Randomization and in vivo selection reveal a GGRG motif essential for packaging human immunodeficiency virus type 2 RNA

The packaging signal (ψ) of human immunodeficiency virus type 2 (HIV-2) is present in the 5′ noncoding region of RNA and contains a 10-nucleotide palindrome (pal; 5′-392-GGAGUGCUCC) located upstream of the dimerization signal stem-loop 1 (SL1). pal has been shown to be functionally important in vitro and in vivo. We previously showed that the 3′ side of pal (GCUCC-3′) is involved in base-pairing interactions with a sequence downstream of SL1 to make an extended SL1, which is important for replication in vivo and the regulation of dimerization in vitro. However, the role of the 5′ side of pal (5′-GGAGU) was less clear. Here, we characterized this role using an in vivo SELEX approach. We produced a population of HIV-2 DNA genomes with random sequences within the 5′ side of pal and transfected these into COS-7 cells. Viruses from COS-7 cells were used to infect C8166 permissive cells. After several weeks of serial passage in C8166 cells, surviving viruses were sequenced. On the 5′ side of pal there was a striking convergence toward a GGRGN consensus sequence. Individual clones with consensus and nonconsensus sequences were tested in infectivity and packaging assays. Analysis of individuals that diverged from the consensus sequence showed normal viral RNA and protein synthesis but had replication defects and impaired RNA packaging. These findings clearly indicate that the GGRG motif is essential for viral replication and genomic RNA packaging.     

Elements located upstream and downstream of the major splice donor site influence the ability of HIV-2 leader RNA to dimerize in vitro

An essential step in the replication cycle of all retroviruses is the dimerization of genomic RNA prior to or during budding and maturation of the viral particle. In HIV-1, a 5' leader region site termed stem-loop 1 (SL1) promotes RNA dimerization in vitro and influences dimerization in vivo. In HIV-2, two sequences promote dimerization of RNA fragments in vitro: the 5'-end of the primer-binding site (PBS) and a stem-loop region homologous to the HIV-1 SL1 sequence. Because HIV-2 RNA constructs of different lengths use these two dimerization signals disproportionately, we hypothesized that other sequences could modulate their relative utilization. Here, we characterized the influence of sequences upstream and downstream of the major splice donor site on the formation of HIV-2 RNA dimers in vitro using a variety of RNA constructs and dimerization and electrophoresis protocols. We first assayed the formation of loose or tight dimers for 1-444 and 1-561 model RNAs. Although both RNAs could form PBS-dependent loose dimers, the 1-561 RNA was unable to make SL1-dependent tight dimers. Using RNAs truncated at their 5'- and/or 3'-ends and by making compensatory base substitutions, we found that two elements interfere with the formation of SL1-dependent tight dimers. The cores of these elements are located at nucleotides 189-196 and 543-550. Our results suggest that base pairing between these sequences prevents the formation of SL1-dependent tight dimers, probably by sequestering SL1 in a stable intramolecular arrangement. Moreover, we found that nucleotides downstream of SL1 decreased the rate of tight dimerization. Interestingly, dimerization at 37 degrees C in the presence of nucleocapsid protein increased the yield of SL1-mediated tight dimerization in vitro, even in the presence of the two interfering elements, suggesting a relationship between the nucleocapsid protein and activation of the SL1 dimerization signal in vivo.     

