An analytical study of the dimerization of in vitro generated RNA of Moloney murine leukemia virus MoMuLV.

The genome of Moloney murine leukemia virus(MoMuLV) is composed of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Recently it was shown that in vitro generated MuLV RNA formed dimeric molecules and that dimerization sequences are located within the Psi encapsidation domain between positions 215 and 420. Conditions for the spontaneous dimerization of a MuLV RNA fragment encompassing the Psi domain have been investigated. The rate of spontaneous MuLV RNA dimer formation is dependent upon RNA, NaCl and MgCl2 concentrations as well as temperature. Thermal denaturation of in vitro generated dimer RNA of 350 nt, from positions 215 to 565, gave a Tm of about 58 degrees C in 100 mM NaCl. This Tm value is very close to that found for RNA corresponding to the 5' 755 nt and to the genomic 70 S RNA isolated from virions. According to thrermodynamic parameters derived from denaturation curves of MuLV dimer RNA generated in vitro, the dimer linkage structure probably involves short sequences.     

cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA.

The genetic material of all retroviruses examined so far consists of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Since the precise location of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analyzed the dimerization process of Moloney murine leukemia virus (MoMuLV) genomic RNA. For this purpose we derived an in vitro model for RNA dimerization. By using this model, murine leukemia virus RNA was shown to form dimeric molecules. Deletion mutagenesis in the 620-nucleotide leader of MoMuLV RNA showed that the dimer promoting sequences are located within the encapsidation element Psi between positions 215 and 420. Furthermore, hybridization assays in which DNA oligomers were used to probe monomer and dimer forms of MoMuLV RNA indicated that the DLS probably maps between positions 280 and 330 from the RNA 5' end. Also, retroviral nucleocapsid protein was shown to catalyze dimerization of MoMuLV RNA and to be tightly bound to genomic dimer RNA in virions. These results suggest that MoMuLV RNA dimerization and encapsidation are probably controlled by the same cis element, Psi, and trans-acting factor, nucleocapsid protein, and thus might be linked during virion formation.     

A small and efficient dimerization/packaging signal of rat VL30 RNA and its use in murine leukemia virus-VL30-derived vectors for gene transfer.

Retroviral genomes consist of two identical RNA molecules associated at their 5' ends by the dimer linkage structure located in the packaging element (Psi or E) necessary for RNA dimerization in vitro and packaging in vivo. In murine leukemia virus (MLV)-derived vectors designed for gene transfer, the Psi + sequence of 600 nucleotides directs the packaging of recombinant RNAs into MLV virions produced by helper cells. By using in vitro RNA dimerization as a screening system, a sequence of rat VL30 RNA located next to the 5' end of the Harvey mouse sarcoma virus genome and as small as 67 nucleotides was found to form stable dimeric RNA. In addition, a purine-rich sequence located at the 5' end of this VL30 RNA seems to be critical for RNA dimerization. When this VL30 element was extended by 107 nucleotides at its 3' end and inserted into an MLV-derived vector lacking MLV Psi +, it directed the efficient encapsidation of recombinant RNAs into MLV virions. Because this VL30 packaging signal is smaller and more efficient in packaging recombinant RNAs than the MLV Psi + and does not contain gag or glyco-gag coding sequences, its use in MLV-derived vectors should render even more unlikely recombinations which could generate replication-competent viruses. Therefore, utilization of the rat VL30 packaging sequence should improve the biological safety of MLV vectors for human gene transfer.     

A study of the dimer formation of Rous sarcoma virus RNA and of its effect on viral protein synthesis in vitro.

