The human immunodeficiency virus type 1 packaging signal and major splice donor region have a conserved stable secondary structure.

Interaction of cis-acting RNA sequences with nucleocapsid proteins is one of the critical events leading to retroviral genomic RNA packaging. We have derived a potentially stable secondary structure for the packaging signal region of human immunodeficiency virus strain IIIB, using a combination of biochemical analysis and computer modelling. This region encompasses the major splice donor (SD), which is found in a highly structured conserved stem-loop. Comparison with other published human immunodeficiency virus type 1 sequences shows almost absolute nucleotide conservation in base-paired regions required to maintain this structure. Presently and previously described packaging-defective mutants would disrupt the structure. The structure depends on base pairing between nucleotide sequences 5' of the major SD which are common to both genomic and subgenomic RNAs and sequences 3' of SD which are unique to the unspliced RNA. This may explain how in retroviruses such as Rous sarcoma virus, mutations in regions common to genomic and subgenomic RNA might prevent packaging of the unspliced mRNA by disrupting a signal structure which can exist only in the genomic RNA species.     

Conserved Region of Mammalian Retrovirus RNA

The viral RNAs of various mammalian retroviruses contain highly conserved sequences close to their 3' ends. This was demonstrated by interviral molecular hybridization between fractionated viral complementary DNA (cDNA) and RNA. cDNA near the 3' end (cDNA(3')) from a rat virus (RPL strain) was fractionated by size and mixed with mouse virus RNA (Rauscher leukemia virus). No hybridization occurred with total cDNA (cDNA(total)), in agreement with previous results, but a cross-reacting sequence was found with the fractionated cDNA(3'). The sequences between 50 to 400 nucleotides from the 3' terminus of heteropolymeric RNA were most hybridizable. The rat viral cDNA(3') hybridized with mouse virus RNA more extensively than with RNA of remotely related retroviruses. The related viral sequence of the rodent viruses (mouse and rat) showed as much divergence in heteroduplex thermal denaturation profiles as did the unique sequence DNA of these two rodents. This suggests that over a period of time, rodent viruses have preserved a sequence with changes correlated to phylogenetic distance of hosts. The cross-reacting sequence of replication-competent retroviruses was conserved even in the genome of the replication-defective sarcoma virus and was also located in these genomes near the 3' end of 30S RNA. A fraction of RD114 cDNA(3'), corresponding to the conserved region, cross-hybridized extensively with RNA of a baboon endogenous virus (M7). Fractions of similar size prepared from cDNA(3') of MPMV, a primate type D virus, hybridized with M7 RNA to a lesser extent. Hybridization was not observed between Mason-Pfizer monkey virus and M7 if total cDNA's were incubated with viral RNAs. The degree of cross-reaction of the shared sequence appeared to be influenced by viral ancestral relatedness and host cell phylogenetic relationships. Thus, the strikingly high extent of cross-reaction at the conserved region between rodent viruses and simian sarcoma virus and between baboon virus and RD114 virus may reflect ancestral relatedness of the viruses. Slight cross-reaction at the site between type B and C viruses of rodents (mouse mammary tumor virus and RPL virus, 58-2T) or type C and D viruses of primates (M7, RD114, and Mason-Pfizer monkey virus) may have arisen at the conserved region through a mechanism that depends more on the phylogenetic relatedness of the host cells than on the viral type or origin. Determining the sequence of the conserved region may help elucidate this mechanism. The conserved sequences in retroviruses described here may be an important functional unit for the life cycle of many retroviruses.     

Identification and characterization of a coronavirus packaging signal.