Role of the 5' TAR stem--loop and the U5-AUG duplex in dimerization of HIV-1 genomic RNA

The HIV-1 genome consists of two identical RNAs that are linked together through noncovalent interactions involving nucleotides from the 5' untranslated region (5' UTR) of each RNA strand. The 5' UTR is the most conserved part of the HIV-1 RNA genome, and its 335 nucleotide residues form regulatory motifs that mediate multiple essential steps in the viral replication cycle. Here, studying the effect of selected mutations both singly and together with mutations disabling SL1 (SL1 is a 5' UTR stem-loop containing a palindrome called the dimerization initiation site), we have done a rather systematic survey of the 5' UTR requirements for full genomic RNA dimerization in grown-up (i.e., predominantly >/=10 h old) HIV-1 viruses produced by transfected human and simian cells. We have identified a role for the 5' transactivation response element (5' TAR) and a contribution of a long-distance base pairing between a sequence located at the beginning of the U5 region and nucleotides surrounding the AUG Gag initiation codon. The resulting intra- or intermolecular duplex is called the U5-AUG duplex. The other regions of the 5' UTR have been shown to play no systematic role in genomic RNA dimerization, except for a sequence located around the 3' end of a large stem-loop enclosing the primer binding site, and the well-documented SL1. Our data are consistent with a direct role for the 5' TAR in genomic RNA dimerization (possibly via a palindrome encompassing the apical loop of the 5' TAR).     

Identification of the in vitro HIV-2/SIV RNA dimerization site reveals striking differences with HIV-1

Although their genomes cannot be aligned at the nucleotide level, the HIV-1/SIVcpz and the HIV-2/SIVsm viruses are closely related lentiviruses that contain homologous functional and structural RNA elements in their 5'-untranslated regions. In both groups, the domains containing the trans-activating region, the 5'-copy of the polyadenylation signal, and the primer binding site (PBS) are followed by a short stem-loop (SL1) containing a six-nucleotide self-complementary sequence in the loop, flanked by unpaired purines. In HIV-1, SL1 is involved in the dimerization of the viral RNA, in vitro and in vivo. Here, we tested whether SL1 has the same function in HIV-2 and SIVsm RNA. Surprisingly, we found that SL1 is neither required nor involved in the dimerization of HIV-2 and SIV RNA. We identified the NarI sequence located in the PBS as the main site of HIV-2 RNA dimerization. cis and trans complementation of point mutations indicated that this self-complementary sequence forms symmetrical intermolecular interactions in the RNA dimer and suggested that HIV-2 and SIV RNA dimerization proceeds through a kissing loop mechanism, as previously shown for HIV-1. Furthermore, annealing of tRNA(3)(Lys) to the PBS strongly inhibited in vitro RNA dimerization, indicating that, in vivo, the intermolecular interaction involving the NarI sequence must be dissociated to allow annealing of the primer tRNA.     

The in vitro loose dimer structure and rearrangements of the HIV-2 leader RNA

RNA dimerization is an essential step in the retroviral life cycle. Dimerization and encapsidation signals, closely linked in HIV-2, are located in the leader RNA region. The SL1 motif and nucleocapsid protein are considered important for both processes. In this study, we show the structure of the HIV-2 leader RNA (+1-560) captured as a loose dimer. Potential structural rearrangements within the leader RNA were studied. In the loose dimer form, the HIV-2 leader RNA strand exists in vitro as a single global fold. Two kissing loop interfaces within the loose dimer were identified: SL1/SL1 and TAR/TAR. Evidence for these findings is provided by RNA probing using SHAPE, chemical reagents, enzymes, non-denaturing PAGE mobility assays, antisense oligonucleotides hybridization and analysis of an RNA mutant. Both TAR and SL1 as isolated domains are bound by recombinant NCp8 protein with high affinity, contrary to the hairpins downstream of SL1. Foot-printing of the SL1/NCp8 complex indicates that the major binding site maps to the SL1 upper stem. Taken together, these data suggest a model in which TAR hairpin III, the segment of SL1 proximal to the loop and the PAL palindromic sequence play specific roles in the initiation of dimerization.     