The genetic material of all retroviruses examined so far is an RNA dimer where two identical RNA subunits are joined at their 5' ends by a structure named dimer linkage structure (DLS). Since the precise location and structure of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analysed the dimerization process of Rous sarcoma virus (RSV) RNA. For this purpose we set up an in vitro model for RSV RNA dimerization. Using this model RSV RNA was shown to form dimeric molecules and this dimerization process was greatly activated by nucleocapsid protein (NCp12) of RSV. Furthermore, RSV RNA dimerization was performed in the presence of complementary 5'32P-DNA oligomers in order to probe the monomer and dimer forms of RSV RNA. Data indicated that the DLS of RSV RNA probably maps between positions 544-564 from the 5' end. In an attempt to define sequences needed for the dimerization of RSV RNA, deletion mutageneses were generated in the 5' 600 nt. The results showed that the dimer promoting sequences probably are located within positions 208-270 and 400-600 from the 5' end and hence possibly encompassing the cis-acting elements needed for the specific encapsidation of RSV genomic RNA. Also it is reported that synthesis of the polyprotein precursor Pr76gag is inhibited upon dimerization of RSV RNA. These results suggest that dimerization and encapsidation of genome length RSV RNA might be linked in the course of virion formation since they appear to be under the control of the same cis elements, E and DLS, and the trans-acting factor nucleocapsid protein NCp12.     

Dimerization of human immunodeficiency virus (type 1) RNA: stimulation by cations and possible mechanism.

The retroviral genome consists of two identical RNA molecules joined close to their 5' ends by the dimer linkage structure. Recent findings indicated that retroviral RNA dimerization and encapsidation are probably related events during virion assembly. We studied the cation-induced dimerization of HIV-1 RNA and results indicate that all in vitro generated HIV-1 RNAs containing a 100 nucleotide domain downstream from the 5' splice site are able to dimerize. RNA dimerization depends on the concentration of RNA, mono- and multivalent cations, the size of the monovalent cation, temperature, and pH. Up to 75% of HIV-1 RNA is dimeric in the presence of spermidine. HIV-1 RNA dimer is fairly resistant to denaturing agents and unaffected by intercalating drugs. Antisense HIV-1 RNA does not dimerize but heterodimers can be formed between HIV-1 RNA and either MoMuLV or RSV RNA. Therefore retroviral RNA dimerization probably does not simply proceed through mechanisms involving Watson-Crick base-pairing. Neither adenine and cytosine protonation, nor quartets containing only guanines appear to determine the stability of the HIV-1 RNA dimer, while quartets involving both adenine(s) and guanine(s) could account for our results. A consensus sequence PuGGAPuA found in the putative dimerization-encapsidation region of all retroviral genomes examined may participate in the dimerization process.     

Human immunodeficiency virus type 1 RNA outside the primary encapsidation and dimer linkage region affects RNA dimer stability in vivo.

To characterize the cis-acting determinants that function in RNA dimer formation and maintenance, we examined the stability of RNA dimers isolated from virus particles containing mutations in the encapsidation region of human immunodeficiency virus type 1 (HIV-1). The genomic RNAs of all mutants containing lesions in elements required for in vitro dimerization exhibited thermal stability similar to that of wild-type (WT) HIV-1. These data indicate that the eventual formation of stable dimeric RNA in vivo is not absolutely dependent on the elements that promote dimer formation in vitro. Surprisingly, mutants that lacked a large segment of the middle portion of the genome, outside the likely primary dimer linkage region, formed RNA dimers that were measurably more stable than WT. In addition, the insertion of one or multiple copies of a foreign gene, which resulted in a series of vectors that approached RNA length similar to that of WT RNA, still exhibited augmented dimer stability. These results suggest that there are regions in the HIV-1 genome outside the primary dimer initiation and dimer linkage regions that can negatively affect dimer stability.     

Alteration of location of dimer linkage sequence in retroviral RNA: little effect on replication or homologous recombination.

Retrovirus particles contain a dimer of retroviral genomic RNA. A defined region of the retrovirus genome has previously been shown to be important for both dimerization and encapsidation. To study the importance of the position of this encapsidation and dimerization signal for retroviral replication and homologous recombination, we used a previously described spleen necrosis virus-based helper cell system. This system allows retroviral vectors with multiple genetic markers to be studied after a single cycle of retroviral replication. The sequence responsible for dimerization, the encapsidation/dimer linkage sequence (E/DLS), was moved from its normal location near the 5' end of the retroviral genome to a location near the 3' end of the genome. We characterized four pairs of retroviral vectors: (i) with both E/DLSs at the 5' ends of the genomes, (ii) with both E/DLSs at the 3' ends of the genomes, and (iii) two with one E/DLS at the 5' end of the genome and one at the 3' end of the genome. We found that moving the E/DLS to the 3' end of the genome resulted in at most an approximately factor of 5 reduction in virus titer in a single cycle of retroviral replication. Furthermore, we found no changes that were attributable to the alteration of the position of the E/DLS in the minus-strand DNA primer transfers or the plus-strand DNA primer transfers, the rate of homologous recombination, or the number of internal template switches in recombinant proviruses. These results indicate that any alignment or conformation necessary for retroviral replication or recombination is not the result of the position of the E/DLS.     

