Murine Leukemia Virus RNA Research Articles

5-Methylcytosine RNA Modifications Promote Retrovirus Replication in an ALYREF Reader Protein-Dependent Manner.

RNA modifications play diverse roles in regulating RNA function, and viruses co-opt these pathways for their own benefit. While recent studies have highlighted the importance of N6-methyladenosine (m6A)-the most abundant mRNA modification-in regulating retrovirus replication, the identification and function of other RNA modifications in viral biology have been largely unexplored. Here, we characterized the RNA modifications present in a model retrovirus, murine leukemia virus (MLV), using mass spectrometry and sequencing. We found that 5-methylcytosine (m5C) is highly enriched in viral genomic RNA relative to uninfected cellular mRNAs, and we mapped at single-nucleotide resolution the m5C sites, which are located in multiple clusters throughout the MLV genome. Further, we showed that the m5C reader protein ALYREF plays an important role in regulating MLV replication. Together, our results provide a complete m5C profile in a virus and its function in a eukaryotic mRNA.IMPORTANCE Over 130 modifications have been identified in cellular RNAs, which play critical roles in many cellular processes, from modulating RNA stability to altering translation efficiency. One such modification, 5-methylcytosine, is relatively abundant in mammalian mRNAs, but its precise location and function are not well understood. In this study, we identified unexpectedly high levels of m5C in the murine leukemia virus RNA, precisely mapped its location, and showed that ALYREF, a reader protein that specifically recognizes m5C, regulates viral production. Together, our findings provide a high-resolution atlas of m5C in murine leukemia virus and reveal a functional role of m5C in viral replication.

An immature retroviral RNA genome resembles a kinetically trapped intermediate state.

Retroviral virions initially assemble in an immature form that differs from that of the mature infectious particle. The RNA genomes in both immature and infectious particles are dimers, and interactions between the RNA dimer and the viral Gag protein ensure selective packaging into nascent immature virions. We used high-sensitivity selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to obtain nucleotide-resolution structural information from scarce, femtomole quantities of Moloney murine leukemia virus (MuLV) RNA inside authentic virions and from viral RNA extracted from immature (protease-minus) virions. Our secondary structure model of the dimerization and packaging domain indicated that a stable intermolecular duplex known as PAL2, previously shown to be present in mature infectious MuLV particles, was sequestered in an alternate stem-loop structure inside immature virions. The intermediate state corresponded closely to a late-folding intermediate that we detected in time-resolved studies of the free MuLV RNA, suggesting that the immature RNA structure reflects trapping of the intermediate folding state by interactions in the immature virion. We propose models for the RNA-protein interactions that trap the RNA in the immature state and for the conformational rearrangement that occurs during maturation of virion particles. The structure of the RNA genome in mature retroviruses has been studied extensively, whereas very little was known about the RNA structure in immature virions. The immature RNA structure is important because it is the form initially selected for packaging in new virions and may have other roles. This lack of information was due to the difficulty of isolating sufficient viral RNA for study. In this work, we apply a high-sensitivity and nucleotide-resolution approach to examine the structure of the dimerization and packaging domain of Moloney murine leukemia virus. We find that the genomic RNA is packaged in a high-energy state, suggesting that interactions within the virion hold or capture the RNA before it reaches its most stable state. This new structural information makes it possible to propose models for the conformational changes in the RNA genome that accompany retroviral maturation.

MLV requires Tap/NXF1-dependent pathway to export its unspliced RNA to the cytoplasm and to express both spliced and unspliced RNAs.

BackgroundEukaryotic cells have evolved stringent proofreading mechanisms to ensure that intron-containing mRNAs do not leave the nucleus. However, all retroviruses must bypass this checkpoint for replication. Indeed, their primary polycistronic transcript (Full-Length) must reach the cytoplasm to be either translated or packaged as genomic RNA in progeny viruses.Murine leukemia virus (MLV) is a prototype of simple retroviruses with only two well-regulated splicing events that directly influence viral leukemogenic properties in mice. Several cis-elements have been identified in the FL RNA that regulate its cytoplasmic accumulation. However, their connection with an export mechanism is yet unknown. Our goal was to identify the cellular pathway used by MLV to export its RNAs into the cytoplasm of the host cells.ResultsSince other retroviruses use the CRM1 and/or the Tap/NXF1 pathways to export their unspliced RNA from the nucleus, we investigated the role of these two pathways in MLV replication by using specific inhibitors. The effects of export inhibition on MLV protein synthesis, RNA levels and RNA localization were studied by Western blotting, RT-qPCR, fluorescence microscopy and ribonucleoprotein immunoprecipitation assays. Taken together, our results show for the first time that MLV requires the Tap/NXF1-mediated export pathway, and not the CRM1 pathway, for the expression of its spliced and unspliced RNAs and for FL RNA nuclear export.ConclusionsBy contrast to HIV-1, MLV recruits the same pathway for the cytoplasmic expression of its spliced and unspliced RNAs. Thus, MLV RNA expression depends upon coordinated splicing/export processes. In addition, FL RNA translation relies on Tap/NXF1-dependent export, raising the critical question of whether the pool of FL RNA to be packaged is also exported by Tap/NXF1.

