Avian retroviral RNA encapsidation: reexamination of functional 5' RNA sequences and the role of nucleocapsid Cys-His motifs.

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.     

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.     

Modular transposition and the dynamical structure of eukaryote regulatory evolution

This paper examines a model in which transposable elements provide a modular architecture for the cellular genome, complemented by cellular recombinational transformations, arising in turn as a dynamical consequence of this modular structure. It is proposed that the ecology of transposable elements in a given organism is a function of recombinational protocols of the evolving cellular genome. In mammals this is proposed to involve coordinated meiosis-phased activation of LINEs, SINEs and retrogenes complemented by endogenous retroviral transfer between cells.     

Targeted modification of mammalian genomes

The stable and site-specific modification of mammalian genomes has a variety of applications in biomedicine and biotechnology. Here we outline two alternative approaches that can be employed to achieve this goal: homologous recombination (HR) or site-specific recombination. Homologous recombination relies on sequence similarity (or rather identity) of a piece of DNA that is introduced into a host cell and the host genome. In most cell types, the frequency of homologous recombination is markedly lower than the frequency of random integration. Especially in somatic cells, homologous recombination is an extremely rare event. However, recent strategies involving the introduction of DNA double-strand breaks, triplex forming oligonucleotides or adeno-associated virus can increase the frequency of homologous recombination.

Site-specific recombination makes use of enzymes (recombinases, transposases, integrases), which catalyse DNA strand exchange between DNA molecules that have only limited sequence homology. The recognition sites of site-specific recombinases (e.g. Cre, Flp or ΦC31 integrase) are usually 30–50 bp. In contrast, retroviral integrases only require a specific dinucleotide sequence to insert the viral cDNA into the host genome. Depending on the individual enzyme, there are either innumerable or very few potential target sites for a particular integrase/recombinase in a mammalian genome. A number of strategies have been utilised successfully to alter the site-specificity of recombinases. Therefore, site-specific recombinases provide an attractive tool for the targeted modification of mammalian genomes.     

Inhibition of HIV derived lentiviral production by TAR RNA binding domain of TAT protein

Background

Retroviruses have a diploid genome and recombine at high frequency. Recombinant proviruses can be generated when two genetically different RNA genomes are packaged into the same retroviral particle. It was shown in several studies that recombinant proviruses could be generated in each round of HIV-1 replication, whereas the recombination rates of SNV and Mo-MuLV are 5 to 10-fold lower. The reason for these differences is not clear. One possibility is that these retroviruses may differ in their ability to copackage genomic RNAs produced at different chromosomal loci.

Results

To investigate whether there is a difference in the efficiency of heterodimer formation when two proviruses have the same or different chromosomal locations, we introduced two different Mo-MuLV-based retroviral vectors into the packaging cell line using either the cotransfection or sequential transfection procedure. The comparative study has shown that the frequency of recombination increased about four-fold when the cotransfection procedure was used. This difference was not associated with possible recombination of retroviral vectors during or after cotransfection and the ratios of retroviral virion RNAs were the same for two variants of transfection.

Conclusions

The results of this study indicate that a mechanism exists to enable the preferential copackaging of Mo-MuLV genomic RNA molecules that are transcribed on the same DNA template. The properties of Mo-MuLV genomic RNAs transport, processing or dimerization might be responsible for this preference. The data presented in this report can be useful when designing methods to study different aspects of replication and recombination of a diploid retroviral genome.     

Characterization of replication-competent retroviruses from nonhuman primates with virus-induced T-cell lymphomas and observations regarding the mechanism of oncogenesis.

