIV RNA packaging. Filter-binding assays performed using purified FIV Pr50Gag on WT and mutant U85CUG88 ψ RNAs led to reduced levels of Pr50Gag binding to mutant U85CUG88 ψ RNAs, indicating that the U85CUG88 stretch is crucial for ψ RNA-Pr50Gag interactions. Delineating sequences important for FIV gRNA encapsidation should enhance our understanding of both gRNA packaging and virion assembly, making them potential targets for novel retroviral therapeutic interventions, as well as the development of FIV-based vectors for human gene therapy.

Expression, purification, and functional characterization of soluble recombinant full-length simian immunodeficiency virus (SIV) Pr55Gag.

The simian immunodeficiency virus (SIV) precursor polypeptide Pr55Gag drives viral assembly and facilitates specific recognition and packaging of the SIV genomic RNA (gRNA) into viral particles. While several studies have tried to elucidate the role of SIV Pr55Gag by expressing its different components independently, studies using full-length SIV Pr55Gag have not been conducted, primarily due to the unavailability of purified and biologically active full-length SIV Pr55Gag. We successfully expressed soluble, full-length SIV Pr55Gag with His6-tag in bacteria and purified it using affinity and gel filtration chromatography. In the process, we identified within Gag, a second in-frame start codon downstream of a putative Shine-Dalgarno-like sequence resulting in an additional truncated form of Gag. Synonymously mutating this sequence allowed expression of full-length Gag in its native form. The purified Gag assembled into virus-like particles (VLPs) in vitro in the presence of nucleic acids, revealing its biological functionality. In vivo experiments also confirmed formation of functional VLPs, and quantitative reverse transcriptase PCR demonstrated efficient packaging of SIV gRNA by these VLPs. The methodology we employed ensured the availability of >95% pure, biologically active, full-length SIV Pr55Gag which should facilitate future studies to understand protein structure and RNA-protein interactions involved during SIV gRNA packaging.

A Stretch of Unpaired Purines in the Leader Region of Simian Immunodeficiency Virus (SIV) Genomic RNA is Critical for its Packaging into Virions.

Simian immunodeficiency virus (SIV) is an important lentivirus used as a non-human primate model to study HIV replication, and pathogenesis of human AIDS, as well as a potential vector for human gene therapy. This study investigated the role of single-stranded purines (ssPurines) as potential genomic RNA (gRNA) packaging determinants in SIV replication. Similar ssPurines have been implicated as important motifs for gRNA packaging in many retroviruses like, HIV-1, MPMV, and MMTV by serving as Gag binding sites during virion assembly. In examining the secondary structure of the SIV 5' leader region, as recently deduced using SHAPE methodology, we identified four specific stretches of ssPurines (I-IV) in the region that harbors major packaging determinants of SIV. The significance of these ssPurine motifs were investigated by mutational analysis coupled with a biologically relevant single round of replication assay. These analyses revealed that while ssPurine II was essential, the others (ssPurines I, III, & IV) did not significantly contribute to SIV gRNA packaging. Any mutation in the ssPurine II, such as its deletion or substitution, or other mutations that caused base pairing of ssPurine II loop resulted in near abrogation of RNA packaging, further substantiating the crucial role of ssPurine II and its looped conformation in SIV gRNA packaging. Structure prediction analysis of these mutants further corroborated the biological results and further revealed that the unpaired nature of ssPurine II is critical for its function during SIV RNA packaging perhaps by enabling it to function as a specific binding site for SIV Gag.

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

Retroviral RNA genome (gRNA) harbors cis-acting sequences that facilitate its specific packaging from a pool of other viral and cellular RNAs by binding with high-affinity to the viral Gag protein during virus assembly. However, the molecular intricacies involved during selective gRNA packaging are poorly understood. Binding and footprinting assays on mouse mammary tumor virus (MMTV) gRNA with purified Pr77Gag along with in cell gRNA packaging study identified two Pr77Gag binding sites constituting critical, non-redundant packaging signals. These included: a purine loop in a bifurcated stem-loop containing the gRNA dimerization initiation site, and the primer binding site (PBS). Despite these sites being present on both unspliced and spliced RNAs, Pr77Gag specifically bound to unspliced RNA, since only that could adopt the native bifurcated stem-loop structure containing looped purines. These results map minimum structural elements required to initiate MMTV gRNA packaging, distinguishing features that are conserved amongst divergent retroviruses from those perhaps unique to MMTV. Unlike purine-rich motifs frequently associated with packaging signals, direct involvement of PBS in gRNA packaging has not been documented in retroviruses. These results enhance our understanding of retroviral gRNA packaging/assembly, making it not only a target for novel therapeutic interventions, but also development of safer gene therapy vectors.

Identification of Pr78Gag Binding Sites on the Mason-Pfizer Monkey Virus Genomic RNA Packaging Determinants.