Zinc-finger antiviral protein mediates retinoic acid inducible gene I–like receptor-independent antiviral response to murine leukemia virus

When host cells are infected by an RNA virus, pattern-recognition receptors (PRRs) recognize the viral RNA and induce the antiviral innate immunity. Toll-like receptor 7 (TLR7) detects the genomic RNA of incoming murine leukemia virus (MLV) in endosomes and mediates the antiviral response. However, the RNA-sensing PRR that recognizes the MLV in the cytosol is not fully understood. Here, we definitively demonstrate that zinc-finger antiviral protein (ZAP) acts as a cytosolic RNA sensor, inducing the degradation of the MLV transcripts by the exosome, an RNA degradation system, on RNA granules. Although the retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 detect various RNA viruses in the cytosol and induce the type I IFN-dependent antiviral response, RLR loss does not alter the replication efficiency of MLV. In sharp contrast, the loss of ZAP greatly enhances the replication efficiency of MLV. ZAP localizes to RNA granules, where the processing-body and stress-granule proteins assemble. ZAP induces the recruitment of the MLV transcripts and exosome components to the RNA granules. The CCCH-type zinc-finger domains of ZAP, which are RNA-binding motifs, mediate its localization to RNA granules and MLV transcripts degradation by the exosome. Although ZAP was known as a regulator of RIG-I signaling in a human cell line, ZAP deficiency does not affect the RIG-I-dependent production of type I IFN in mouse cells. Thus, ZAP is a unique member of the cytosolic RNA-sensing PRR family that targets and eliminates intracellular RNA viruses independently of TLR and RLR family members.

Applications of RNA structure analysis to retroviral packaging and anti-retroviral therapeutic discovery

Our laboratory is blending new RNA chemistries, largely directed at the ribose 2'-hydroxyl group, with quantitative interpretation to create new kinds of chemical microscopes for accurate RNA structure analysis. This seminar will focus on two examples from retroviral biology. All retroviral RNAs contain a packaging element that directs the genome into nascent virus particles through a mechanism that has been unclear. Using SHAPE, we probed the structure of murine leukemia virus RNA inside virus particles and identified the binding site for the nucleocapsid domain of Gag. The RNA structural context is critical: High-affinity binding requires two specific UCUG-UR-UCUG motifs flanked by base-paired elements. Mutation of four guanosine residues in these two motifs - only 4 nucleotides per genomic RNA - reduced packaging to non-specific levels. These motifs only form in genomic RNA dimers; thus analysis of authentic full-length RNA was critical. This work illustrates how local context and RNA structure can create information-rich recognition signals from simple single-stranded sequence elements. In the second example, we discovered strong correlations between shRNA-mediated inhibition of HIV-1 replication in a cell-based assay and both the energetic cost of breaking secondary structures and the overall annealing energy between guide strand and folded target RNA. These correlations were only apparent when the energetic cost of disrupting pre-existing structure in the HIV-1 RNA was calculated using a secondary structure model derived from SHAPE probing information. We verified these correlations by design and evaluation of additional shRNAs and could achieve consistent inhibition of HIV-1 production by 90%. This approach emphasizes the profound, but ultimately predictable, role of RNA structure in tuning RNAi function and is likely to prove broadly useful in understanding large-scale biological recognition involving RNA.