Previously, a mouse hepatitis virus (MHV) genomic sequence necessary for defective interfering (DI) RNA packaging into MHV particles (packaging signal) was mapped to within a region of 1,480 nucleotides in the MHV polymerase gene by comparison of two DI RNAs. One of these, DIssF, is 3.6 kb in size and exhibits efficient packaging, whereas the other, DIssE, which is 2.3 kb, does not. For more precise mapping, a series of mutant DIssF RNAs with deletions within this 1,480-nucleotide region were constructed. After transfection of in vitro-synthesized mutant DI RNA in MHV-infected cells, the virus product was passaged several times. The efficiency of DI RNA packaging into MHV virions was then estimated by viral homologous interference activity and by analysis of intracellular virus-specific RNAs and virion RNA. The results indicated that an area of 190 nucleotides was necessary for packaging. A computer-generated secondary structural analysis of the A59 and JHM strains of MHV demonstrated that within this 190-nucleotide region a stable stem-loop of 69 nucleotides was common between the two viruses. A DIssE-derived DI DNA which had these 69 nucleotides inserted into the DIssE sequence demonstrated efficient DI RNA packaging. Site-directed mutagenic analysis showed that of these 69 nucleotides, the minimum sequence of the packaging signal was 61 nucleotides and that destruction of the secondary structure abolished packaging ability. These studies demonstrated that an MHV packaging signal was present within the 61 nucleotides, which are located on MHV genomic RNA 1,381 to 1,441 nucleotides upstream of the 3' end of gene 1.     

Structural and thermodynamic studies on mutant RNA motifs that impair the specificity between a viral replicase and its promoter

The 3'-end region of the genomic RNA of brome mosaic virus forms a tRNA-like structure that is critical for its replication. Previous studies have shown that in this region, a stem-loop structure, called SLC, is necessary and sufficient for the binding of the RNA replicase, and for RNA replication. Recently, we determined the high-resolution NMR structure of SLC, which demonstrated that a 5'-AUA-3' triloop region is an important structural element for the enzymatic recognition. We proposed that the 5'-adenine of the triloop, which is rigidly fixed ("clamped") to the stem, is a key recognition element for the replicase. To elucidate the role of this "clamped base motif" for the enzymatic recognition, we have now investigated the solution conformations of several stem-loop molecules with mutant triloops, 5'-UUA-3', 5'-GUA-3', 5'-CUA-3' and 5'-UUU-3', that destroy the enzymatic recognition. For the GUA and UUA mutants, we have obtained high-resolution solution structures using 2D NMR. All four mutants have very similar thermodynamic stabilities, and all have the same secondary structures, a triloop with a five base-paired stem helix. In addition, they have quite similar sugar puckering patterns in the triloop region. The NMR structures of the GUA and UUA show that the 5' nucleotide of the triloop (G6 in GUA or U6 in UUA) lacks the strong interactions that hold its base in a fixed position. In particular, the U6 of UUA is found in two different conformations. Neither of these two mutants has the clamped base motif that was observed in the wild-type. While UUA also shows global change in the overall triloop conformation, GUA shows a very similar triloop conformation to the wild-type except for the lack of this motif. The absence of the clamped base motif is the only common structural difference between these two mutants and the wild-type. These results clearly indicate that the loss of function of the UUA and GUA mutants comes mainly from the destruction of a small key recognition motif rather than from global changes in their triloop conformations. Based on this study, we conclude that the key structural motif in the triloop recognized by the replicase is a solution-exposed, 5'-adenine base in the triloop that is clamped to the stem helix, which is called a clamped adenine motif.     

Secondary structure and mutational analysis of the Mason-Pfizer monkey virus RNA constitutive transport element.

The Mason-Pfizer monkey virus (MPMV) genome contains a cis-acting element that serves to facilitate nucleocytoplasmic export of intron-containing RNA. This element, known as the constitutive transport element (CTE), has been mapped to a 154-nt region close to the 3' end of the MPMV genome. The CTE contains a degenerate direct repeat of approximately 70 nt. We have probed the secondary structure of the CTE using double-strand- and single-strand-specific ribonucleases and chemical modification agents. A mutational analysis was also performed to confirm critical features of the CTE structure, as well as to identify regions that contain sequence-specific information required for function. Our results indicate that the CTE forms a long stem structure that contains a 9-nt terminal hairpin loop as well as two identical 16-nt inner loops. The inner loop sequences are rotated 180 degrees relative to each other within the structure. The mutational analysis shows that primary sequences in the loop regions are important for function, suggesting that they may contain binding sites for cellular proteins involved in RNA export. Interestingly, sequences with significant homology to the inner loop regions are found in the genomes of spumaviruses and mouse intracisternal A particles.     

