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.     

The brain-specific double-stranded RNA-binding protein Staufen2 is required for dendritic spine morphogenesis

Mammalian Staufen2 (Stau2) is a member of the double-stranded RNA-binding protein family. Its expression is largely restricted to the brain. It is thought to play a role in the delivery of RNA to dendrites of polarized neurons. To investigate the function of Stau2 in mature neurons, we interfered with Stau2 expression by RNA interference (RNAi). Mature neurons lacking Stau2 displayed a significant reduction in the number of dendritic spines and an increase in filopodia-like structures. The number of PSD95-positive synapses and miniature excitatory postsynaptic currents were markedly reduced in Stau2 down-regulated neurons. Akin effects were caused by overexpression of dominant-negative Stau2. The observed phenotype could be rescued by overexpression of two RNAi cleavage-resistant Stau2 isoforms. In situ hybridization revealed reduced expression levels of beta-actin mRNA and fewer dendritic beta-actin mRNPs in Stau2 down-regulated neurons. Thus, our data suggest an important role for Stau2 in the formation and maintenance of dendritic spines of hippocampal neurons.     

The human immunodeficiency virus type 1 encapsidation site is a multipartite RNA element composed of functional hairpin structures.

We analyzed the leader region of human immunodeficiency virus type 1 (HIV-1) RNA to decipher the nature of the cis-acting E/psi element required for encapsidation of viral RNA into virus particles. Our data indicate that, for RNA encapsidation, there are at least two functional subregions in the leader region. One subregion is located at a position immediately proximal to the major splice donor, and the second is located between the splice donor and the beginning of the gag gene. This suggests that at least two discrete cis-acting elements are recognition signals for encapsidation. To determine whether specific putative RNA secondary structures serve as the signal(s) for encapsidation, we constructed primary base substitution mutations that would be expected to destabilize these potential structures and second-site compensatory mutations that would restore secondary structure. Analysis of these mutants allowed the identification of two discrete hairpins that facilitate RNA encapsidation in vivo. Thus, the HIV-1 E/psi region is a multipartite element composed of specific and functional RNA secondary structures. Compensation of the primary mutations by the second-site mutations could not be attained in trans. This indicates that interstrand base pairing between these two stem regions within the hairpins does not appear to be the basis for HIV-1 RNA dimer formation. Comparison of the hypothetical RNA secondary structures from 10 replication-competent HIV-1 strains suggests that a subset of the hydrogen-bonded base pairs within the stems of the hairpins is likely to be required for function in cis.     

The double-stranded RNA-binding protein,Staufen1, is an IRES-transacting factor regulating HIV-1 cap-independent translation initiation

Translation initiation of the viral genomic mRNA (vRNA) of human immunodeficiency virus-type 1 (HIV-1) can be mediated by a cap- or an internal ribosome entry site (IRES)-dependent mechanism. A previous report shows that Staufen1, a cellular double-stranded (ds) RNA-binding protein (RBP), binds to the 5’untranslated region (5′UTR) of the HIV-1 vRNA and promotes its cap-dependent translation. In this study, we now evaluate the role of Staufen1 as an HIV-1 IRES-transacting factor (ITAF). We first confirm that Staufen1 associates with both the HIV-1 vRNA and the Gag protein during HIV-1 replication. We found that in HIV-1-expressing cells, siRNA-mediated depletion of Staufen1 reduces HIV-1 vRNA translation. Using dual-luciferase bicistronic mRNAs, we show that the siRNA-mediated depletion and cDNA-mediated overexpression of Staufen1 acutely regulates HIV-1 IRES activity. Furthermore, we show that Staufen1-vRNA interaction is required for the enhancement of HIV-1 IRES activity. Interestingly, we find that only Staufen1 harboring an intact dsRNA-binding domain 3 (dsRBD3) rescues HIV-1 IRES activity in Staufen1 CRISPR-Cas9 gene edited cells. Finally, we show that the expression of Staufen1-dsRBD3 alone enhances HIV-1 IRES activity. This study provides evidence of a novel role for Staufen1 as an ITAF promoting HIV-1 vRNA IRES activity.     

Noninfectious human immunodeficiency virus type 1 mutants deficient in genomic RNA.

All retroviruses contain, in the nucleocapsid domain of the Gag protein, one or two copies of the sequence Cys-X2-Cys-X4-His-X4-Cys. We have generated a series of mutants in the two copies of this motif present in human immunodeficiency virus type 1. These mutants encoded virus particles that were apparently composed of the normal complement of viral proteins but contained only 2 to 20% of the normal level of genomic RNA. No infectivity could be detected in the mutant particles, while 10(5) infectious U were present in an equivalent amount of wild-type particles. Thus, the mutants have another defect in addition to the inefficiency with which they encapsidate genomic RNA. Our results show that both copies of the motif are required for normal RNA packaging and for infectivity. Mutants of this type may have important applications, including nonhazardous materials for research, immunogens in vaccine and immunotherapy studies, and diagnostic reagents.     