An Endogenous Murine Leukemia Viral Genome Contaminant in a Commercial RT-PCR Kit is Amplified Using Standard Primers for XMRV

During pilot studies to investigate the presence of viral RNA of xenotropic murine leukemia virus (MLV)-related virus (XMRV) infection in sera from chronic fatigue syndrome (CFS) patients in Japan, a positive band was frequently detected at the expected product size in negative control samples when detecting a partial gag region of XMRV using a one-step RT-PCR kit. We suspected that the kit itself might have been contaminated with small traces of endogenous MLV genome or XMRV and attempted to evaluate the quality of the kit in two independent laboratories. We purchased four one-step RT-PCR kits from Invitrogen, TaKaRa, Promega and QIAGEN in Japan. To amplify the partial gag gene of XMRV or other MLV-related viruses, primer sets (419F and 1154R, and GAG-I-F and GAG-I-R) which have been widely used in XMRV studies were employed. The nucleotide sequences of the amplicons were determined and compared with deposited sequences of a polytropic endogenous MLV (PmERV), XMRV and endogenous MLV-related viruses derived from CFS patients. We found that the enzyme mixtures of the one-step RT-PCR kit from Invitrogen were contaminated with RNA derived from PmERV. The nucleotide sequence of a partial gag region of the contaminant amplified by RT-PCR was nearly identical (99.4% identity) to a PmERV on chromosome 7 and highly similar (96.9 to 97.6%) to recently identified MLV-like viruses derived from CFS patients. We also determined the nucleotide sequence of a partial env region of the contaminant and found that it was almost identical (99.6%) to the PmERV. In the investigation of XMRV infection in patients of CFS and prostate cancer, researchers should prudently evaluate the test kits for the presence of endogenous MLV as well as XMRV genomes prior to PCR and RT-PCR tests.

Definition of a high-affinity Gag recognition structure mediating packaging of a retroviral RNA genome

All retroviral genomic RNAs contain a cis-acting packaging signal by which dimeric genomes are selectively packaged into nascent virions. However, it is not understood how Gag (the viral structural protein) interacts with these signals to package the genome with high selectivity. We probed the structure of murine leukemia virus RNA inside virus particles using SHAPE, a high-throughput RNA structure analysis technology. These experiments showed that NC (the nucleic acid binding domain derived from Gag) binds within the virus to the sequence UCUG-UR-UCUG. Recombinant Gag and NC proteins bound to this same RNA sequence in dimeric RNA in vitro; in all cases, interactions were strongest with the first U and final G in each UCUG element. The RNA structural context is critical: High-affinity binding requires base-paired regions flanking this motif, and two UCUG-UR-UCUG motifs are specifically exposed in the viral RNA dimer. Mutating the guanosine residues in these two motifs--only four nucleotides per genomic RNA--reduced packaging 100-fold, comparable to the level of nonspecific packaging. These results thus explain the selective packaging of dimeric RNA. This paradigm has implications for RNA recognition in general, illustrating how local context and RNA structure can create information-rich recognition signals from simple single-stranded sequence elements in large RNAs.

Murine leukemia virus RNA dimerization is coupled to transcription and splicing processes

Most of the cell biological aspects of retroviral genome dimerization remain unknown. Murine leukemia virus (MLV) constitutes a useful model to study when and where dimerization occurs within the cell. For instance, MLV produces a subgenomic RNA (called SD') that is co-packaged with the genomic RNA predominantly as FLSD' heterodimers. This SD' RNA is generated by splicing of the genomic RNA and also by direct transcription of a splice-associated retroelement of MLV (SDARE). We took advantage of these two SD' origins to study the effects of transcription and splicing events on RNA dimerization. Using genetic approaches coupled to capture of RNA heterodimer in virions, we determined heterodimerization frequencies in different cellular contexts. Several cell lines were stably established in which SD' RNA was produced by either splicing or transcription from SDARE. Moreover, SDARE was integrated into the host chromosome either concomitantly or sequentially with the genomic provirus. Our results showed that transcribed genomic and SD' RNAs preferentially formed heterodimers when their respective proviruses were integrated together. In contrast, heterodimerization was strongly affected when the two proviruses were integrated independently. Finally, dimerization was enhanced when the transcription sites were expected to be physically close. For the first time, we report that splicing and RNA dimerization appear to be coupled. Indeed, when the RNAs underwent splicing, the FLSD' dimerization reached a frequency similar to co-transcriptional heterodimerization. Altogether, our results indicate that randomness of heterodimerization increases when RNAs are co-expressed during either transcription or splicing. Our results strongly support the notion that dimerization occurs in the nucleus, at or near the transcription and splicing sites, at areas of high viral RNA concentration.