Interaction of a cellular 57-kilodalton protein with the internal translation initiation site of foot-and-mouth disease virus.

A cellular 57-kDa protein (p57) that binds specifically to the internal translation initiation site in the 5' untranslated region of foot-and-mouth disease virus RNA was detected in cell extracts of different mammalian species by UV cross-linking. The protein binds to two distinct sites of the translation control region which have as the only common sequence a UUUC motif. The first binding site consists of a conserved hairpin structure, whereas the second binding site contains an essential pyrimidine-rich region without obvious secondary structure. Competition experiments indicate that the complexes with the two binding sites were formed by a single p57 species. The protein binds also to the 5' untranslated region of other picornaviruses. Results from footprint analyses with foot-and-mouth disease RNA suggest the participation of additional cellular factors in the translation initiation complex.     

Dimer initiation sequence of HIV-1Lai genomic RNA: NMR solution structure of the extended duplex.

The genome of all retrovirus consists of two copies of genomic RNA which are noncovalently linked near their 5' end. A sequence localized immediately upstream from the splice donor site inside the HIV-1 psi-RNA region was identified as the domain responsible for the dimerization initiation. It was shown that a kissing complex and a stable dimer are both involved in the HIV-1Lai RNA dimerization process in vitro. The NCp7 protein activates the dimerization by converting a transient loop-loop complex into a more stable dimer. The structure of this transitory loop-loop complex was recently elucidated by Mujeeb et al. In work presented here, we use NMR spectroscopy to determine the stable extended dimer structure formed from a 23 nucleotides RNA fragment, part of the 35 nucleotides SL1 sequence. By heating to 90 degrees C, then slowly cooling this sequence, we were able to show that an extended dimer is formed. We present evidence for the three dimensional structure of this dimer. NMR data yields evidence for a zipper like motif A8A9.A16 existence. This motif enables the surrounding bases to be positioned more closely and permit the G7 and C17 bases to be paired. This is different to other related sequences where only the kissing complex is observed, we suggest that the zipper like motif AA.A could be an important stabilization factor of the extended duplex.     

A mutant viral RNA promoter with an altered conformation retains efficient recognition by a viral RNA replicase through a solution-exposed adenine.

Brome mosaic virus (BMV) genomic minus-strand RNA synthesis requires an RNA motif named stem-loop C (SLC). An NMR-derived solution structure of SLC was reported by Kim et al. (Nature Struc Biol, 2000, 7:415-423) to contain three replicase-recognition elements, the most important of which is a stable stem with a terminal trinucleotide loop, 5'AUA3'. The 5'-most adenine of the triloop is rigidly fixed to the stem helix by interactions that require the 3'-most adenine, which is called a clamped adenine motif. However, a change of the 3' adenine to guanine (5'AUG3') unexpectedly directed RNA synthesis at 130% of wild type (Kim et al., Nature Struc Biol, 2000, 7:415-423). To understand how RNA with the AUG mutation maintains interaction with the BMV replicase, we used NMR and other biophysical techniques to elucidate the solution conformation of a 13-nt RNA containing the AUG triloop, called S-AUG. We found that S-AUG has a drastically different loop conformation in comparison to the wild type, as evidenced by an unusual C x G loop-closing base pair. Despite the conformational change, S-AUG maintains a solution-exposed adenine similar to the clamped adenine motif found in the wild type. Biochemical studies of the 5'AUG3' loop with various substitutions in the context of the whole SLC construct confirm that the clamped adenine motif exists in S-AUG remains a primary structural feature required for RNA synthesis by the BMV replicase.     