Palindromic sequence plays a critical role in human foamy virus dimerization

The retroviral RNA genome is dimeric, consisting of two identical strands of RNA linked near their 5' ends by a dimer linkage structure. Previously it was shown that human foamy virus (HFV) RNA transcribed in vitro contained three sites, designated SI, SII, and SIII, which contributed to the dimerization process (O. Erlwein, D. Cain, N. Fischer, A. Rethwilm, and M. O. McClure, Virology 229:251-258, 1997). To characterize these sites further, a series of mutants were designed and tested for their ability to dimerize in vitro. The primer binding site and a G tetrad in SI were dispensable for dimerization. However, a mutant that changed the 3' end of SI migrated slower on nondenaturing gels than wild-type RNA dimers. The sequence composition of the SII palindrome, consisting of 10 nucleotides, proved to be critical for in vitro dimerization, since mutations within this sequence or replacement of the sequence with a different palindrome of equal length impaired in vitro dimerization. The length of the palindrome also seems to play an important role. A moderate extension to 12 nucleotides was tolerated, whereas an extension to 16 nucleotides or more impaired dimerization. When nucleotides flanking the palindrome were mutated in a random fashion, dimerization was unaffected. Changing the SIII sequence also led to decreased dimer formation, confirming its contribution to the dimerization process. Interesting mutants were cloned into the infectious molecular clone of HFV, HSRV-2, and were transfected into BHK-21 cells. Mutations in SII that reduced dimerization in vitro also abolished virus replication. In contrast, constructs containing mutations in SI and SIII replicated to some extent in cell culture after an initial drop in viral replication. Analysis of the SIM1 mutant revealed reversion to the wild type but with the insertion of an additional two nucleotides. Analysis of cell-free virions demonstrated that both replication-competent and replication-defective mutants packaged nucleic acid. Thus, efficient dimerization is a critical step for HFV to generate infectious virus, but HFV RNA dimerization is not a prerequisite for packaging.     

An additional dimer linkage structure in Moloney murine leukemia virus RNA.

We have identified an additional dimerization linkage structure in the genome of Moloney murine leukemia virus (MoMLV). Retroviral genomes have long been known to be linked at their 5' ends to form dimers. In MoMLV, a hairpin loop functioning as a dimer linkage structure (DLS) has previously been identified at nucleotides 278-303. Here, we describe RNA dimers formed from sections of the MoMLV 5' untranslated region that do not contain the previously described MoMLV DLS. These dimers exhibit the distinctive characteristics previously described for whole genome dimers. We have mapped this novel region to nucleotides 199-243. This sequence contains a stem-loop structure (nucleotides 204-227) much like the 278-303 region. We describe the chemical and thermal stability of dimers containing the 204-227 stem-loop as well as kinetics and salt-dependence of dimer formation. Our results show that dimerization of MoMLV RNA can be nucleated at multiple sites and suggest that the 5' untranslated region may contain separately folding and dimerizing domains.     

Structural requirements for nucleocapsid protein-mediated dimerization of avian leukosis virus RNA