An extended stem-loop 1 is necessary for human immunodeficiency virus type 2 replication and affects genomic RNA encapsidation

Genomic RNA encapsidation in lentiviruses is a highly selective and regulated process. The unspliced RNA molecules are selected for encapsidation from a pool of many different viral and cellular RNA species. Moreover, two molecules are encapsidated per viral particle, where they are found associated as a dimer. In this study, we demonstrate that a 10-nucleotide palindromic sequence (pal) located at the 3' end of the psi encapsidation signal is critical for human immunodeficiency virus type 2 (HIV-2) replication and affects genomic RNA encapsidation. We used short-term and long-term culture of pal-mutated viruses in permissive C8166 cells and their phenotypic reversion to show the existence of a structurally extended SL1 during HIV-2 replication, formed by the interaction of the 3' end of the pal within psi with a motif located downstream of SL1. The stem extending HIV-2 SL1 is structurally similar to stem B described for HIV-1 SL1. Despite the high degree of phylogenetic conservation, these results show that mutant viruses are viable when the autocomplementary nature of the pal sequence is disrupted, but not without a stable stem B. Our observations show that formation of the extended SL1 is necessary during viral replication and positively affects HIV-2 genomic RNA encapsidation. Sequestration of part of the packaging signal into SL1 may be a means of regulating its presentation during the replication cycle.     

Sequence and structural elements at the 3' terminus of bovine viral diarrhea virus genomic RNA: functional role during RNA replication

Bovine viral diarrhea virus (BVDV), a member of the genus Pestivirus in the family Flaviviridae, has a positive-stranded RNA genome consisting of a single open reading frame and untranslated regions (UTRs) at the 5' and 3' ends. Computer modeling suggested the 3' UTR comprised single-stranded regions as well as stem-loop structures-features that were suspected of being essentially implicated in the viral RNA replication pathway. Employing a subgenomic BVDV RNA (DI9c) that was shown to function as an autonomous RNA replicon (S.-E. Behrens, C. W. Grassmann, H. J. Thiel, G. Meyers, and N. Tautz, J. Virol. 72:2364-2372, 1998) the goal of this study was to determine the RNA secondary structure of the 3' UTR by experimental means and to investigate the significance of defined RNA motifs for the RNA replication pathway. Enzymatic and chemical structure probing revealed mainly the conserved terminal part (termed 3'C) of the DI9c 3' UTR containing distinctive RNA motifs, i.e., a stable stem-loop, SL I, near the RNA 3' terminus and a considerably less stable stem-loop, SL II, that forms the 5' portion of 3'C. SL I and SL II are separated by a long single-stranded intervening sequence, denoted SS. The 3'-terminal four C residues of the viral RNA were confirmed to be single stranded as well. Other intramolecular interactions, e.g., with upstream DI9c RNA sequences, were not detected under the experimental conditions used. Mutagenesis of the DI9c RNA demonstrated that the SL I and SS motifs do indeed play essential roles during RNA replication. Abolition of RNA stems, which ought to maintain the overall folding of SL I, as well as substitution of certain single-stranded nucleotides located in the SS region or SL I loop region, gave rise to DI9c derivatives unable to replicate. Conversely, SL I stems comprising compensatory base exchanges turned out to support replication, but mostly to a lower degree than the original structure. Surprisingly, replacement of a number of residues, although they were previously defined as constituents of a highly conserved stretch of sequence of the SS motif, had little effect on the replication ability of DI9c. In summary, these results indicate that RNA structure as well as sequence elements harbored within the 3'C region of the BVDV 3' UTR create a common cis-acting element of the replication process. The data further point at possible interaction sites of host and/or viral proteins and thus provide valuable information for future experiments intended to identify and characterize these factors.     

A heterologous, high-affinity RNA ligand for human immunodeficiency virus Gag protein has RNA packaging activity

Retroviral RNA encapsidation depends on the specific binding of Gag proteins to packaging (psi) signals in genomic RNA. We investigated whether an in vitro-selected, high-affinity RNA ligand for the nucleocapsid (NC) portion of the Gag protein from human immunodeficiency virus type 1 (HIV-1) could mediate packaging into HIV-1 virions. We find that this ligand can functionally substitute for one of the Gag-binding elements (termed SL3) in the HIV-1 psi locus to support packaging and viral infectivity in cis. By contrast, this ligand, which fails to dimerize spontaneously in vitro, is unable to replace a different psi element (termed SL1) which is required for both Gag binding and dimerization of the HIV-1 genome. A single point mutation within the ligand that eliminates high-affinity in vitro Gag binding also abolishes its packaging activity at the SL3 position. These results demonstrate that specific binding of Gag or NC protein is a critical determinant of genomic RNA packaging.     

