scholarly journals Effect of the Murine Leukemia Virus Extended Packaging Signal on the Rates and Locations of Retroviral Recombination

2000 ◽  
Vol 74 (15) ◽  
pp. 6953-6963 ◽  
Author(s):  
Jeffrey A. Anderson ◽  
Vinay K. Pathak ◽  
Wei-Shau Hu
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Template Switching ◽  
Packaging Signal ◽  
Lower Efficiency ◽  
Selectable Markers ◽  
Negative Interference ◽  
Dimer Linkage ◽  
Retroviral Recombination ◽  
Murine Leukemia

ABSTRACT Reverse transcriptase (RT) switches templates frequently during DNA synthesis; the acceptor template can be the same RNA (intramolecular) or the copackaged RNA (intermolecular). Previous results indicated that intramolecular template switching occurred far more frequently than intermolecular template switching. We hypothesized that intermolecular template-switching events (recombination) occurred at a lower efficiency because the copackaged RNA was not accessible to the RT. To test our hypothesis, the murine leukemia virus (MLV) extended packaging signal (Ψ+) containing a dimer linkage structure (DLS) was relocated from the 5′ untranslated region (UTR) to between selectable markers, allowing the two viral RNAs to interact closely in this region. It was found that the overall maximum recombination rates of vectors with Ψ+ in the 5′ UTR or Ψ+ between selectable markers were not drastically different. However, vectors with Ψ+ located between selectable markers reached a plateau of recombination rate at a shorter distance. This suggested a limited enhancement of recombination by Ψ+. The locations of the recombination events were also examined by using restriction enzyme markers. Recombination occurred in all four regions between the selectable markers; the region containing 5′ Ψ+ including DLS did not undergo more recombination than expected from the size of the region. These experiments indicated that although the accessibility of the copackaged RNA was important in recombination, other factors existed to limit the number of viruses that were capable of undergoing intermolecular template switching. In addition, recombinants with multiple template switches were observed at a frequency much higher than expected, indicating the presence of high negative interference in the MLV-based system. This extends our observation with the spleen necrosis virus system and suggests that high negative interference may be a common phenomenon in retroviral recombination.

scholarly journals mRNA Molecules Containing Murine Leukemia Virus Packaging Signals Are Encapsidated as Dimers

2004 ◽  
Vol 78 (20) ◽  
pp. 10927-10938 ◽  
Author(s):  
Catherine S. Hibbert ◽  
Jane Mirro ◽  
Alan Rein
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Genomic Rna ◽  
Packaging Signal ◽  
Electron Microscopic ◽  
Genomic Rnas ◽  
Microscopic Studies ◽  
The Stability ◽  
Murine Leukemia

ABSTRACT Prior work by others has shown that insertion of ψ (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 ψ or with ψ sequences from MLV-related acute transforming viruses. The results indicate that the dimeric linkage in genomic RNA can be completely attributed to the ψ 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 Ψ+ 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.

scholarly journals The Nucleocapsid Domain Is Responsible for the Ability of Spleen Necrosis Virus (SNV) Gag Polyprotein To Package both SNV and Murine Leukemia Virus RNA

1999 ◽  
Vol 73 (11) ◽  
pp. 9170-9177 ◽  
Author(s):  
Jeanine L. Certo ◽  
Timur O. Kabdulov ◽  
Michelle L. Paulson ◽  
Jeffrey A. Anderson ◽  
Wei-Shau Hu
Keyword(s):  
Murine Leukemia Virus ◽  
Viral Vector ◽  
Leukemia Virus ◽  
Gene Products ◽  
Rna Packaging ◽  
Packaging Signal ◽  
Necrosis Virus ◽  
Spleen Necrosis Virus ◽  
Spleen Necrosis ◽  
Murine Leukemia

ABSTRACT 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 chimericgag-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 (Ψ) and another viral vector RNA containing the SNV packaging signal (E). The chimericgag-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 Ψ. 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-MLVpol 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 SNVcis-acting elements are not ideal substrates for MLVpol 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 othercis-acting elements.

scholarly journals Structure-Based Moloney Murine Leukemia Virus Reverse Transcriptase Mutants with Altered Intracellular Direct-Repeat Deletion Frequencies

2000 ◽  
Vol 74 (20) ◽  
pp. 9629-9636 ◽  
Author(s):  
Julie K. Pfeiffer ◽  
Millie M. Georgiadis ◽  
Alice Telesnitsky
Keyword(s):  
Reverse Transcriptase ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Direct Repeat ◽  
Moloney Murine Leukemia Virus ◽  
Wild Type Virus ◽  
Template Switching ◽  
Wild Type ◽  
Catalytic Core ◽  
Murine Leukemia

ABSTRACT Template switching rates of Moloney murine leukemia virus reverse transcriptase mutants were tested using a retroviral vector-based direct-repeat deletion assay. The reverse transcriptase mutants contained alterations in residues that modeling of substrates into the catalytic core had suggested might affect interactions with primer and/or template strands. As assessed by the frequency of functionallacZ gene generation from vectors in which lacZwas disrupted by insertion of a sequence duplication, the frequency of template switching varied more than threefold among fully replication-competent mutants. Some mutants displayed deletion rates that were lower and others displayed rates that were higher than that of wild-type virus. Replication for the mutants with the most significant alterations in template switching frequencies was similar to that of the wild type. These data suggest that reverse transcriptase template switching rates can be altered significantly without destroying normal replication functions.

scholarly journals Recombination in the 5′ Leader of Murine Leukemia Virus Is Accurate and Influenced by Sequence Identity with a Strong Bias toward the Kissing-Loop Dimerization Region

1998 ◽  
Vol 72 (9) ◽  
pp. 6967-6978 ◽  
Author(s):  
Jacob Giehm Mikkelsen ◽  
Anders H. Lund ◽  
Mogens Duch ◽  
Finn Skou Pedersen
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Marker Gene ◽  
Template Switching ◽  
Target Sequence ◽  
Sequence Identity ◽  
Crossover Site ◽  
Palindromic Sequences ◽  
Murine Leukemia ◽  
Kissing Loop

ABSTRACT Retroviral recombination occurs frequently during reverse transcription of the dimeric RNA genome. By a forced recombination approach based on the transduction of Akv murine leukemia virus vectors harboring a primer binding site knockout mutation and the entire 5′ untranslated region, we studied recombination between two closely related naturally occurring retroviral sequences. On the basis of 24 independent template switching events within a 481-nucleotide target sequence containing multiple sequence identity windows, we found that shifting from vector RNA to an endogenous retroviral RNA template during minus-strand DNA synthesis occurred within defined areas of the genome and did not lead to misincorporations at the crossover site. The nonrandom distribution of recombination sites did not reflect a bias for specific sites due to selection at the level of marker gene expression. We address whether template switching is affected by the length of sequence identity, by palindromic sequences, and/or by putative stem-loop structures. Sixteen of 24 sites of recombination colocalized with the kissing-loop dimerization region, and we propose that RNA-RNA interactions between palindromic sequences facilitate template switching. We discuss the putative role of the dimerization domain in the overall structure of the reverse-transcribed RNA dimer and note that related mechanisms of template switching may be found in remote RNA viruses.

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