The avian leukosis virus (ALV) belongs to the alpha group of retroviruses that are widespread in nature. The 5'-untranslated region of ALV genome contains the L3 element that is important for virus infectivity and the formation of an unstable RNA dimer in vitro. The L3 sequence is predicted to fold into a long stem-loop structure with two internal loops and an apical one. Phylogenetic analysis predicts that the L3 stem-loop is conserved in alpharetroviruses. Furthermore, a significant selection mechanism maintains a palindrome in the apical loop. The nucleocapsid protein of the alpharetroviruses (NCp12) is required for RNA dimer formation and replication in vivo. It is not known whether L3 can be an NCp12-mediated RNA dimerization site able to bind NCp12 with high affinity. Here, we report that NCp12 chaperones formation of a stable ALV RNA dimer through L3. To investigate the NCp12-mediated L3 dimerization reaction, we performed site-directed mutagenesis, gel retardation and heterodimerization assays and analysis of thermostability of dimeric RNAs. We show that the affinity of NCp12 for L3 is lower than its affinity for the microPsi RNA packaging signal. Results show that conservation of a long stem-loop structure and a loop-loop interaction are not required for NCp12-mediated L3 dimerization. We show that the L3 apical stem-loop is sufficient to form an extended duplex and the whole stem-loop L3 cannot be converted by NCp12 into a duplex extending throughout L3. Three-dimensional modelling of the stable L3 dimer supports the notion that the extended duplex may represent the minimal dimer linkage structure found in the genomic RNA.     

Architecture of a gamma retroviral genomic RNA dimer

Retroviral genomes contain two sense-strand RNAs that are noncovalently linked at their 5' ends, forming a dimer. Establishing a structure for this dimer is an obligatory first step toward understanding the fundamental role of the dimeric RNA in retroviral biology. We developed a secondary structure model for the minimal dimerization active sequence (MiDAS) for the Moloney murine sarcoma virus in the final dimer state using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). In this model, two self-complementary, or palindromic, sequences (PAL1 and PAL2) form extended intermolecular duplexes of 10 and 16 base pairs, respectively. The monomeric starting state was shown previously to contain a flexible domain in which nucleotides do not form stable interactions with other parts of the RNA. In the final dimer state, portions of this initial flexible domain form stable base pairs, while previously base-paired elements lie in a new flexible domain. Thus, partially overlapping and structurally well-defined flexible domains are prominent features of both monomer and dimer states. We then used hydroxyl radical cleavage experiments to characterize the global architecture of the dimer state. Extensive regions, including portions of both PAL1 and PAL2, are occluded from solvent-based cleavage indicating that the MiDAS domain does not function simply as a collection of autonomous secondary structure elements. Instead, the retroviral dimerization domain adopts a compact architecture characterized by close packing of its constituent helices.     

Preliminary physical mapping of RNA-RNA linkages in the genomic RNA of Moloney murine leukemia virus

Retrovirus particles contain two copies of their genomic RNA, held together in a dimer by linkages which presumably consist of a limited number of base pairs. In an effort to localize these linkages, we digested deproteinized RNA from Moloney murine leukemia virus (MLV) particles with RNase H in the presence of oligodeoxynucleotides complementary to specific sites in viral RNA. The cleaved RNAs were then characterized by nondenaturing gel electrophoresis. We found that fragments composed of nucleotides 1 to 754 were dimeric, with a linkage as thermostable as that between dimers of intact genomic RNA. In contrast, there was no stable linkage between fragments consisting of nucleotides 755 to 8332. Thus, the most stable linkage between monomers is on the 5' side of nucleotide 754. This conclusion is in agreement with earlier electron microscopic analyses of partially denatured viral RNAs and with our study (C. S. Hibbert, J. Mirro, and A. Rein, J. Virol. 78:10927-10938, 2004) of encapsidated nonviral mRNAs containing inserts of viral sequence. We obtained similar results with RNAs from immature MLV particles, in which the dimeric linkage is different from that in mature particles and has not previously been localized. The 5' and 3' fragments of cleaved RNA are all held together by thermolabile linkages, indicating the presence of tethering interactions between bases 5' and bases 3' of the cleavage site. When RNAs from mature particles were cleaved at nucleotide 1201, we detected tethering interactions spanning the cleavage site which are intramonomeric and are as strong as the most stable linkage between the monomers.     