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.

Packaging of heterologous RNAs by a minimal bovine leukemia virus RNA packaging signal into virus particles

A minimal bovine leukemia virus (BLV) RNA packaging sequence (E) required for heterologous RNAs to be packaged into BLV particles was analyzed. The BLV E was inserted into a non-viral vector, pLacZ, in order to determine if packaging of the non-viral vector RNA would occur. The construct was transfected into cells chronically infected with BLV in order to produce virus particles. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of viral RNA from virus particles revealed that non-viral RNA containing the BLV E was packaged into BLV particles, indicating that the BLV E is necessary and sufficient to allow for packaging of a non-viral vector RNA. We also analyzed the ability of a chimeric murine leukemia virus (MLV) retroviral vector (pLN) containing BLV E to be packaged into BLV particles. Interestingly, it was found that pLNDelta (which does not possess psi+) could be packaged into BLV particles. This indicates that a MLV RNA region outside of psi+ allows for packaging of the MLV RNA into BLV particles.

Detection of reverse transcriptase activity in human melanoma cell lines and identification of a murine leukemia virus contaminant

Stimulated by earlier reports on the presence of retroviruses in mouse and hamster melanoma cell lines, we addressed the question as to whether human melanoma cell lines might also harbour a retrovirus. The melanoma cell lines SK-MEL-25, SK-MEL-28, MEL-JUSO, MML-I, MeWo, A-375, Colo-38, BS-780 were confirmed to be human by human leucocyte antigen (HLA) typing, and supernatants were tested by the product-enhanced reverse transcriptase (PERT) assay for reverse transcriptase (RT) activity. Cell lines SK-MEL-25, SK-MEL-28, MEL-JUSO and MML-I were positive, whereas cell lines MeWo, A-375, Colo-38 and BS-780 were negative. The RT activity peaked at a buoyant density in sucrose typical for retroviruses. From this peak fraction an R-U5 sequence indistinguishable from murine leukemia virus (MLV) was identified by particle-associated retrovirus RNA amplification (PARRA). Semiquantitative MLV-specific RNA-PCR demonstrated colocalization of the MLV-like RNA and RT activity on the sucrose gradient of SK-Mel-25. MLV RNA and DNA were also detectable in culture supernatants of SK-MEL-28, MEL-JUSO and MML-I, but not of MeWo, A-375, Colo-38 and BS-780 by semiquantitative polymerase chain reaction (PCR). Sequence comparison revealed highest homology with the RET sequence previously identified in mouse myeloma SP2/0-AG14 cells. Taken together, our data strongly suggest that certain human melanoma cell lines are productively infected by a MLV which was probably introduced during tumour passage in mice or by laboratory contamination many years ago and subsequently spread to other lines. We recommend mandatory testing of melanoma and other human cell lines for contamination with infectious MLV or other animal retroviruses, similar to mycoplasma screening, in order to avoid artificial experimental data.

Fundamentals

Friend murine erythroleukemia is a model of virus-induced acute leukemia. RNA and protein viral expression were simultaneously studied in a Friend erythroleukemia murine cell line (HFL/b cells, Freedmanet al. (1975) J. expl. Med.142, 212–223), which had lost its capacity of producing infectious virus and was found to contain neither structural p30 nor gp70 envelope-glycoprotein by radioimmunoassay. Presence of the spleen focus-forming virus (SFFV) in these cells was demonstrated by its biological rescue in vivo and in vitro with Moloney-helper leukemia virus (M-MuLV). Molecular hybridization using a DNA complementary to Friend virus complex revealed the presence in cytoplasmic Poly(A) RNA of a major species with the size of the 32S SFFV genome. No. 38S helper Friend Murine Leukemia Virus (F-MuLV) RNA was detected. Viral-encoded polypeptide expression was studied by immunoprecipitation of cell extracts. A first class of polypeptides constituted by two species of 50,000 and 52,000 daltons was precipitated by a goat anti Rauscher gp70 serum and corresponded to the glycoprotein encoded by the SFFV genome. A second class of polypeptides with apparent mobilities of 48,000 and 50,000 daltons was precipitated by anti p15 and anti p12 sera and to a lesser extent by an anti p30 serum. These products were glycosylated in part. Moreover two p37 and p39 products, which disappeared more rapidly than p48gag + p50gag in pulse-chase labeling experiments were also precipitated by anti-gag sera. No other gag-product such as pr65gag was detected, confirming a negative expression of the F-MuLV genome and supporting the idea that all the gag-related products expressed in these cells were coded by SFFV virus.