Position dependence of functional hairpins important for human immunodeficiency virus type 1 RNA encapsidation in vivo.

At least two hairpins in the 5' untranslated leader region, stem-loops 1 and 3 (SL1 and SL3), contribute to human immunodeficiency virus type 1 RNA encapsidation in vivo. We used a competitive assay, which measures the relative encapsidation efficiency of mutant viral RNA in the presence of competing wild-type RNA, to compare the contributions of SL1, SL3, and two adjacent secondary structures, SL2 and SL4, to encapsidation. SL2 is not required for RNA encapsidation, while SL1, SL3, and SL4 all contribute approximately equally to encapsidation. To determine whether these hairpins function in a position-dependent manner, we interchanged the positions of two of these stem-loop structures. This resulted in substantial diminution of encapsidation, indicating that the secondary structures that comprise E, the encapsidation signal, function only in their correct contexts. Mutation of nucleotides flanking SL1 and SL3 had little effect on encapsidation. We also showed that SL1, while present on both genomic and subgenomic viral RNAs, nonetheless contributes to selective encapsidation of genomic RNA. Taken together, these data are consistent with the formation of a higher-order RNA structure, partially composed of SL1, SL3, and SL4, that functions to effect concurrent encapsidation of full-length RNA and exclusion of subgenomic RNA. Finally, it has been reported that E is required for efficient translation of Gag mRNA in vivo. However, we have found that a variety of mutants, including a mutant lacking the entire region encompassing SL1, SL2, and SL3, still produce RNAs that are efficiently translated. These data indicate that E is unlikely to contribute to efficient Gag mRNA translation in vivo.     

trans-acting proteins involved in RNA encapsidation and viral assembly in human immunodeficiency virus type 1.

The human immunodeficiency virus type 1 gag gene product Pr55gag self-assembles when expressed on its own in a variety of eukaryotic systems. Assembly in T lymphocytes has not previously been studied, nor is it clear whether Pr55gag particles can package genomic RNA or if the Gag-Pol polyprotein is required. We have used a series of constructs that express Gag or Gag-Pol proteins with or without the viral protease in transient transfections in COS-1 cells and also expressed stably in CD4+ T cells to study this. Deletion of the p6 domain at the C terminus of protease-negative Pr55gag did not abolish particle release, while truncation of the nucleocapsid protein reduced it significantly, particularly in lymphocytes. Gag-Pol polyprotein was released from T cells in the absence of Pr55gag but did not encapsidate RNA. Pr55gag encapsidated human immunodeficiency virus type 1 RNA whether expressed in a protease-positive or protease-negative context. p6 was dispensable for RNA encapsidation. Marked differences in the level of RNA export were noted between the different cell lines.     

Determination of the structure of the RNA complex of a double-stranded RNA-binding domain from Drosophila Staufen protein

We have determined using NMR the structure of the complex between the third double-stranded RNA-binding domain (dsRBD3) of Drosophila Staufen protein and a RNA stem-loop with optimal binding properties in vitro. This work was designed to understand how dsRBD proteins bind RNA and to investigate the role of Staufen dsRBDs in the localization of maternal RNAs during early embryonic development. The structure determination was challenging, because of weak, nonsequence specific binding and residual conformational flexibility at the RNA-protein interface. In order to overcome the problems originated by the weak interaction, we used both new and more traditional approaches to obtain distance and orientation information for the protein and RNA components of the complex. The resulting structure allowed the verification of aspects of RNA recognition by dsRBDs matching the information obtained by a related crystallographic study. We were also able to generate new observations that are likely to be relevant to dsRBD-RNA binding and to the physiological role of Staufen protein.Copyright 2001 John Wiley & Sons, Inc.     

Simian immunodeficiency virus RNA is efficiently encapsidated by human immunodeficiency virus type 1 particles.