Rapidly progressive T-cell lymphomas were observed in 3 of 10 rhesus monkeys several months after autologous transplantation of enriched bone marrow stem cells that had been transduced with a retroviral vector preparation containing replication-competent virus (R. E. Donahue, S. W. Kessler, D. Bodice, K. McDonagh, C. Dunbar, S. Goodman, B. Agricola, E. Byrne, M. Raffeld, R. Moen, J. Bacher, K. M. Zsebo, and A. W. Nienhuis, J. Exp. Med. 176:1124-1135, 1992). The animals with lymphoma appeared to be tolerant to retroviral antigens in that their sera lacked antibodies reactive with viral proteins and contained 10(4) to 10(5) infectious virus particles per ml. By molecular cloning and DNA sequencing, we have now demonstrated that the serum from one of the monkeys contained a replication-competent retrovirus that arose by recombination between vector and packaging encoding sequences (vector/helper [V/H] recombinant) in the producer clone used for transduction of bone marrow stem cells. Southern blot analysis demonstrated 14 or 25 copies of this genome per cell where present in two animals. The genome of a second replication-competent virus was also recovered by molecular cloning; it arose by recombination involving the genome of the V/H recombinant and endogenous murine retroviral genomes in the producer clone. Twelve copies of this amphotropic virus/mink cell focus-forming virus genome were present in tumor DNA of one animal, but it was not found in tumor DNA of the other two animals with lymphoma. Southern blot analysis of DNA from various tissues demonstrated common insertion site bands in several samples of tumor DNA from one animal, suggesting clonal origin of the lymphoma. Our data are most consistent with a pathogenic mechanism in which chronic productive retroviral infection allowed insertional mutagenesis of critical growth control genes, leading to cell transformation and clonal tumor evolution.     

Polydnavirus genome segment families in the ichneumonid parasitoid Hyposoter fugitivus.

Sequences homologous to encapsidated polydnavirus genome segments are routinely detected in parasitoid chromosomal DNA; typically, each viral genome segment hybridizes to a single cognate chromosomal locus. In the present study, we show that in some cases, two or more viral genome segments may hybridize to the same chromosomal locus. Genome segments of this type invariably share a majority of restriction enzyme sites, a fact suggesting derivation from a common template. Families of viral genome segments appear to be relatively common in the Hyposoter fugitivus polydnavirus genome.     

Analysis of new retrogenes provides insight into dog adaptive evolution

The origin and subsequent evolution of new genes have been considered as an important source of genetic and phenotypic diversity in organisms. Dog breeds show great phenotypic diversity for morphological, physiological, and behavioral traits. However, the contributions of newly originated retrogenes, which provide important genetic bases for dog species differentiation and adaptive traits, are largely unknown. Here, we analyzed the dog genome to identify new RNA‐based duplications and comprehensively investigated their origin, evolution, functions in adaptive traits, and gene movement processes. First, we totally identified 3,025 retrocopies including 476 intact retrogenes, 2,518 retropseudogenes, and 31 chimerical retrogenes. Second, selective pressure along with ESTs expression analysis showed that most of the intact retrogenes were significantly under stronger purifying selection and subjected to more functional constraints when compared to retropseudogenes. Furthermore, a large number of retrocopies and chimerical retrogenes that occurred approximately 22 million years ago implied a burst of retrotransposition in the dog genome after the divergence time between dog and its closely related species red fox. Interestingly, GO and pathway analyses showed that new retrogenes had expanded in glutathione biosynthetic/metabolic process which likely provided important genetic basis for dogs' adaptation to scavenge human waste dumps. Finally, consistent with the results in human and mouse, a significant excess of functional retrogenes movement on and off the X chromosome in the dog confirmed a general pattern of gene movement process in mammals which was likely driven by natural selection or sexual antagonism. Together, these results increase our understanding that new retrogenes can reshape the dog genome and provide further exploration of the molecular mechanisms underlying the dogs' adaptive evolution.     

Requirement for double-strand breaks but not for specific DNA sequences in herpes simplex virus type 1 genome isomerization events.

