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1.
The virion incorporation of 7SL, the RNA component of the host signal recognition particle (SRP), has been shown for several simple retroviruses. Data here demonstrate that 7SL is also packaged by HIV-1, in sevenfold molar excess of genomic RNA. Viral determinants of HIV-1 genome and primer tRNA packaging were not required for 7SL incorporation, as virus-like particles with only minimal assembly components efficiently packaged 7SL. The majority of 7SL within cells resides in ribonucleoprotein complexes bound by SRP proteins, and most SRP protein exists in signal recognition particles. However, Western blot comparison of virion and cell samples revealed that there is at least 25-fold less SRP p54 protein per 7SL RNA in HIV-1 particles than in cells. Comparing 7SL:actin mRNA ratios in virions and cells revealed that 7SL RNA appears selectively enriched in virions. 相似文献
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HIV controls the selective packaging of genomic, spliced viral and cellular RNAs into virions through different mechanisms 总被引:5,自引:2,他引:3
Houzet L Paillart JC Smagulova F Maurel S Morichaud Z Marquet R Mougel M 《Nucleic acids research》2007,35(8):2695-2704
In addition to genomic RNA, HIV-1 particles package cellular and spliced viral RNAs. In order to determine the encapsidation mechanisms of these RNAs, we determined the packaging efficiencies and specificities of genomic RNA, singly and fully spliced HIV mRNAs and different host RNAs species: 7SL RNA, U6 snRNA and GAPDH mRNA using RT-QPCR. Except GAPDH mRNA, all RNAs are selectively encapsidated. Singly spliced RNAs, harboring the Rev-responsible element, and fully spliced viral RNAs, which do not contain this motif, are enriched in virions to similar levels, even though they are exported from the nucleus by different routes. Deletions of key motifs (SL1 and/or SL3) of the packaging signal of genomic RNA indicate that HIV and host RNAs are encapsidated through independent mechanisms, while genomic and spliced viral RNA compete for the same trans-acting factor due to the presence of the 5′ common exon containing the TAR, poly(A) and U5-PBS hairpins. Surprisingly, the RNA dimerization initiation site (DIS/SL1) appears to be the main packaging determinant of genomic RNA, but is not involved in packaging of spliced viral RNAs, suggesting a functional interaction with intronic sequences. Active and selective packaging of host and spliced viral RNAs provide new potential functions to these RNAs in the early stages of the virus life cycle. 相似文献
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mRNA molecules containing murine leukemia virus packaging signals are encapsidated as dimers
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Prior work by others has shown that insertion of psi (i.e., leader) sequences from the Moloney murine leukemia virus (MLV) genome into the 3' untranslated region of a nonviral mRNA leads to the specific encapsidation of this RNA in MLV particles. We now report that these RNAs are, like genomic RNAs, encapsidated as dimers. These dimers have the same thermostability as MLV genomic RNA dimers; like them, these dimers are more stable if isolated from mature virions than from immature virions. We characterized encapsidated mRNAs containing deletions or truncations of MLV psi or with psi sequences from MLV-related acute transforming viruses. The results indicate that the dimeric linkage in genomic RNA can be completely attributed to the psi region of the genome. While this conclusion agrees with earlier electron microscopic studies on mature MLV dimers, it is the first evidence as to the site of the linkage in immature dimers for any retrovirus. Since the Psi(+) mRNA is not encapsidated as well as genomic RNA, it is only present in a minority of virions. The fact that it is nevertheless dimeric argues strongly that two of these molecules are packaged into particles together. We also found that the kissing loop is unnecessary for this coencapsidation or for the stability of mature dimers but makes a major contribution to the stability of immature dimers. Our results are consistent with the hypothesis that the packaging signal involves a dimeric structure in which the RNAs are joined by intermolecular interactions between GACG loops. 相似文献
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Avian retroviral RNA encapsidation: reexamination of functional 5' RNA sequences and the role of nucleocapsid Cys-His motifs. 总被引:35,自引:31,他引:4
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RNA packaging signals (psi) from the 5' ends of murine and avian retroviral genomes have previously been shown to direct encapsidation of heterologous mRNA into the retroviral virion. The avian 5' packaging region has now been further characterized, and we have defined a 270-nucleotide sequence, A psi, which is sufficient to direct packaging of heterologous RNA. Identification of the A psi sequence suggests that several retroviral cis-acting sequences contained in psi+ (the primer binding site, the putative dimer linkage sequence, and the splice donor site) are dispensable for specific RNA encapsidation. Subgenomic env mRNA is not efficiently encapsidated into particles, even though the A psi sequence is present in this RNA. In contrast, spliced heterologous psi-containing RNA is packaged into virions as efficiently as unspliced species; thus splicing per se is not responsible for the failure of env mRNA to be encapsidated. We also found that an avian retroviral mutant deleted for both nucleocapsid Cys-His boxes retains the capacity to encapsidate RNA containing psi sequences, although this RNA is unstable and is thus difficult to detect in mature particles. Electron microscopy reveals that virions produced by this mutant lack a condensed core, which may allow the RNA to be accessible to nucleases. 相似文献
5.