Packaging of retroviral RNA is attained through the specific recognition of a cis-acting encapsidation site (located near the 5' end of the viral RNA) by components of the Gag precursor protein. Human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) are two lentiviruses that lack apparent sequence similarity in their putative encapsidation regions. We used SIV vectors to determine whether HIV-1 particles can recognize the SIV encapsidation site and functionally propagate SIV nucleic acid. SIV nucleic acid was replicated by HIV-1 proteins. Thus, efficient lentivirus pseudotyping can take place at the RNA level. Direct examination of the RNA contents of virus particles indicated that encapsidation of this heterologous RNA is efficient. Characterization of deletion mutants in the untranslated leader region of SIV RNA indicates that only a very short region at the 5' end of the SIV RNA is needed for packaging. Comparison of this region with the corresponding region of HIV-1 reveals that both are marked by secondary structures that are likely to be similar. Thus, it is likely that a similar higher-order RNA structure is required for encapsidation.     

Cytoplasmic utilization of human immunodeficiency virus type 1 genomic RNA is not dependent on a nuclear interaction with gag

In some retroviruses, such as Rous sarcoma virus and prototype foamy virus, Gag proteins are known to shuttle between the nucleus and the cytoplasm and are implicated in nuclear export of the viral genomic unspliced RNA (gRNA) for subsequent encapsidation. A similar function has been proposed for human immunodeficiency virus type 1 (HIV-1) Gag based on the identification of nuclear localization and export signals. However, the ability of HIV-1 Gag to transit through the nucleus has never been confirmed. In addition, the lentiviral Rev protein promotes efficient nuclear gRNA export, and previous reports indicate a cytoplasmic interaction between Gag and gRNA. Therefore, functional effects of HIV-1 Gag on gRNA and its usage were explored. Expression of gag in the absence of Rev was not able to increase cytoplasmic gRNA levels of subgenomic, proviral, or lentiviral vector constructs, and gene expression from genomic reporter plasmids could not be induced by Gag provided in trans. Furthermore, Gag lacking the reported nuclear localization and export signals was still able to mediate an efficient packaging process. Although small amounts of Gag were detectable in the nuclei of transfected cells, a Crm1-dependent nuclear export signal in Gag could not be confirmed. Thus, our study does not provide any evidence for a nuclear function of HIV-1 Gag. The encapsidation process of HIV-1 therefore clearly differs from that of Rous sarcoma virus and prototype foamy virus.     

The brain-specific double-stranded RNA-binding protein Staufen2: nucleolar accumulation and isoform-specific exportin-5-dependent export

The mammalian double-stranded RNA-binding proteins Staufen (Stau1 and Stau2) are involved in RNA localization in polarized neurons. In contrast to the more ubiquitously expressed Stau1, Stau2 is mainly expressed in the nervous system. In Drosophila, the third double-stranded RNA-binding domain (RBD3) of Staufen is essential for RNA interaction. When conserved amino acids within the RBD3 of Stau2 were mutated to render Stau2 defective for RNA binding, the mutant Stau2 proteins accumulate predominantly in the nucleolus. This is in contrast to wild type Stau2 that mostly localizes in the cytosol. The nuclear import is dependent on a nuclear localization signal in close proximity to the RBD3. The nuclear export of Stau2 is not dependent on CRM1 but rather on Exportin-5. We show that Exportin-5 interacts with the RBD3 of wild type Stau2 in an RNA-dependent manner in vitro but not with mutant Stau2. When Exportin-5 is down-regulated by RNA interference, only the largest isoform of Stau2 (Stau2(62)) preferentially accumulates in the nucleolus. It is tempting to speculate that Stau2(62) binds RNA in the nucleus and assembles into ribonucleoparticles, which are then exported via the Exportin-5 pathway to their final destination.     

Distinct functions and requirements for the Cys-His boxes of the human immunodeficiency virus type 1 nucleocapsid protein during RNA encapsidation and replication.

The process of retroviral RNA encapsidation involves interaction between trans-acting viral proteins and cis-acting RNA elements. The encapsidation signal on human immunodeficiency virus type 1 (HIV-1) RNA is a multipartite structure composed of functional stem-loop structures. The nucleocapsid (NC) domain of the Gag polyprotein precursor contains two copies of a Cys-His box motif that have been demonstrated to be important in RNA encapsidation. To further characterize the role of the Cys-His boxes of the HIV-1 NC protein in RNA encapsidation, the relative efficiency of RNA encapsidation for virus particles that contained mutations within the Cys-His boxes was measured. Mutations that disrupted the first Cys-His box of the NC protein resulted in virus particles that encapsidated genomic RNA less efficiently and subgenomic RNA more efficiently than did wild-type virus. Mutations within the second Cys-His box did not significantly affect RNA encapsidation. In addition, a full complement of wild-type NC protein in virus particles is not required for efficient RNA encapsidation or virus replication. Finally, both Cys-His boxes of the NC protein play additional roles in virus replication.     