A novel subgenomic murine leukemia virus RNA transcript results from alternative splicing.

Here we show the existence of a novel subgenomic 4.4-kb RNA in cells infected with the prototypic replication-competent Friend or Moloney murine leukemia viruses (MuLV). This RNA derives by splicing from an alternative donor site (SD') within the capsid-coding region to the canonical envelope splice acceptor site. The position and the sequence of SD' was highly conserved among mammalian type C and D oncoviruses. Point mutations used to inactivate SD' without changing the capsid-coding ability affected viral RNA splicing and reduced viral replication in infected cells.

Characterization of an internal ribosomal entry segment in the 5' leader of murine leukemia virus env RNA.

The 5' untranslated region, also called the leader, of oncoretroviruses and lentiviruses is long and is formed of several structured domains critically important in virus replication. The 5' leader of murine leukemia virus (MLV) RNA contains an internal ribosomal entry segment (IRES) which promotes synthesis of Gag and glyco-Gag polyprotein precursors. In the present study we investigated the translational features of the 5' leader of MLV subgenomic RNA (env RNA) encoding the Env polyprotein precursor. When the env leader was inserted between two genes, such as lacZ and the neomycin resistance cassette, in a dicistronic vector, it allowed IRES-dependent translation of the 3' cistron in the rabbit reticulocyte lysate system and in murine cells. The drug rapamycin and the foot-and-mouth disease virus L protease, known to inhibit cap-dependent translation, caused an enhancement of the translation driven by the env leader sequence, consistent with an IRES activity promoting Env expression. Analysis of several deletion mutants led us to localize the minimal env IRES between the splice junction and the env AUG start codon.

The nucleocapsid domain is responsible for the ability of spleen necrosis virus (SNV) Gag polyprotein to package both SNV and murine leukemia virus RNA.

Murine leukemia virus (MLV)-based vector RNA can be packaged and propagated by the proteins of spleen necrosis virus (SNV). We recently demonstrated that MLV proteins cannot support the replication of an SNV-based vector; RNA analysis revealed that MLV proteins cannot efficiently package SNV-based vector RNA. The domain in Gag responsible for the specificity of RNA packaging was identified using chimeric gag-pol expression constructs. A competitive packaging system was established by generating a cell line that expresses one viral vector RNA containing the MLV packaging signal (Psi) and another viral vector RNA containing the SNV packaging signal (E). The chimeric gag-pol expression constructs were introduced into the cells, and vector titers as well as the efficiency of RNA packaging were examined. Our data confirm that Gag is solely responsible for the selection of viral RNAs. Furthermore, the nucleocapsid (NC) domain in the SNV Gag is responsible for its ability to interact with both SNV E and MLV Psi. Replacement of the SNV NC with the MLV NC generated a chimeric Gag that could not package SNV RNA but retained its ability to package MLV RNA. A construct expressing SNV gag-MLV pol supported the replication of both MLV and SNV vectors, indicating that the gag and pol gene products from two different viruses can functionally cooperate to perform one cycle of retroviral replication. Viral titer data indicated that SNV cis-acting elements are not ideal substrates for MLV pol gene products since infectious viruses were generated at a lower efficiency. These results indicate that the nonreciprocal recognition between SNV and MLV extends beyond the Gag-RNA interaction and also includes interactions between Pol and other cis-acting elements.

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.

Cis Elements required for high-level expression of unspliced Gag-containing message in Moloney murine leukemia virus.

The 441-nucleotide (nt) region (nt 5325 to 5766) around the splice acceptor (SA) site (nt 5491) was found to be necessary for high-level expression of gag-containing unspliced RNA of Moloney murine leukemia virus (M. Oshima, T. Odawara, T. Matano, H. Sakahira, K. Kuchino, A. Iwamoto, and H. Yoshikura, J. Virol. 70:2286-2295, 1996). Detailed genetic dissection of the 441-nt region revealed that the 5'-end 64 nt (nt 5325 to 5389) were necessary for high-level expression of the unspliced RNA when the spliced RNA was not produced, while the 3'-side 301 nt (nt 5466 to 5766) containing the SA site were necessary for producing spliced RNA. When the spliced RNA was produced, the unspliced RNA could be expressed at a high level even when the 5'-end 64 nt were absent. Probably the virus sequence ensuring the splicing could produce an RNA structure able to compensate for the function of the 5'-end 64-nt region responsible for the expression of the unspliced RNA.