Herpes simplex virus type 1 (HSV-1) genome isomerization occurs as a result of DNA replication-mediated homologous recombination between several sets of inverted repeat sequences present in the viral DNA. The frequency with which this recombination occurs has been demonstrated to be dependent upon DNA homology length rather than specific sequences. However, the smallest of the viral inverted repeats, the alpha sequence, has been shown to function as a recombinational hot spot, leading to speculation that this sequence may represent a specific element through which genome isomerization is mediated. To investigate this apparent paradox, a quantitative transient recombination assay system was developed and used to examine the recombinogenic properties of a panel of alpha sequence mutants. This analysis revealed that the presence of both the pac1 and pac2 elements was both necessary and sufficient for the induction of high-frequency recombination events by the alpha sequence. However, it was the double-strand break promoted by pac1 and pac2 during cleavage and packaging at the alpha sequence, and not the DNA sequences of the elements themselves, which appeared to be critical for recombination. This was illustrated (i) by the inability of the same pac1 and pac2 sequences to mediate inversion events in cells infected with an HSV-1 mutant which was competent for DNA replication-dependent recombination but defective for the cleavage and packaging process and (ii) by the ability of double-strand breaks generated in non-HSV-1 DNA by an in vivo-expressed restriction endonuclease to significantly stimulate the initiation of recombination events in virus-infected cells. Thus, the alpha sequence appears to act as a hot spot for homologous recombination simply because it happens to coincide with the site of the double-strand break which is generated during the cleavage and packaging process, not because it contains discrete sequences which are required for this activity. However, it was found that this enhanced recombinogenicity disappeared when the element was flanked by regions of extensive sequence homology, particularly that of the large inverted repeats which flank the alpha sequence at its natural site in the HSV-1 genome. These findings are consistent with a model for HSV-1 genome isomerization in which recombination is initiated primarily by multiple random double-strand breaks which arise during DNA replication across the inverted repeats of the genome, rather than by a single specific break which occurs at the alpha sequence during the cleavage and packaging process.     

Human endogenous retroviral elements as indicators of ectopic recombination events in the primate genome

HERV elements make up a significant fraction of the human genome and, as interspersed repetitive elements, have the capacity to provide substrates for ectopic recombination and gene conversion events. To understand the extent to which these events occur and gain further insight into the complex evolutionary history of these elements in our genome, we undertook a phylogenetic study of the long terminal repeat sequences of 15 HERV-K(HML-2) elements in various primate species. This family of human endogenous retroviruses first entered the primate genome between 35 and 45 million years ago. Throughout primate evolution, these elements have undergone bursts of amplification. From this analysis, which is the largest-scale study of HERV sequence dynamics during primate evolution to date, we were able to detect intraelement gene conversion and recombination at five HERV-K loci. We also found evidence for replacement of an ancient element by another HERV-K provirus, apparently reflecting an occurrence of retroviral integration by homologous recombination. The high frequency of these events casts doubt on the accuracy of integration time estimates based only on divergence between retroelement LTRs.     

Repair of a specific double-strand break generated within a mammalian chromosome by yeast endonuclease I-SceI.

We established a mouse Ltk- cell line that contains within its genome a herpes simplex virus thymidine kinase gene (tk) that had been disrupted by the insertion of the recognition sequence for yeast endonuclease I-SceI. The artificially introduced 18 bp I-SceI recognition sequence was likely a unique sequence in the genome of the mouse cell line. To assess whether an induced double-strand break (DSB) in the genomic tk gene would be repaired preferentially by gene targeting or non-homologous recombination, we electroporated the mouse cell line with endonuclease I-SceI alone, one of two different gene targeting constructs alone, or with I-SceI in conjunction with each of the two targeting constructs. Each targeting construct was, in principle, capable of correcting the defective genomic tk sequence via homologous recombination. tk+ colonies were recovered following electroporation of cells with I-SceI in the presence or absence of a targeting construct. Through the detection of small deletions at the I-SceI recognition sequence in the mouse genome, we present evidence that a specific DSB can be introduced into the genome of a living mammalian cell by yeast endonuclease I-SceI. We further report that a DSB in the genome of a mouse Ltk- cell is repaired preferentially by non-homologous end-joining rather than by targeted homologous recombination with an exogenous donor sequence. The potential utility of this system is discussed.