Visualization of Retroviral Gag-Genomic RNA Cellular Interactions Leading to Genome Encapsidation and Viral Assembly: An Overview.

Retroviruses must selectively recognize their unspliced RNA genome (gRNA) among abundant cellular and spliced viral RNAs to assemble into newly formed viral particles. Retroviral gRNA packaging is governed by Gag precursors that also orchestrate all the aspects of viral assembly. Retroviral life cycles, and especially the HIV-1 one, have been previously extensively analyzed by several methods, most of them based on molecular biology and biochemistry approaches. Despite these efforts, the spatio-temporal mechanisms leading to gRNA packaging and viral assembly are only partially understood. Nevertheless, in these last decades, progress in novel bioimaging microscopic approaches (as FFS, FRAP, TIRF, and wide-field microscopy) have allowed for the tracking of retroviral Gag and gRNA in living cells, thus providing important insights at high spatial and temporal resolution of the events regulating the late phases of the retroviral life cycle. Here, the implementation of these recent bioimaging tools based on highly performing strategies to label fluorescent macromolecules is described. This report also summarizes recent gains in the current understanding of the mechanisms employed by retroviral Gag polyproteins to regulate molecular mechanisms enabling gRNA packaging and the formation of retroviral particles, highlighting variations and similarities among the different retroviruses.

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.

The Life-Cycle of the HIV-1 Gag-RNA Complex.

Human immunodeficiency virus type 1 (HIV-1) replication is a highly regulated process requiring the recruitment of viral and cellular components to the plasma membrane for assembly into infectious particles. This review highlights the recent process of understanding the selection of the genomic RNA (gRNA) by the viral Pr55(Gag) precursor polyprotein, and the processes leading to its incorporation into viral particles.

APOBEC3G impairs the multimerization of the HIV-1 Vif protein in living cells.

The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Vif allows productive infection in nonpermissive cells, including most natural HIV-1 target cells, by counteracting the cellular cytosine deaminases APOBEC3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G [A3G]) and A3F. Vif is also associated with the viral assembly complex and packaged into viral particles through interactions with the viral genomic RNA and the nucleocapsid domain of Pr55(Gag). Recently, we showed that oligomerization of Vif into high-molecular-mass complexes induces Vif folding and influences its binding to high-affinity RNA binding sites present in the HIV genomic RNA. To get further insight into the role of Vif multimerization in viral assembly and A3G repression, we used fluorescence lifetime imaging microscopy (FLIM)- and fluorescence resonance energy transfer (FRET)-based assays to investigate Vif-Vif interactions in living cells. By using two N-terminally tagged Vif proteins, we show that Vif-Vif interactions occur in living cells. This oligomerization is strongly reduced when the putative Vif multimerization domain ((161)PPLP(164)) is mutated, indicating that this domain is crucial, but that regions outside this motif also participate in Vif oligomerization. When coexpressed together with Pr55(Gag), Vif is largely relocated to the cell membrane, where Vif oligomerization also occurs. Interestingly, wild-type A3G strongly interferes with Vif multimerization, contrary to an A3G mutant that does not bind to Vif. These findings confirm that Vif oligomerization occurs in living cells partly through its C-terminal motif and suggest that A3G may target and perturb the Vif oligomerization state to limit its functions in the cell.

RNA and DNA binding properties of HIV-1 Vif protein: a fluorescence study.

The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Some "non-permissive" cell lines cannot sustain replication of Vif(-) HIV-1 virions. In these cells, Vif counteracts the natural antiretroviral activity of the DNA-editing enzymes APOBEC3G/3F. Moreover, Vif is packaged into viral particles through a strong interaction with genomic RNA in viral nucleoprotein complexes. To gain insights into determinants of this binding process, we performed the first characterization of Vif/nucleic acid interactions using Vif intrinsic fluorescence. We determined the affinity of Vif for RNA fragments corresponding to various regions of the HIV-1 genome. Our results demonstrated preferential and moderately cooperative binding for RNAs corresponding to the 5'-untranslated region of HIV-1 (5'-untranslated region) and gag (cooperativity parameter omega approximately 65-80, and K(d) = 45-55 nM). In addition, fluorescence spectroscopy allowed us to point out the TAR apical loop and a short region in gag as primary strong affinity binding sites (K(d) = 9.5-14 nM). Interestingly, beside its RNA binding properties, the Vif protein can also bind the corresponding DNA oligonucleotides and their complementary counterparts with an affinity similar to the one observed for the RNA sequences, while other DNA sequences displayed reduced affinity. Taken together, our results suggest that Vif binding to RNA and DNA offers several non-exclusive ways to counteract APOBEC3G/3F factors, in addition to the well documented Vif-induced degradation by the proteasome and to the Vif-mediated repression of translation of these antiviral factors.