Nonreciprocal pseudotyping: murine leukemia virus proteins cannot efficiently package spleen necrosis virus-based vector RNA.

It has been documented that spleen necrosis virus (SNV) can package murine leukemia virus (MLV) RNA efficiently and propagate MLV vectors to the same titers as it propagates SNV-based vectors. Although the SNV packaging signal (E) and MLV packaging signal (Psi) have little sequence homology, similar double-hairpin RNA structures were predicted and supported by experimental evidence. To test whether SNV RNA can be packaged by MLV proteins, we modified an SNV vector to be expressed in an MLV-based murine helper cell line. Surprisingly, we found that MLV proteins could not support the replication of SNV vectors. The decrease in titer was approximately 2,000- to 20,000-fold in one round of retroviral replication. RNA analysis revealed that SNV RNA was not efficiently packaged by MLV proteins. RNA hybridization of the cellular and viral RNAs indicated that SNV RNA was packaged at least 25-fold less efficiently than MLV RNA, which was the sensitivity limit of the hybridization assay. The contrast between the MLV and SNV packaging specificity is striking. SNV proteins can recognize both SNV E and MLV Psi, but MLV can recognize only MLV Psi. This is the first demonstration of two retroviruses with nonreciprocal packaging specificities.

CIS elements and Trans-acting factors required for minus strand DNA transfer during reverse transcription of the genomic RNA of murine leukemia virus

During reverse transcription of the retroviral genomic RNA, two obligatory DNA strand transfers take place to synthesize the complete proviral DNA with two LTRs. We have previously shown that using an in vitro system made up of two viral RNAs mimicking the 5′ and 3′ regions of the retroviral genome, both nucleocapsid protein and the repeat (R) sequences were necessary for minus strong-stop cDNA (ss-cDNA) transfer and elongation by reverse transcriptase (RT). In this paper we show that the basic residues of nucleocapsid protein NCp10 of Moloney murine leukemia virus (MoMuLV), but not the zinc finger, are necessary for minus strand transfer. In order to examine the role of the R sequence repeated at the 5′ and 3′ ends of the genome in minus strand DNA transfer, the MoMuLV R sequence of 68 nt was replaced by either HIV-1 R of 96 nt, or RSV R of 21 nt, or by an artificial sequence of 21 nt. Analysis of MoMuLV DNA strand transfer from the 5′ RNA to the 3’ RNA and elongation in the presence of NCp10 and RT showed that it was high with control MoMuLV R, high with RSV R, reduced with HIV-1 R, and undetectable with the artificial R sequence. These results suggest that minus strand DNA transfer is a process more complex than simple hybridization of ss-cDNA to the 3′ R sequence of the genomic RNA.

Modified nucleotides of tRNAPro restrict interactions in the binary Primer/Template complex of M-MuLV

In all retroviruses, reverse transcription is primed by a cellular tRNA, which is base-paired through its 3′-terminal 18 nucleotides to a complementary sequence on the viral RNA genome termed the primer binding site (PBS). Evidence for specific primer-template interactions in addition to this standard interaction has recently been demonstrated for several retroviruses. Here, we used chemical and enzymatic probing to investigate the interactions between Moloney murine leukemia virus (M-MuLV) RNA and its natural primer tRNAPro. The existence of extended interactions was further tested by comparing the viral RNA/tRNAProcomplex with simplified complexes in which viral RNA or tRNA were reduced to the 18 nt of the PBS or to the complementary tRNA sequence. These data, combined with computer modeling provide important clues on the secondary structure and three-dimensional folding of the M-MuLV RNA/tRNAProcomplex. In contrast with other retroviruses, we found that the interaction between tRNAProand the M-MuLV RNA template is restricted to the standard PBS interaction. In this binary complex, the viral RNA is highly constrained and the rest of tRNAProis rearranged, with the exception of the anticodon arm, leading to a very compact structure. Unexpectedly, when a synthetic tRNAProlacking the post-transcriptional modifications is substituted for the natural tRNAProprimer, the interactions between the primer and the viral RNA are extended. Hence, our data suggest that the post-transcriptional modifications of natural tRNAProprevent additional contacts between tRNAProand the U5 region of M-MuLV RNA.