scholarly journals Nonreciprocal Pseudotyping: Murine Leukemia Virus Proteins Cannot Efficiently Package Spleen Necrosis Virus-Based Vector RNA

1998 ◽  
Vol 72 (7) ◽  
pp. 5408-5413 ◽  
Author(s):  
Jeanine L. Certo ◽  
Betsy F. Shook ◽  
Philip D. Yin ◽  
John T. Snider ◽  
Wei-Shau Hu
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Rna Structures ◽  
Packaging Signal ◽  
Necrosis Virus ◽  
Hairpin Rna ◽  
Spleen Necrosis Virus ◽  
Retroviral Replication ◽  
Spleen Necrosis ◽  
Murine Leukemia

ABSTRACT 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 (Ψ) 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 Ψ, but MLV can recognize only MLV Ψ. This is the first demonstration of two retroviruses with nonreciprocal packaging specificities.

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 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 Isolation of Cell Lines That Show Novel, Murine Leukemia Virus-Specific Blocks to Early Steps of Retroviral Replication

2005 ◽  
Vol 79 (20) ◽  
pp. 12969-12978 ◽  
Author(s):  
James W. Bruce ◽  
Kenneth A. Bradley ◽  
Paul Ahlquist ◽  
John A. T. Young
Keyword(s):  
Cell Lines ◽  
Reverse Transcription ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
High Volume ◽  
Early Stages ◽  
Retroviral Replication ◽  
Murine Leukemia

ABSTRACT In order to identify cellular proteins required for early stages of retroviral replication, a high volume screening with mammalian somatic cells was performed. Ten pools of chemically mutagenized Chinese hamster ovary (CHO-K1) cells were challenged with a murine leukemia virus (MLV) vector pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G), and cells that failed to be transduced were enriched by cell sorting. Each pool yielded a clonally derived cell line with a 5-fold or greater resistance to virus infection, and five cell lines exhibited a >50-fold resistance. These five cell lines were efficiently infected by a human immunodeficiency virus vector pseudotyped with VSV-G. When engineered to express the TVA receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A), the five cell lines were resistant to infection with a MLV vector pseudotyped with the ASLV-A envelope protein but were fully susceptible to infection with an ASLV-A vector. Thus, the defect in these cells resides after virus-cell membrane fusion and, unlike those in other mutant cell lines that have been described, is specific for the MLV core. To identify the specific stages of MLV infection that are impaired in the resistant cell lines, real-time quantitative PCR analyses were employed and two phenotypic groups were identified. Viral infection of three cell lines was restricted before reverse transcription; in the other two cell lines, it was blocked after reverse transcription, nuclear localization, and two-long terminal repeat circle formation but before integration. These data provide genetic evidence that at least two distinct intracellular gene products are required specifically for MLV infection. These cell lines are important tools for the biochemical and genetic analysis of early stages in retrovirus infection.

scholarly journals Spliced Spleen Necrosis Virus Vector RNA Is Not Encapsidated: Implications for Retroviral Replication and Vector Design

2004 ◽  
Vol 9 (4) ◽  
pp. 557-565 ◽  
Author(s):  
Adrienne Goodrich ◽  
Zahida Parveen ◽  
Ralph Dornburg ◽  
Matthias J Schnell ◽  
Roger J Pomerantz
Keyword(s):  
Virus Vector ◽  
Necrosis Virus ◽  
Spleen Necrosis Virus ◽  
Retroviral Replication ◽  
Spleen Necrosis

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 Cooperative Effect of Gag Proteins p12 and Capsid during Early Events of Murine Leukemia Virus Replication

2005 ◽  
Vol 79 (7) ◽  
pp. 4159-4169 ◽  
Author(s):  
Sook-Kyung Lee ◽  
Kunio Nagashima ◽  
Wei-Shau Hu
Keyword(s):  
Virus Replication ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Virus Production ◽  
Cooperative Effect ◽  
Gag Proteins ◽  
Rous Sarcoma ◽  
Reverse Transcription Step ◽  
Spleen Necrosis ◽  
Murine Leukemia

ABSTRACT The Gag polyprotein of murine leukemia virus (MLV) is processed into matrix (MA), p12, capsid (CA), and nucleocapsid (NC) proteins. p12 affects early events of virus replication and contains a PPPY motif important for virus release. To probe the functions of p12 in the early steps of MLV replication, we tested whether p12 can be replaced by spleen necrosis virus (SNV) p18, human immunodeficiency virus type 1 p6, or Rous sarcoma virus p2b. Analyses revealed that all chimeras generated virions at levels similar to that of MLV gag-pol; however, none of them could support MLV vector replication, and all of them exhibited severely reduced DNA synthesis upon virus infection. Because a previously reported SNV gag-MLV pol chimera, but not the MLV hybrid with SNV p18, can support replication of an MLV vector, we hypothesized that other Gag proteins act cooperatively with p12 during the early phase of virus replication. To test this hypothesis, we generated three more MLV-based chimeras containing SNV CA, p18-CA, or p18-CA-NC. We found that the MLV chimera containing SNV p18-CA or p18-CA-NC could support MLV vector replication, but the chimera containing SNV CA could not. Furthermore, viruses derived from the MLV chimera with SNV CA could synthesize viral DNA upon infection but were blocked at a post-reverse-transcription step and generated very little two long terminal repeat circle DNA, thereby producing a phenotype similar to that of the provirus formation-defective p12 mutants. Taken together, our data indicate that when p12/p18 or CA was from different viruses, despite abundant virus production and proper Gag processing, the resulting viruses were not infectious. However, when p12/p18 and CA were from the same virus, even though they were from SNV and not MLV, the resulting viruses were infectious. Therefore, these results suggest a cooperative effect of p12 and CA during the early events of MLV replication.

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