Impact of the terminal bulges of HIV-1 cTAR DNA on its stability and the destabilizing activity of the nucleocapsid protein NCp7.

Reverse transcription of HIV-1 genomic RNA to double-stranded DNA by reverse transcriptase (RT) is a critical step in HIV-1 replication. This process relies on two viral proteins, the RT enzyme and nucleocapsid protein NCp7 that has well documented nucleic acid chaperone properties. At the beginning of the linear DNA synthesis, the newly made minus-strand strong-stop DNA ((-)ssDNA) is transferred to the 3'end of the genomic RNA by means of an hybridization reaction between transactivation response element (TAR) RNA and cTAR DNA sequences. Since both TAR sequences exhibit stable hairpin structures, NCp7 needs to destabilize the TAR structures in order to chaperone their hybridization. To further characterize the relationships between TAR stability and NC-mediated destabilization, the role of the A(49) and G(52) bulged residues in cTAR DNA stability was investigated. The stability of cTAR and mutants where one or the two terminal bulges were replaced by base-pairs as well as the NCp7-mediated destabilization of these cTAR sequences were examined. Thermodynamic data indicate that the two bulges cooperatively destabilize cTAR by reducing the stacking interactions between the bases. This causes a free energy change of about 6.4 kcal/mol and seems to be critical for NC activity. Time-resolved fluorescence data of doubly labelled cTAR derivatives suggest that NC-mediated melting of cTAR ends propagates up to the 10C.A(44) mismatch or T(40) bulge. Fluorescence correlation spectroscopy using two-photon excitation was also used to monitor cTAR ends fraying by NC. Results show that NC causes a very significant increase of cTAR ends fraying, probably limited to the terminal base-pair in the case of cTAR mutants. Since the TAR RNA and cTAR DNA bulges or mismatches appear well conserved among all HIV-1 strains, the present data support the notion of a co-evolutionary relationship between TAR and NC activity.

HIV-1 nucleocapsid protein activates transient melting of least stable parts of the secondary structure of TAR and its complementary sequence.

The nucleocapsid protein NCp7 of HIV-1 possesses a nucleic acid chaperone activity that is critical in minus and plus strand transfer during reverse transcription. The minus strand transfer notably relies on the ability of NCp7 to destabilize the stable stem with five contiguous, double-stranded segments of both the TAR sequence at the 3' end of the viral genome and the complementary sequence, cTAR, in minus strong-stop DNA. In order to examine the nature and the extent of NCp7 destabilizing activity, we investigated, by absorbance and fluorescence spectroscopy, the interaction of TAR and cTAR with a (12-55)NCp7 peptide containing the zinc-finger motifs but lacking the ability to aggregate the oligonucleotides. The absorbance changes in the UV band of cTAR show that seven to eight base-pairs, on average, are melted per oligonucleotide at a ratio of one peptide to 7.5 nucleotides. In contrast, the melting of TAR does not exceed an average of one base-pair per oligonucleotide. This may be linked to the greater stability of TAR, since a strong correlation between NCp7 destabilizing effect and oligonucleotide stability was observed. The effect of (12-55)NCp7 on the stem terminus was investigated by using a cTAR molecule doubly labeled at the 3' and 5' ends by a donor/acceptor couple. In the absence of the peptide, about 80 % of the oligonucleotides are in a dark non-fluorescent state, having a close proximity of the two dyes. The remaining 20 % are distributed between three fluorescent species, having either the terminal segment, the two terminal segments or all segments of the stem melted. This is in line with a fraying mechanism wherein the stem terminus fluctuates rapidly between open and closed states. Addition of (12-55)NCp7 shifts the equilibrium toward the open species, suggesting that NC enhances fraying of the stem terminus. Taken together, our data suggest that NCp7 activates the transient opening of base-pairs in the least stable parts of the stem. Also, this activity of NCp7 was found to be dependent on the zinc-finger motifs, since no melting was observed with a fingerless NCp7 peptide.