A 5′UTR-Spliced mRNA Isoform Is Specialized for Enhanced HIV-2 gag Translation

Full-length unspliced genomic RNA plays critical roles in HIV replication, serving both as mRNA for the synthesis of the key viral polyproteins Gag and Gag-Pol and as genomic RNA for encapsidation into assembling viral particles. We show that a second gag mRNA species that differs from the genomic RNA molecule by the absence of an intron in the 5′ untranslated region (5′UTR) is produced during HIV-2 replication in cell culture and in infected patients. We developed a cotransfection system in which epitopically tagged Gag proteins can be traced back to their mRNA origins in the translation pool. We show that a disproportionate amount of Gag is translated from 5′UTR intron-spliced mRNAs, demonstrating a role for the 5′UTR intron in the regulation of gag translation. To further characterize the effects of the HIV-2 5′UTR on translation, we fused wild-type, spliced, or mutant leader RNA constructs to a luciferase reporter gene and assayed their translation in reticulocyte lysates. These assays confirmed that leaders lacking the 5′UTR intron increased translational efficiency compared to that of the unspliced leader. In addition, we found that removal or mutagenesis of the C-box, a pyrimidine-rich sequence located in the 5′UTR intron and previously shown to affect RNA dimerization, also strongly influenced translational efficiency. These results suggest that the splicing of both the 5′UTR intron and the C-box element have key roles in regulation of HIV-2 gag translation in vitro and in vivo.     

Optimal Packaging of FIV Genomic RNA Depends upon a Conserved Long-range Interaction and a Palindromic Sequence within gag

The feline immunodeficiency virus (FIV) is a lentivirus that is related to human immunodeficiency virus (HIV), causing a similar pathology in cats. It is a potential small animal model for AIDS and the FIV-based vectors are also being pursued for human gene therapy. Previous studies have mapped the FIV packaging signal (ψ) to two or more discontinuous regions within the 5′ 511 nt of the genomic RNA and structural analyses have determined its secondary structure. The 5′ and 3′ sequences within ψ region interact through extensive long-range interactions (LRIs), including a conserved heptanucleotide interaction between R/U5 and gag. Other secondary structural elements identified include a conserved 150 nt stem-loop (SL2) and a small palindromic stem-loop within gag open reading frame that might act as a viral dimerization initiation site. We have performed extensive mutational analysis of these sequences and structures and ascertained their importance in FIV packaging using a trans-complementation assay. Disrupting the conserved heptanucleotide LRI to prevent base pairing between R/U5 and gag reduced packaging by 2.8-5.5 fold. Restoration of pairing using an alternative, non-wild type (wt) LRI sequence restored RNA packaging and propagation to wt levels, suggesting that it is the structure of the LRI, rather than its sequence, that is important for FIV packaging. Disrupting the palindrome within gag reduced packaging by 1.5-3-fold, but substitution with a different palindromic sequence did not restore packaging completely, suggesting that the sequence of this region as well as its palindromic nature is important. Mutation of individual regions of SL2 did not have a pronounced effect on FIV packaging, suggesting that either it is the structure of SL2 as a whole that is necessary for optimal packaging, or that there is redundancy within this structure. The mutational analysis presented here has further validated the previously predicted RNA secondary structure of FIV ψ.     

A novel role for gag as a cis-acting element regulating RNA structure,dimerization and packaging in HIV-1 lentiviral vectors

Clinical usage of lentiviral vectors is now established and increasing but remains constrained by vector titer with RNA packaging being a limiting factor. Lentiviral vector RNA is packaged through specific recognition of the packaging signal on the RNA by the viral structural protein Gag. We investigated structurally informed modifications of the 5′ leader and gag RNA sequences in which the extended packaging signal lies, to attempt to enhance the packaging process by facilitating vector RNA dimerization, a process closely linked to packaging. We used in-gel SHAPE to study the structures of these mutants in an attempt to derive structure-function correlations that could inform optimized vector RNA design. In-gel SHAPE of both dimeric and monomeric species of RNA revealed a previously unreported direct interaction between the U5 region of the HIV-1 leader and the downstream gag sequences. Our data suggest a structural equilibrium exists in the dimeric viral RNA between a metastable structure that includes a U5–gag interaction and a more stable structure with a U5–AUG duplex. Our data provide clarification for the previously unexplained requirement for the 5′ region of gag in enhancing genomic RNA packaging and provide a basis for design of optimized HIV-1 based vectors.     