Peng Ni Robert C. VaughanBrady Tragesser Haley HooverC. Cheng Kao 《Journal of molecular biology》2014
Brome mosaic virus (BMV) packages its genomic and subgenomic RNAs into three separate viral particles. BMV purified from barley, wheat, and tobacco have distinct relative abundances of the encapsidated RNAs. We seek to identify the basis for the host-dependent differences in viral RNA encapsidation. Sequencing of the viral RNAs revealed recombination events in the 3′ untranslated region of RNA1 of BMV purified from barley and wheat, but not from tobacco. However, the relative amounts of the BMV RNAs that accumulated in barley and wheat are similar and RNA accumulation is not sufficient to account for the difference in RNA encapsidation. Virions purified from barley and wheat were found to differ in their isoelectric points, resistance to proteolysis, and contacts between the capsid residues and the RNA. Mass spectrometric analyses revealed that virions from the three hosts had different post-translational modifications that should impact the physiochemical properties of the virions. Another major source of variation in RNA encapsidation was due to the purification of BMV particles to homogeneity. Highly enriched BMV present in lysates had a surprising range of sizes, buoyant densities, and distinct relative amounts of encapsidated RNAs. These results show that the encapsidated BMV RNAs reflect a combination of host effects on the physiochemical properties of the viral capsids and the enrichment of a subset of virions. The previously unexpected heterogeneity in BMV should influence the timing of the infection and also the host innate immune responses. 相似文献
6.
Retroviral genomes consist of two unspliced RNAs linked noncovalently in a dimer. Although these two RNAs are generally identical, two different RNAs can be copackaged when virions are produced by coinfected cells. It has been assumed, but not tested, that copackaging results from random RNA associations in the cytoplasm to yield encapsidated RNA homodimers and heterodimers in Hardy-Weinberg proportions. Here, virion RNA homo- and heterodimerization were examined for Moloney murine leukemia virus (MLV) using nondenaturing Northern blotting and a novel RNA dimer capture assay. The results demonstrated that coexpressed MLV RNAs preferentially self-associated, even when RNAs were identical in known packaging and dimerization sequences or when they differed overall by less than 0.1%. In contrast, HIV-1 RNAs formed homo- and heterodimers in random proportions. We speculate that these species-specific differences in RNA dimer partner selection may at least partially explain the higher frequency of genetic recombination observed for human immunodeficiency virus type 1 than for MLV. 相似文献
7.