Moloney Murine Sarcoma Virus Genomic RNAs Dimerize via a Two-Step Process: a Concentration-Dependent Kissing-Loop Interaction Is Driven by Initial Contact between Consecutive Guanines

Retroviruses contain two plus-strand genomic RNAs, which are stably but noncovalently joined in their 5' regions by a dimer linkage structure (DLS). Two models have been put forward to explain the mechanisms by which the RNAs dimerize; each model emphasizes the role of specific molecular determinants. The kissing-loop model implicates interactions between palindromic sequences in the DLS region. The second model proposes that purine-rich stretches in the region form purine quartet structures. Here, we present an examination of the in vitro dimerization of Moloney murine sarcoma virus (MuSV) RNA in the context of these two models. Dimers were found to form spontaneously in a temperature-, time-, concentration-, and salt-dependent manner. In contrast to earlier reports, we found that deletion of neither the palindrome nor the consensus purine motifs (PuGGAPuA) affected the level of dimer formation at low concentrations of RNA. Rather, different purine-rich sequences, i.e., consecutive stretches of guanines, were found to enhance both in vitro RNA dimerization and in vivo viral replication. Biochemical evidence further suggests that these guanine-rich (G-rich) stretches form guanine quartet structures. We also found that the palindromic sequences could support dimerization at significantly higher RNA concentrations. In addition, the G-rich stretches were as important as the palindromic sequence for maintaining efficient viral replication. Overall, our data support a model that entails contributions from both of the previously proposed mechanisms of retroviral RNA dimerization.     

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.     

Duplication of the primary encapsidation and dimer linkage region of human immunodeficiency virus type 1 RNA results in the appearance of monomeric RNA in virions

The dimerization initiation site (DIS) and the dimer linkage sequences (DLS) of human immunodeficiency virus type 1 have been shown to mediate in vitro dimerization of genomic RNA. However, the precise role of the DIS-DLS region in virion assembly and RNA dimerization in virus particles has not been fully elucidated, since deletion or mutation of the DIS-DLS region also abolishes the packaging ability of genomic RNA. To characterize the DIS-DLS region without altering packaging ability, we generated mutant constructs carrying a duplication of approximately 1,000 bases including the encapsidation signal and DIS-DLS (E/DLS) region. We found that duplication of the E/DLS region resulted in the appearance of monomeric RNA in virus particles. No monomers were observed in virions of mutants carrying the E/DLS region only at ectopic positions. Monomers were not observed when pol or env regions were duplicated, indicating an absolute need for two intact E/DLS regions on the same RNA for generating particles with monomeric RNA. These monomeric RNAs were most likely generated by intramolecular interaction between two E/DLS regions on one genome. Moreover, incomplete genome dimerization did not affect RNA packaging and virion formation. Examination of intramolecular interaction between E/DLS regions could be a convenient tool for characterizing the E/DLS region in virion assembly and RNA dimerization within virus particles.     

Modulation of homo- and heterodimerization of Harvey sarcoma virus RNA by GACG tetraloops and point mutations in palindromic sequences

Retroviruses harbour a diploid genome of two plus-strand RNAs linked non-covalently at the dimer linkage structure. Co-packaging of two parental RNAs is a prerequisite for recombination in retroviruses, but formation of heterodimers has not been demonstrated directly in vivo. Here, we explore elements in Harvey sarcoma virus (HaSV) RNA involved in homodimerization and heterodimerization with RNA of Moloney (Mo) and Akv murine leukemia viruses (MLV).By an in vitro assay, we found that HaSV dimerization specificity could be modulated by mutations in a decanucleotide palindrome (Pal) probably folded into a kissing-loop. Autocomplementary and non-autocomplementary sequences introduced into the putative loop directed the specificity towards formation of homodimers and heterodimers, respectively. Two stem-loop (SL) structures, both exposing a GACG tetraloop, enhanced the formation of stable HaSV dimers.A similar decanucleotide palindrome has been implicated in homodimerization of MLVs. Heterodimers between HaSV RNA and Mo- or Akv MLV were unstable, but could be stabilized by introduction of two point mutations in the putative HaSV kissing-loop, creating exact complementarity with Mo/Akv MLV palindromes. Moreover, such changes increased the HaSV RNA affinity for the two MLV RNAs. Similar to HaSV RNA homodimers, formation of heterodimers with Mo- or Akv MLV RNAs was induced by the presence of GACG loops.On the basis of these results, we propose that palindromic sequences act as variable determinants of specificity and GACG tetraloops as conserved determinants in the formation of homodimers and heterodimers of gamma-retrovirus retroviral RNAs in vivo. The complementarity of loop sequences in the packaging signal upstream of the GACG tetraloops might therefore determine homo- and heterodimerization specificity and recombination activity of these viruses.