Comparative mutational analysis of cis-acting RNA signals for translational frameshifting in HIV-1 and HTLV-2

Human immunodeficiency virus type 1 (HIV-1) and human T cell leukemia virus type II (HTLV-2) use a similar mechanism for –1 translational frameshifting to overcome the termination codon in viral RNA at the end of the gag gene. Previous studies have identified two important RNA signals for frameshifting, the slippery sequence and a downstream stem–loop structure. However, there have been somewhat conflicting reports concerning the individual contributions of these sequences. In this study we have performed a comprehensive mutational analysis of the cis-acting RNA sequences involved in HIV-1 gag–pol and HTLV-2 gag–pro frameshifting. Using an in vitro translation system we determined frameshifting efficiencies for shuffled HIV-1/HTLV-2 RNA elements in a background of HIV-1 or HTLV-2 sequences. We show that the ability of the slippery sequence and stem–loop to promote ribosomal frameshifting is influenced by the flanking upstream sequence and the nucleotides in the spacer element. A wide range of frameshift efficiency rates was observed for both viruses when shuffling single sequence elements. The results for HIV-1/HTLV-2 chimeric constructs represent strong evidence supporting the notion that the viral wild-type sequences are not designed for maximal frameshifting activity but are optimized to a level suited to efficient viral replication.     

Effect of Canavanine on Murine Retrovirus Polypeptide Formation

Canavanine is an arginine analog which is widely used to inhibit proteolytic processing of viral polyproteins. Certain results obtained with canavanine have suggested that it may have other effects. Therefore, we examined the effects of canavanine on the cell-free synthesis of murine retrovirus proteins. It was found that the electrophoretic mobility of the major gag-related cell-free product of both Rauscher murine leukemia virus (R-MuLV) and Moloney murine sarcoma virus 124 (Mo-MuSV-124) RNA was dependent on the concentration of canavanine used during translation. As the canavanine concentration was increased up to 4 mM, the apparent size of the major gag-related polypeptide also increased from 65,000 (R-MuLV RNA) or 63,000 (Mo-MuSV-124 RNA) to approximately 80,000 daltons. Additional increases in the canavanine concentration up to 12 mM did not increase the size of the gag gene product beyond 80,000 daltons. This change in electrophoretic mobility appeared to be due to a substitution of canavanine for arginine residues in the polypeptides, not to a change in their actual size. If amber suppressor tRNA and canavanine were used together during translation of Mo-MuSV-124 RNA and Mo-MuLV RNA, the results were also in agreement with this proposal. Translation experiments done with ovalbumin mRNA and mengovirus 35S RNA indicated that canavanine incorporation caused a shift in the electrophoretic mobility of ovalbumin from 43,000 to 45,000 daltons and caused the appearance of two slightly larger polypeptides in the 155,000- and 115,000- dalton regions of the mengovirus RNA cell-free product.     

Suppression of Murine Retrovirus Polypeptide Termination: Effect of Amber Suppressor tRNA on the Cell-Free Translation of Rauscher Murine Leukemia Virus, Moloney Murine Leukemia Virus, and Moloney Murine Sarcoma Virus 124 RNA

The effect of suppressor tRNA's on the cell-free translation of several leukemia and sarcoma virus RNAs was examined. Yeast amber suppressor tRNA (amber tRNA) enhanced the synthesis of the Rauscher murine leukemia virus and clone 1 Moloney murine leukemia virus Pr200^gag-pol polypeptides by 10- to 45-fold, but at the same time depressed the synthesis of Rauscher murine leukemia virus Pr65^gag and Moloney murine leukemia virus Pr63^gag. Under suppressor-minus conditions, Moloney murine leukemia virus Pr70^gag was present as a closely spaced doublet. Amber tRNA stimulated the synthesis of the “upper” Moloney murine leukemia virus Pr70^gag polypeptide. Yeast ochre suppressor tRNA appeared to be ineffective. Quantitative analyses of the kinetics of viral precursor polypeptide accumulation in the presence of amber tRNA showed that during linear protein synthesis, the increase in accumulated Moloney murine leukemia virus Pr200^gag-pol coincided closely with the molar loss of Pr63^gag. Enhancement of Pr200^gag-pol and Pr70^gag by amber tRNA persisted in the presence of pactamycin, a drug which blocks the initiation of protein synthesis, thus arguing for the addition of amino acids to the C terminus of Pr63^gag as the mechanism behind the amber tRNA effect. Moloney murine sarcoma virus 124 30S RNA was translated into four major polypeptides, Pr63^gag, P42, P38, and P23. In the presence of amber tRNA, a new polypeptide, Pr67^gag, appeared, whereas Pr63^gag synthesis was decreased. Quantitative estimates indicated that for every 1 mol of Pr67^gag which appeared, 1 mol of Pr63^gag was lost.     