RNA pseudoknots downstream of the frameshift sites of retroviruses.

RNA pseudoknot structural motifs could have implications for a wide range of biological processes of RNAs. In this study, the potential RNA pseudoknots just downstream from the known and suspected retroviral frame-shift sites were predicted in the Rous sarcoma virus, primate immunodeficiency viruses (HIV-1, HIV-2, and SIV), equine infectious anemia virus, visna virus, bovine leukemia virus, human T-cell leukemia virus (types I and II), mouse mammary tumor virus, Mason-Pfizer monkey virus, and simian SRV-1 type-D retrovirus. Also, the putative RNA pseudoknots were detected in the gag-pol overlaps of two retrotransposons of Drosophila, 17.6 and gypsy, and the mouse intracisternal A particle. For each sequence, the thermodynamic stability and statistical significance of the secondary structure involved in the predicted tertiary structure were assessed and compared. Our results show that the stem-loop structures in the pseudoknots are both thermodynamically highly stable and statistically significant relative to other such configurations that potentially occur in the gag-pol or gag-pro and pro-pol junction domains of these viruses (300 nucleotides upstream and downstream from the possible frameshift sites are included). Moreover, the structural features of the predicted pseudoknots following the frameshift site of pro-pol overlaps of the HTLV-1 and HTLV-2 retroviruses are structurally well conserved. The occurrence of eight compensatory base changes in the tertiary interaction of the two related sequences allow the conservation of their tertiary structures in spite of the sequence divergence. The results support the possible control mechanism for frameshifting proposed by Brierley et al. and Jacks et al.     

Transcription of 70S RNA by DNA polymerases from mammalian RNA viruses.

DNA polymerases purified by the same procedure from four mammalian RNA viruses, simian sarcoma virus type 1, gibbon ape lymphoma virus, Mason-Pfizer monkey virus, and Rauscher murine leukemia virus are capable of transcribing heteropolymeric regions of viral 70S RNA without any other primer. In this reconstituted system the enzymes from simian sarcoma virus type 1, Mason-Pfizer monkey virus, and Rauscher murine leukemia virus transcribe viral 70S RNA almost as efficiently as the DNA polymerase from the avian myeloblastosis virus, but gibbon ape lymphoma virus DNA polymerase is approximately three-to fivefold less efficient. Although there is a substantial difference among the sizes of these DNA polymerases (160,000 daltons for the avian myeloblastosis virus enzyme, 110,000 daltons for the Mason-Pfizer monkey virus enzyme, and 70,000 daltons for the mammalian type C viral polymerases), the ability to transcribe viral 70S RNA is a characteristic common to these enzymes.     

Bipartite 3'-cis-acting signal for replication in yeast 23 S RNA virus and its repair

23 S RNA narnavirus is a persistent positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome is small (2.9 kb) and only encodes its RNA-dependent RNA polymerase. Recently, we have succeeded in generating 23 S RNA virus from an expression vector containing the entire viral cDNA sequence. Using this in vivo launching system, we analyzed the 3'-cis-acting signals for replication. The 3'-non-coding region of 23 S RNA contains two cis-elements. One is a stretch of 4 Cs at the 3' end, and the other is a mismatched pair in a stem-loop structure that partially overlaps the terminal 4 Cs. In the latter element, the loop or stem sequence is not important but the stem structure with the mismatch pair is essential. The mismatched bases should be purines. Any combination of purines at the mismatch pair bestowed capability of replication on the RNA, whereas converting it to a single bulge at either side of the stem abolished the activity. The terminal and penultimate Cs at the 3' end could be eliminated or modified to other nucleotides in the launching plasmid without affecting virus generation. However, the viruses generated regained or restored these Cs at the 3' terminus. Considering the importance of the viral 3' ends in RNA replication, these results suggest that this 3' end repair may contribute to the persistence of 23 S RNA virus in yeast by maintaining the genomic RNA termini intact. We discuss possible mechanisms for this 3' end repair in vivo.     