Possible role of dimerization in human immunodeficiency virus type 1 genome RNA packaging

The dimer initiation site/dimer linkage sequence (DIS/DLS) region in the human immunodeficiency virus type 1 (HIV-1) RNA genome is suggested to play important roles in various steps of the virus life cycle. However, due to the presence of a putative DIS/DLS region located within the encapsidation signal region (E/psi), it is difficult to perform a mutational analysis of DIS/DLS without affecting the packaging of RNA into virions. Recently, we demonstrated that duplication of the DIS/DLS region in viral RNA caused the production of partially monomeric RNAs in virions, indicating that the region indeed mediated RNA-RNA interaction. We utilized this system to assess the precise location of DIS/DLS in the 5' region of the HIV-1 genome with minimum effect on RNA packaging. We found that the entire lower stem of the U5/L stem-loop was required for packaging, whereas the region important for dimer formation was only 10 bases long within the lower stem of the U5/L stem-loop. The R/U5 stem-loop was required for RNA packaging but was completely dispensable for dimer formation. The SL1 lower stem was important for both dimerization and packaging, but surprisingly, deletion of the palindromic sequence at the top of the loop only partially affected dimerization. These results clearly indicated that the E/psi of HIV-1 is much larger than the DIS/DLS and that the primary DIS/DLS is completely included in the E/psi. Therefore, it is suggested that RNA dimerization is a part of RNA packaging, which requires multiple steps.     

RNA interactions in the 5' region of the HIV-1 genome

The untranslated leader of the dimeric HIV-1 RNA genome is folded into a complex structure that plays multiple and essential roles in the viral replication cycle. Here, we have investigated secondary and tertiary structural elements within the 5' 744 nucleotides of the HIV-1 genome using a combination of bioinformatics, enzymatic probing, native gel electrophoresis, and UV-crosslinking experiments. We used a recently developed RNA folding algorithm (Pfold) to predict the common secondary structure of an alignment of 20 divergent HIV-1 sequences. Combining this analysis with biochemical data, we present a secondary structure model for the entire 744 nucleotide fragment, which incorporates previously recognized and novel structural elements. In particular, our data provided strong evidence for a long-distance interaction between the region encompassing the AUG Gag initiation codon and an upstream region and we demonstrate that this feature is highly conserved in distantly related human and animal retroviruses. To obtain information about tertiary interactions we applied an intramolecular UV-crosslinking strategy and identified a novel tertiary interaction within the PBS hairpin structure.     

RNA structure and packaging signals in the 5' leader region of the human immunodeficiency virus type 1 genome

The leader region of the human immunodeficiency virus type 1 (HIV-1) genome has a highly folded structure, comprising at least two RNA stem-loops [the transactivation response (TAR) and poly(A) hairpins] near its 5' end and four others (SL1 to SL4) downstream. Each of these stem-loops contributes to the function of the HIV-1 packaging signal, which efficiently targets genomic RNA into nascent virions. The central 140-base region of the leader, which includes the U5 and primer binding site (PBS) sequences, is also believed to adopt a complex structure, but the nature of this structure and its possible role in RNA packaging have not been extensively explored. Here we report a mutational analysis identifying at least three separate loci within the U5-PBS region which, when mutated, impair both HIV-1 packaging specificity and infectivity in a single-round proviral assay. In common with those of all previously described packaging signals in the leader, the function of one of these loci appeared to depend on secondary structure rather than on sequence alone. By contrast, the activity of the other two loci did not correlate with any predicted conformations. Moreover, unlike SL1 to SL4, the TAR, poly(A), and U5-PBS hairpins were not bound with high affinity by the nucleocapsid portion of the HIV-1 Gag protein in vitro, implying that they contribute to packaging through a mechanism distinct from that of SL1 to SL4. Our findings confirm the existence and importance of secondary structure around the PBS and demonstrate that functional packaging signals are distributed across the entire HIV-1 leader.     