7SL RNA mediates virion packaging of the antiviral cytidine deaminase APOBEC3G 总被引:4,自引:2,他引:2
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Wang T Tian C Zhang W Luo K Sarkis PT Yu L Liu B Yu Y Yu XF 《Journal of virology》2007,81(23):13112-13124
Cytidine deaminase APOBEC3G (A3G) has broad antiviral activity against diverse retroviruses and/or retrotransposons, and its antiviral functions are believed to rely on its encapsidation into virions in an RNA-dependent fashion. However, the cofactors of A3G virion packaging have not yet been identified. We demonstrate here that A3G selectively interacts with certain polymerase III (Pol III)-derived RNAs, including Y3 and 7SL RNAs. Among A3G-binding Pol III-derived RNAs, 7SL RNA was preferentially packaged into human immunodeficiency virus type 1 (HIV-1) particles. Efficient packaging of 7SL RNA, as well as A3G, was mediated by the RNA-binding nucleocapsid domain of HIV-1 Gag. A3G mutants that had reduced 7SL RNA binding but maintained wild-type levels of mRNA and tRNA binding were packaged poorly and had impaired antiviral activity. Reducing 7SL RNA packaging by overexpression of SRP19 proteins inhibited 7SL RNA and A3G virion packaging and impaired its antiviral function. Thus, 7SL RNA that is encapsidated into diverse retroviruses is a key cofactor of the antiviral A3G. This selective interaction of A3G with certain Pol III-derived RNAs raises the question of whether A3G and its cofactors may have as-yet-unidentified cellular functions. 相似文献
8.
Hema M Murali A Ni P Vaughan RC Fujisaki K Tsvetkova I Dragnea B Kao CC 《Molecular plant-microbe interactions : MPMI》2010,23(11):1433-1447
Brome mosaic virus (BMV) packages its genomic RNAs (RNA1, RNA2, and RNA3) and subgenomic RNA4 into three different particles. However, since the RNAs in the virions have distinct lengths and electrostatic charges, we hypothesize that subsets of the virions should have distinct properties. A glutamine to cysteine substitution at position 120 of the capsid protein (CP) was found to result in a mutant virus named QC that exhibited a dramatically altered ratio of the RNAs in virions. RNA2 was far more abundant than the other RNAs, although the ratios could be affected by the host plant species. RNAs with the QC mutation were competent for replication early in the infection, suggesting that they were either selectively packaged or degraded after packaging. In support of the latter idea, low concentrations of truncated RNA1 that co-migrated with RNA2 were found in the QC virions. Spectroscopic analysis and peptide fingerprinting experiments showed that the QC virus capsid interacted with the encapsidated RNAs differently than did the wild type. Furthermore, wild-type BMV RNA1 was found to be more susceptible to nuclease digestion relative to RNA2 as a function of the buffer pH. Other BMV capsid mutants also had altered ratios of packaged RNAs. 相似文献
9.
Didierlaurent L Racine PJ Houzet L Chamontin C Berkhout B Mougel M 《Nucleic acids research》2011,39(20):8915-8927
HIV-1 particles contain RNA species other than the unspliced viral RNA genome. For instance, viral spliced RNAs and host 7SL and U6 RNAs are natural components that are non-randomly incorporated. To understand the mechanism of packaging selectivity, we analyzed the content of a large panel of HIV-1 variants mutated either in the 5'UTR structures of the viral RNA or in the Gag-nucleocapsid protein (GagNC). In parallel, we determined whether the selection of host 7SL and U6 RNAs is dependent or not on viral RNA and/or GagNC. Our results reveal that the polyA hairpin in the 5'UTR is a major packaging determinant for both spliced and unspliced viral RNAs. In contrast, 5'UTR RNA structures have little influence on the U6 and 7SL RNAs, indicating that packaging of these host RNAs is independent of viral RNA packaging. Experiments with GagNC mutants indicated that the two zinc-fingers and N-terminal basic residues restrict the incorporation of the spliced RNAs, while favoring unspliced RNA packaging. GagNC through the zinc-finger motifs also restricts the packaging of 7SL and U6 RNAs. Thus, GagNC is a major contributor to the packaging selectivity. Altogether our results provide new molecular insight on how HIV selects distinct RNA species for incorporation into particles. 相似文献