Bovine leukemia virus matrix-associated protein MA(p15): further processing and formation of a specific complex with the dimer of the 5''-terminal genomic RNA fragment.

The retrovirus precursor protein has an arrangement of several characteristic domains with which it achieves selective and efficient packaging of the genome RNA during particle assembly. In this study, we analyzed the composition of the bovine leukemia virus (BLV) gag proteins and examined their RNA-binding properties in gel mobility shift assays, using various genomic RNA probes synthesized in vitro. Results obtained in amino acid sequence and composition analyses indicate that the matrix-associated protein MA(p15) is further processed by the BLV protease (PR) to generate MA(p10), a short peptide of seven amino acid residues, and p4. The gag precursor is now mapped as NH2-MA(p10)-p4-CA(p24)-NC(p12)-COOH. MA(p15) formed a specific complex with the dimer RNA of the U5-5' gag region presumed to contain the BLV packaging signal but not with other RNAs. The NH2-terminal cleavage product, MA(p10), bound all RNA fragments tested, while the COOH-terminal peptides with a sequence common to mammalian type C retroviruses had little affinity for RNA. The nucleocapsid protein NC(p12) bound to RNAs nonspecifically and randomly in the presence or absence of zinc ions. These results suggest a possible interaction of the NH2 terminus of the gag precursor with the 5' terminus of the genomic RNA in an early phase of particle assembly, when the conserved structure between the MA and CA domains might be involved.     

A base-paired structure in the avian sarcoma virus 5' leader is required for efficient encapsidation of RNA.

Selective encapsidation of avian sarcoma-leukosis virus genomic RNA within virions requires recognition of a cis-acting signal (termed psi) located in the 5' leader of the RNA between the primer binding site and the splice donor site. Computer analyses indicate the potential for numerous secondary structure interactions within this region, including alternative conformations with similar free energy levels. We have constructed mutations designed to disrupt and restore potential secondary structure interactions within psi to investigate the role of these structures in RNA packaging. To test for the ability of psi mutants to package a heterologous reporter gene into virions, chimeric constructs bearing avian sarcoma virus 5' sequences fused to lacZ were transiently cotransfected with a nonpackageable helper construct into chicken embryo fibroblasts. lacZ virions produced from cotransfected cells were used to infect new cultures of chicken embryo fibroblasts, and then an in situ assay for individual cells expressing lacZ was done. Results obtained with this assay were confirmed in direct analyses of isolated virion RNA by RNase protection assays. Two mutations, predicted to disrupt a potential stem structure forming between elements located at nucleotides 160 to 167 and 227 to 234, severely inhibited packaging when either element was mutated. A construct in which these mutations were combined to restore potential base pairing between the two elements displayed a partially restored packaging phenotype. These results strongly suggest that the structure, referred to as the O3 stem, is required for efficient encapsidation of avian sarcoma virus RNA. Site-directed mutagenesis of additional sequence elements located in the O3 loop reduced packaging as measured by the indirect assay, suggesting that these sequences may also be components of the encapsidation signal. The possible implications of the O3 stem structure with regard to translation of avian sarcoma-leukosis virus short upstream open reading frames are discussed.     