How retroviral Gag proteins recognize the packaging signals (Psi) on their genomic RNA (gRNA) is a key question that we addressed here using Mason-Pfizer monkey virus (MPMV) as a model system by combining band-shift assays and footprinting experiments. Our data show that Pr78Gag selects gRNA against spliced viral RNA by simultaneously binding to two single stranded loops on the MPMV Psi RNA: (1) a large purine loop (ssPurines), and (2) a loop which partially overlaps with a mostly base-paired purine repeat (bpPurines) and extends into a GU-rich binding motif. Importantly, this second Gag binding site is located immediately downstream of the major splice donor (mSD) and is thus absent from the spliced viral RNAs. Identifying elements crucial for MPMV gRNA packaging should help in understanding not only the mechanism of virion assembly by retroviruses, but also facilitate construction of safer retroviral vectors for human gene therapy.

Role of Purine-Rich Regions in Mason-Pfizer Monkey Virus (MPMV) Genomic RNA Packaging and Propagation.

A distinguishing feature of the Mason-Pfizer monkey virus (MPMV) packaging signal RNA secondary structure is a single-stranded purine-rich sequence (ssPurines) in close vicinity to a palindromic stem loop (Pal SL) that functions as MPMV dimerization initiation site (DIS). However, unlike other retroviruses, MPMV contains a partially base-paired repeat sequence of ssPurines (bpPurines) in the adjacent region. Both purine-rich sequences have earlier been proposed to act as potentially redundant Gag binding sites to initiate the process of MPMV genomic RNA (gRNA) packaging. The objective of this study was to investigate the biological significance of ssPurines and bpPurines in MPMV gRNA packaging by systematic mutational and biochemical probing analyses. Deletion of either ssPurines or bpPurines individually had no significant effect on MPMV gRNA packaging, but it was severely compromised when both sequences were deleted simultaneously. Selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) analysis of the mutant RNAs revealed only mild effects on structure by deletion of either ssPurines or bpPurines, while the structure was dramatically affected by the two simultaneous deletions. This suggests that ssPurines and bpPurines play a redundant role in MPMV gRNA packaging, probably as Gag binding sites to facilitate gRNA capture and encapsidation. Interestingly, the deletion of bpPurines revealed an additional severe defect on RNA propagation that was independent of the presence or absence of ssPurines or the gRNA structure of the region. These findings further suggest that the bpPurines play an additional role in the early steps of MPMV replication cycle that is yet to be identified.

Purification and Functional Characterization of a Biologically Active Full-Length Feline Immunodeficiency Virus (FIV) Pr50Gag.

The feline immunodeficiency virus (FIV) full-length Pr50Gag precursor is a key player in the assembly of new viral particles. It is also a critical component of the efficient selection and packaging of two copies of genomic RNA (gRNA) into the newly formed virus particles from a wide pool of cellular and spliced viral RNA. To understand the molecular mechanisms involved during FIV gRNA packaging, we expressed the His6-tagged and untagged recombinant FIV Pr50Gag protein both in eukaryotic and prokaryotic cells. The recombinant Pr50Gag-His6-tag fusion protein was purified from soluble fractions of prokaryotic cultures using immobilized metal affinity chromatography (IMAC). This purified protein was able to assemble in vitro into virus-like particles (VLPs), indicating that it preserved its ability to oligomerize/multimerize. Furthermore, VLPs formed in eukaryotic cells by the FIV full-length Pr50Gag both in the presence and absence of His6-tag could package FIV sub-genomic RNA to similar levels, suggesting that the biological activity of the recombinant full-length Pr50Gag fusion protein was retained in the presence of His6-tag at the carboxy terminus. Successful expression and purification of a biologically active, recombinant full-length Pr50Gag-His6-tag fusion protein will allow study of the intricate RNA-protein interactions involved during FIV gRNA encapsidation.

Stabilizing role of structural elements within the 5´ Untranslated Region (UTR) and gag sequences in Mason-Pfizer monkey virus (MPMV) genomic RNA packaging.

The Mason-Pfizer monkey virus (MPMV) genomic RNA (gRNA) packaging signal is a highly-structured element with several stem-loops held together by two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences. These LRIs play a critical role in maintaining the structure of the 5´ end of the MPMV gRNA. Thus, one could hypothesize that the overall RNA secondary structure of this region is further architecturally held together by three other stem loops (SL3, Gag SL1, and Gag SL2) comprising of sequences from the distal parts of the 5´untranslated region (5' UTR) to ~ 120 nucleotides into gag, excluding gag sequences involved in forming the U5-Gag LRIs. To provide functional evidence for the biological significance of these stem loops during gRNA encapsidation, these structural motifs were mutated and their effects on MPMV RNA packaging and propagation were tested in a single round trans-complementation assay. The mutant RNA structures were further studied by high throughput SHAPE (hSHAPE) assay. Our results reveal that sequences involved in forming these three stem loops do not play crucial roles at an individual level during MPMV gRNA packaging or propagation. Further structure-function analysis indicates that the U5-Gag LRIs have a more important architectural role in stabilizing the higher order structure of the 5´ UTR than the three stem loops which have a more secondary and perhaps indirect role in stabilizing the overall RNA secondary structure of the region. Our work provides a better understanding of the molecular interactions that take place during MPMV gRNA packaging.