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Diverse retroviruses have been shown to package host SRP (7SL) RNA. However, little is known about the viral determinants of 7SL RNA packaging. Here we demonstrate that 7SL RNA is more selectively packaged into HIV-1 virions than are other abundant Pol-III-transcribed RNAs, including Y RNAs, 7SK RNA, U6 snRNA and cellular mRNAs. The majority of the virion-packaged 7SL RNAs were associated with the viral core structures and could be reverse-transcribed in HIV-1 virions and in virus-infected cells. Viral Pol proteins influenced tRNAlys,3 packaging but had little influence on virion packaging of 7SL RNA. The N-terminal basic region and the basic linker region of HIV-1 NCp7 were found to be important for efficient 7SL RNA packaging. Although Alu RNAs are derived from 7SL RNA and share the Alu RNA domain with 7SL RNA, the packaging of Alu RNAs was at least 50-fold less efficient than that of 7SL RNA. Thus, 7SL RNAs are selectively packaged into HIV-1 virions through mechanisms distinct from those for viral genomic RNA or primer tRNAlys,3. Virion packaging of both human cytidine deaminase APOBEC3G and cellular 7SL RNA are mapped to the same regions in HIV-1 NC domain. 相似文献
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Ni P Wang Z Ma X Das NC Sokol P Chiu W Dragnea B Hagan M Kao CC 《Journal of molecular biology》2012,419(5):284-300
The coat protein of positive-stranded RNA viruses often contains a positively charged tail that extends toward the center of the capsid and interacts with the viral genome. Electrostatic interaction between the tail and the RNA has been postulated as a major force in virus assembly and stabilization. The goal of this work is to examine the correlation between electrostatic interaction and amount of RNA packaged in the tripartite Brome Mosaic Virus (BMV). Nanoindentation experiment using atomic force microscopy showed that the stiffness of BMV virions with different RNAs varied by a range that is 10-fold higher than that would be predicted by electrostatics. BMV mutants with decreased positive charges encapsidated lower amounts of RNA while mutants with increased positive charges packaged additional RNAs up to ~900 nt. However, the extra RNAs included truncated BMV RNAs, an additional copy of RNA4, potential cellular RNAs, or a combination of the three, indicating that change in the charge of the capsid could result in several different outcomes in RNA encapsidation. In addition, mutant with specific arginines changed to lysines in the capsid also exhibited defects in the specific encapsidation of BMV RNA4. The experimental results indicate that electrostatics is a major component in RNA encapsidation but was unable to account for all of the observed effects on RNA encapsidation. Thermodynamic modeling incorporating the electrostatics was able to predict the approximate length of the RNA to be encapsidated for the majority of mutant virions, but not for a mutant with extreme clustered positive charges. Cryo-electron microscopy of virions that encapsidated an additional copy of RNA4 revealed that, despite the increase in RNA encapsidated, the capsid structure was minimally changed. These results experimentally demonstrated the impact of electrostatics and additional restraints in the encapsidation of BMV RNAs, which could be applicable to other viruses. 相似文献
20.
Splicing affects presentation of RNA dimerization signals in HIV-2 in vitro 总被引:1,自引:0,他引:1
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During retroviral replication, full-length viral RNAs are encapsidated into new virus particles, while spliced RNAs are excluded. The Retroviridae are unique among viruses in that infectious viral particles contain a dimer of two identical genomic RNA strands. A variety of experimental data has suggested that dimerization and encapsidation of full-length viral RNAs are linked processes, although whether dimerization is a prerequisite for encapsidation, or conversely, dimerization follows encapsidation, has not been firmly established. If dimerization was the sole determinant for encapsidation, then spliced viral RNAs might be expected to display a reduced capacity for dimerization, resulting in their exclusion from the dimerization pool. Here, we studied the in vitro dimerization properties of unspliced and spliced HIV-2 RNA. We find that the rate and yield of dimerization of Nef, Rev and Tat spliced RNAs exceeded those of unspliced RNA. Although these data do not support a simple correlation between dimerization efficiency and the presence of introns, they establish that splicing affects the presentation of dimerization signal(s), which we corroborate with structure probing. This change in RNA conformation likely affects the RNA's suitability for packaging. Furthermore, the presence of upstream and downstream elements that affect the conformation of the packaging signal represents a potentially efficient viral strategy for correctly sorting spliced versus unspliced RNAs. 相似文献