A stem-loop motif formed by the immediate 5' terminus of the bovine viral diarrhea virus genome modulates translation as well as replication of the viral RNA

Bovine viral diarrhea virus (BVDV), a Pestivirus member of the Flaviviridae family, has a positive-stranded RNA genome which consists of a single open reading frame (ORF) and untranslated regions (UTRs) at the 5' and 3' ends. The 5' UTR harbors extensive RNA structure motifs; most of them were shown to contribute to an internal ribosomal entry site (IRES), which mediates cap-independent translation of the ORF. The extreme 5'-terminal region of the BVDV genome had so far been believed not to be required for IRES function. By structure probing techniques, we initially verified the existence of a computer-predicted stem-loop motif at the 5' end of the viral genome (hairpin Ia) as well as at the 3' end of the complementary negative-strand replication intermediate [termed hairpin Ia (-)]. While the stem of this structure is mainly constituted of nucleotides that are conserved among pestiviruses, the loop region is predominantly composed of variable residues. Taking a reverse genetics approach to a subgenomic BVDV replicon RNA (DI9c) which could be equally employed in a translation as well as replication assay system based on BHK-21 cells, we obtained the following results. (i) Proper folding of the Ia stem was found to be crucial for efficient translation. Thus, in the context of an authentic replication-competent viral RNA, the 5'-terminal motif operates apparently as an integral functional part of the ribosome entry. (ii) An intact loop structure and a stretch of nucleotide residues that constitute a portion of the stem of the Ia or the Ia (-) motif, respectively, were defined to represent important determinants of the RNA replication pathway. (iii) Formation of the stem structure of the Ia (-) motif was determined to be not critical for RNA replication. In summary, our findings affirmed that the 5'-terminal region of the BVDV genome encodes a bifunctional secondary structure motif which may enable the viral RNA to switch from the translation to the replicative cycle and vice versa.     

Primate retroviruses: envelope glycoproteins of endogenous type C and type D viruses possess common interspecies antigenic determinants.

The major 70,000- to 80,000-molecular-weight envelope glycoproteins of the squirrel monkey retrovirus, Mason-Pfizer monkey virus, and M7 baboon virus and the related endogenous feline virus, RD114, were isolated and immunologically characterized. Immunoprecipitation and competition immunoassay analysis revealed these viral envelope glycoproteins to possess several distinct classes of immunological determinants. These include species-specific determinants, group-specific antigenic determinants unique to endogenous primate type C viruses, and group-specific determinants for type D viruses such as Mason-Pfizer monkey virus and squirrel monkey retrovirus. In addition, a class of broadly reactive antigenic determinants shared by envelope glycoproteins of both type C viruses of the baboon/RD114 group and type D viruses of the Mason-Pfizer monkey virus/squirrel monkey virus group are described. Other mammalian oncornaviruses tested, including isolates of nonprimate origin and representative type B viruses, lacked these determinants. The demonstration of antigenic determinants specific to envelope glycoproteins of type C and type D primate viruses indicates either that these viruses are evolutionarily related or that genetic recombination occurred between their progenitors. Alternatively, endogenous type D oncornaviruses may be replication defective, and acquisition of endogenous type C viral genetic sequences coding for envelope glycoprotein determinants may be necessary for their isolation as infectious virus.     

A bulged stem-loop structure in the 3' untranslated region of the genome of the coronavirus mouse hepatitis virus is essential for replication.

The 3' untranslated region (UTR) of the positive-sense RNA genome of the coronavirus mouse hepatitis virus (MHV) contains sequences that are necessary for the synthesis of negative-strand viral RNA as well as sequences that may be crucial for both genomic and subgenomic positive-strand RNA synthesis. We have found that the entire 3' UTR of MHV could be replaced by the 3' UTR of bovine coronavirus (BCV), which diverges overall by 31% in nucleotide sequence. This exchange between two viruses that are separated by a species barrier was carried out by targeted RNA recombination. Our results define regions of the two 3' UTRs that are functionally equivalent despite having substantial sequence substitutions, deletions, or insertions with respect to each other. More significantly, our attempts to generate an unallowed substitution of a particular portion of the BCV 3' UTR for the corresponding region of the MHV 3' UTR led to the discovery of a bulged stem-loop RNA secondary structure, adjacent to the stop codon of the nucleocapsid gene, that is essential for MHV viral RNA replication.