Sequences within Both the 5′ UTR and Gag Are Required for Optimal In Vivo Packaging and Propagation of Mouse Mammary Tumor Virus (MMTV) Genomic RNA

Background

This study mapped regions of genomic RNA (gRNA) important for packaging and propagation of mouse mammary tumor virus (MMTV). MMTV is a type B betaretrovirus which preassembles intracellularly, a phenomenon distinct from retroviruses that assemble the progeny virion at cell surface just before budding such as the type C human and feline immunodeficiency viruses (HIV and FIV). Studies of FIV and Mason-Pfizer monkey virus (MPMV), a type D betaretrovirus with similar intracellular virion assembly processes as MMTV, have shown that the 5′ untranslated region (5′ UTR) and 5′ end of gag constitute important packaging determinants for gRNA.

Methodology

Three series of MMTV transfer vectors containing incremental amounts of gag or 5′ UTR sequences, or incremental amounts of 5′ UTR in the presence of 400 nucleotides (nt) of gag were constructed to delineate the extent of 5′ sequences that may be involved in MMTV gRNA packaging. Real time PCR measured the packaging efficiency of these vector RNAs into MMTV particles generated by co-transfection of MMTV Gag/Pol, vesicular stomatitis virus envelope glycoprotein (VSV-G Env), and individual transfer vectors into human 293T cells. Transfer vector RNA propagation was monitored by measuring transduction of target HeLaT4 cells following infection with viral particles containing a hygromycin resistance gene expression cassette on the packaged RNA.

Principal Findings

MMTV requires the entire 5′ UTR and a minimum of ∼120 nucleotide (nt) at the 5′ end of gag for not only efficient gRNA packaging but also propagation of MMTV-based transfer vector RNAs. Vector RNAs without the entire 5′ UTR were defective for both efficient packaging and propagation into target cells.

Conclusions/Significance

These results reveal that the 5′ end of MMTV genome is critical for both gRNA packaging and propagation, unlike the recently delineated FIV and MPMV packaging determinants that have been shown to be of bipartite nature.     

Analysis of Minimal Human Immunodeficiency Virus Type 1 gag Coding Sequences Capable of Virus-Like Particle Assembly and Release

We have constructed a series of human immunodeficiency virus (HIV) gag mutants by progressive truncation of the gag coding sequence from the C terminus and have combined these mutants with an assembly-competent matrix domain deletion mutation (ΔMA). By using several methods, the particle-producing capabilities of each mutant were examined. Our analysis indicated that truncated Gag precursors lacking most of C-terminal gag gene products assembled and were released from 293T cells. Additionally, a mutant with a combined deletion of the MA (ΔMA) and p6 domains even produced particles at levels comparable to that of the wild-type (wt) virus. However, most mutants derived from combination of the ΔMA and the C-terminal truncation mutations did not release particles as well as the wt. Our smallest HIV gag gene product capable of virus-like particle formation was a 28-kDa protein which consists of a few MA amino acids and the CA-p2 domain. Sucrose density gradient fractionation analysis indicated that most mutants exhibited a wt retrovirus particle density. Exceptions to this rule were mutants with an intact MA domain but deleted downstream of the p2 domains. These C-terminal truncation mutants possessed particle densities of 1.13 to 1.15 g/ml, lower than that of the wt. The N-terminal portions of the CA domain, which have been shown to be dispensable for core assembly, became critical when most of the MA domain was deleted, suggesting a requirement for an intact CA domain to assemble and release particles.     

A premature termination codon mutation at the C terminus of foamy virus Gag downregulates the levels of spliced pol mRNA

Foamy viruses (FV) comprise a subfamily of retroviruses. Orthoretroviruses, such as human immunodeficiency virus type 1, synthesize Gag and Pol from unspliced genomic RNA. However, FV Pol is expressed from a spliced mRNA independently of Gag. FV pol splicing uses a 3′ splice site located at the 3′ end of gag, resulting in a shared exon between gag and pol. Previously, our laboratory showed that C-terminal Gag premature termination codon (PTC) mutations in the 3′ shared exon led to greatly decreased levels of Pol protein (C. R. Stenbak and M. L. Linial, J. Virol. 78:9423-9430, 2004). To further characterize these mutants, we quantitated the levels of unspliced gag and spliced pol mRNAs using a real-time PCR assay. In some of the PTC mutants, the levels of spliced pol mRNA were reduced as much as 30-fold, whereas levels of unspliced gag RNA were not affected. Substitutions of a missense codon in place of a PTC restored normal levels of spliced pol mRNA. Disrupting Upf proteins involved in nonsense-mediated mRNA decay (NMD) did not affect Pol protein expression. Introduction of an exonic splicing enhancer downstream of the PTC mutation restored pol splicing to the wild-type level. Taken together, our results show that the PTC mutation itself is responsible for decreased levels of pol mRNA but that mechanisms other than NMD might be involved in downregulating Pol expression. The results also suggest that normal pol splicing utilizes a suboptimal splice site seen for other spliced mRNAs in most retroviruses, in that introduced exonic enhancer elements can increase splicing efficiency.     