The host protein Staufen1 participates in human immunodeficiency virus type 1 assembly in live cells by influencing pr55Gag multimerization

Human immunodeficiency virus type 1 (HIV-1) requires the sequential activities of virus-encoded proteins during replication. The activities of several host cell proteins and machineries are also critical to the completion of virus assembly and the release of infectious virus particles from cells. One of these proteins, the double-stranded RNA-binding protein Staufen1 (Stau1), selectively associates with the HIV-1 genomic RNA and the viral precursor Gag protein, pr55Gag. In this report, we tested whether Stau1 modulates pr55Gag assembly using a new and specific pr55Gag oligomerization assay based on bioluminescence resonance energy transfer (BRET) in both live cells and extracts after cell fractionation. Our results show that both the overexpression and knockdown of Stau1 increase the pr55Gag-pr55Gag BRET levels, suggesting a role for Stau1 in regulating pr55Gag oligomerization during assembly. This effect of Stau1 on pr55Gag oligomerization was observed only in membranes, a cellular compartment in which pr55Gag assembly primarily occurs. Consistently, expression of Stau1 harboring a vSrc myristylation signal led to a 6.5-fold enrichment of Stau1 in membranes and a corresponding enhancement in the Stau1-mediated effect on pr55Gag-pr55Gag BRET, demonstrating that Stau1 acts on assembly when targeted to membranes. A role for Stau1 in the formation of particles is further supported by the detection of membrane-associated detergent-resistant pr55Gag complexes and the increase of virus-like particle release when Stau1 expression levels are modulated. Our results indicate that Stau1 influences HIV-1 assembly by modulating pr55Gag-pr55Gag interactions, as shown in a live cell interaction assay. This likely occurs when Stau1 interacts with membrane-associated assembly intermediates.     

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.     

The double-stranded RNA-binding protein X1rbpa promotes RNA strand annealing.

RNA-annealing activity is a common feature of several RNA-binding proteins. The Xenopus RNA-binding protein X1rbpa is composed of three tandemly arranged double-stranded RNA-binding domains (dsRBDs) but lacks any other catalytic or functional domains, therefore making the assessment of biological functions of this protein rather difficult. Here we show that full-length X1rbpa but also isolated dsRBDs from this protein can facilitate RNA strand annealing. RNA annealing can be efficiently inhibited by heparin. However, dsRBDs with a neutral pI still promote strand annealing, suggesting that charged residues within the dsRBD are important for strand annealing. Additionally, mutant versions of the dsRBD, unable to bind dsRNA in northwestern assays, were tested. Of these, some show RNA-annealing activity while others fail to do so, indicating that RNA annealing and dsRNA binding are separable functions. Our data, together with the previously reported association of the protein with most cellular RNAs, suggests an RNA chaperone-like function of X1rbpa.     

Efficient encapsidation of human immunodeficiency virus type 1 vectors and further characterization of cis elements required for encapsidation.

To determine whether there is a cis-acting effect of translational expression of gag on RNA encapsidation, we compared the encapsidation of wild-type RNA with that of a mutant in which the translation of gag was ablated. This comparison indicated that there is not such a cis effect. To determine what is necessary and sufficient for encapsidation, we measured the relative encapsidation efficiencies of human immunodeficiency virus type 1 vector RNAs containing mutations in domains proximal to the canonical encapsidation signal or containing large deletions in the remainder of the genome. These data indicate that TAR and two additional regions are required for encapsidation and that the 5' end of the genome is sufficient for encapsidation. The Rev-responsive element is required mainly for efficient RNA transport from the nucleus to the cytoplasm. A foreign sequence was found to have a negative effect on encapsidation upon placement within the parental vector. Interestingly, this negative effect was compounded by multiple copies of the sequence.     

Human immunodeficiency virus types 1 and 2 differ in the predominant mechanism used for selection of genomic RNA for encapsidation

Retroviral RNA encapsidation is a highly selective process mediated through recognition by the viral Gag proteins of cis-acting RNA packaging signals in genomic RNA. This RNA species is also translated, producing the viral gag gene products. The relationship between these processes is poorly understood. Unlike that of human immunodeficiency virus type 1 (HIV-1), the dominant packaging signal of HIV-2 is upstream of the major splice donor and present in both unspliced and spliced viral RNAs, necessitating additional mechanisms for preferential packaging of unspliced genomic RNA. Encapsidation studies of a series of HIV-2-based vectors showed efficient packaging of viral genomes only if the unspliced, encapsidated RNA expressed full-length Gag protein, including functional nucleocapsid. We propose a novel encapsidation initiation mechanism, providing selectivity, in which unspliced HIV-2 RNA is captured in cis by the Gag protein. This has implications for the use of HIV-2 and other lentiviruses as vectors.