Sequences downstream of the 5' splice donor site are required for both packaging and dimerization of human immunodeficiency virus type 1 RNA

Two copies of human immunodeficiency virus type 1 RNA are incorporated into each virus particle and are further converted to a stable dimer as the virus particle matures. Several RNA segments that flank the 5' splice donor site at nucleotide (nt) 289 have been shown to act as packaging signals. Among these, RNA stem-loop 1 (SL1) (nt 243 to 277) can trigger RNA dimerization through a "kissing-loop" mechanism and thus is termed the dimerization initiation site. However, it is unknown whether other packaging signals are also needed for dimerization. To pursue this subject, we mutated stem-loop 3 (SL3) (nt 312 to 325), a GA-rich region (nt 325 to 336), and two G-rich repeats (nt 363 to 367 and nt 405 to 409) in proviral DNA and assessed the effects on RNA dimerization by performing native Northern blot analyses. Our results show that the structure but not the specific RNA sequence of SL3 is needed not only for efficient viral RNA packaging but also for dimerization. Mutations of the GA-rich sequence severely diminished viral RNA dimerization as well as packaging; the combination of mutations in both SL3 and the GA-rich region led to further decreases, implying independent roles for each of these two RNA motifs. Compensation studies further demonstrated that the RNA-packaging and dimerization activity of the GA-rich sequence may not depend on a putative interaction between this region and a CU repeat sequence at nt 227 to 233. In contrast, substitutions in the two G-rich sequences did not cause any diminution of viral RNA packaging or dimerization. We conclude that both the SL3 motif and GA-rich RNA sequences, located downstream of the 5' splice donor site, are required for efficient RNA packaging and dimerization.     

Inhibition of 5′-UTR RNA Conformational Switching in HIV-1 Using Antisense PNAs

Background

The genome of retroviruses, including HIV-1, is packaged as two homologous (+) strand RNA molecules, noncovalently associated close to their 5′-end in a region called dimer linkage structure (DLS). Retroviral HIV-1 genomic RNAs dimerize through complex interactions between dimerization initiation sites (DIS) within the (5′-UTR). Dimer formation is prevented by so calledLong Distance Interaction (LDI) conformation, whereas Branched Multiple Hairpin (BMH) conformation leads to spontaneous dimerization.

Methods and Results

We evaluated the role of SL1 (DIS), PolyA Hairpin signal and a long distance U5-AUG interaction by in-vitro dimerization, conformer assay and coupled dimerization and template-switching assays using antisense PNAs. Our data suggests evidence that PNAs targeted against SL1 produced severe inhibitory effect on dimerization and template-switching processes while PNAs targeted against U5 region do not show significant effect on dimerization and template switching, while PNAs targeted against AUG region showed strong inhibition of dimerization and template switching processes.

Conclusions

Our results demonstrate that PNA can be used successfully as an antisense to inhibit dimerization and template switching process in HIV -1 and both of the processes are closely linked to each other. Different PNA oligomers have ability of switching between two thermodynamically stable forms. PNA targeted against DIS and SL1 switch, LDI conformer to more dimerization friendly BMH form. PNAs targeted against PolyA haipin configuration did not show a significant change in dimerization and template switching process. The PNA oligomer directed against the AUG strand of U5-AUG duplex structure also showed a significant reduction in RNA dimerization as well as template- switching efficiency.The antisense PNA oligomers can be used to regulate the shift in the LDI/BMH equilibrium.