A new type of IRES within gag coding region recruits three initiation complexes on HIV-2 genomic RNA

Genomic RNA of primate lentiviruses serves both as an mRNA that encodes Gag and Gag-Pol polyproteins and as a propagated genome. Translation of this RNA is initiated by standard cap dependant mechanism or by internal entry of the ribosome. Two regions of the genomic RNA are able to attract initiation complexes, the 5′ untranslated region and the gag coding region itself. Relying on probing data and a phylogenetic study, we have modelled the secondary structure of HIV-1, HIV-2 and SIV_Mac coding region. This approach brings to light conserved secondary-structure elements that were shown by mutations to be required for internal entry of the ribosome. No structural homologies with other described viral or cellular IRES can be identified and lentiviral IRESes show many peculiar properties. Most notably, the IRES present in HIV-2 gag coding region is endowed with the unique ability to recruit up to three initiation complexes on a single RNA molecule. The structural and functional properties of gag coding sequence define a new type of IRES. Although its precise role is unknown, the conservation of the IRES among fast evolving lentiviruses suggests an important physiological role.     

Dexamethasone Induction of the Intracellular RNAs of Mouse Mammary Tumor Virus

We have studied the kinetics of dexamethasone induction of mouse mammary tumor virus (MMTV) RNAs and proteins in virus-infected rat XC cells and GR mouse mammary tumor cells. A detectable increase in viral RNA in infected XC cells was present within 10 min after hormone addition, and half-maximal induction was achieved in less than 2 h. The increase in viral RNA concentration was apparent first in nuclear RNA and later in the cytoplasm. Within the first 15 min of induction, only genome-sized RNA (35S, 7.8 kilobases) was present in augmented amounts, whereas the major subgenomic RNA (24S, 3.8 kilobases) did not appear until at least 30 to 60 min postinduction. The sequential appearance of these RNAs, the probable mRNA's for the gag and env proteins, paralleled the order of appearance of the gag and env proteins, respectively, after hormone treatment. An additional species of viral RNA (20S, 2.5 kilobases) was detected during these induction experiments, but the role of this RNA is not known. Both subgenomic RNAs contain sequences derived from both the 5′ and 3′ termini of genomic RNA and are presumably spliced. After dexamethasone induction of infected XC cells, we detected two smaller env-related proteins which were not found in full hormone induction. The functional role of these smaller proteins is not known. A previously reported smaller species of RNA (13S, 1.0 kilobase) did not appear to be induced and was shown to be cellular rather than viral in origin. In the fully induced infected XC and GR mammary tumor cells, the only viral RNAs present were the 35S and 24S RNAs. In addition, mammary tumors contained only these two viral RNAs. Thus, tumor cells appear to contain only the viral RNAs which direct the synthesis of the gag, pol, and env proteins of the virion.     

Targeting HIV mRNA for Degradation: 2,5-A Antisense Chimeras as Potential Chemotherapeutic Agents for AIDS

Abstract

We have identified a region within a 1 kb HIV gag RNA which can be ablated in vitro using a 2,5-A antisense chimera. The cleavage was specific and almost complete at a concentration of 100 nM chimera.     

Safety and immunogenicity of an HIV-1 gag DNA vaccine with or without IL-12 and/or IL-15 plasmid cytokine adjuvant in healthy, HIV-1 uninfected adults

Background

DNA vaccines are a promising approach to vaccination since they circumvent the problem of vector-induced immunity. DNA plasmid cytokine adjuvants have been shown to augment immune responses in small animals and in macaques.

Methodology/Principal Findings

We performed two first in human HIV vaccine trials in the US, Brazil and Thailand of an RNA-optimized truncated HIV-1 gag gene (p37) DNA derived from strain HXB2 administered either alone or in combination with dose-escalation of IL-12 or IL-15 plasmid cytokine adjuvants. Vaccinations with both the HIV immunogen and cytokine adjuvant were generally well-tolerated and no significant vaccine-related adverse events were identified. A small number of subjects developed asymptomatic low titer antibodies to IL-12 or IL-15. Cellular immunogenicity following 3 and 4 vaccinations was poor, with response rates to gag of 4.9%/8.7% among vaccinees receiving gag DNA alone, 0%/11.5% among those receiving gag DNA+IL-15, and no responders among those receiving DNA+high dose (1500 ug) IL-12 DNA. However, after three doses, 44.4% (4/9) of vaccinees receiving gag DNA and intermediate dose (500 ug) of IL-12 DNA demonstrated a detectable cellular immune response.

Conclusions/Significance

This combination of HIV gag DNA with plasmid cytokine adjuvants was well tolerated. There were minimal responses to HIV gag DNA alone, and no apparent augmentation with either IL-12 or IL-15 plasmid cytokine adjuvants. Despite the promise of DNA vaccines, newer formulations or methods of delivery will be required to increase their immunogenicity.

Trial Registration

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