Defective retrovirus-like 30S RNA species of rat and mouse cells are infectious if packaged by type C helper virus

248

149

To study the function of the retroviral nucleocapsid protein (NC), we have constructed point mutations in the gag gene of Moloney murine leukemia virus (MuLV) that affect a conserved cysteine-histidine motif of NC. The mutants were characterized biologically and biochemically. Cell lines producing the mutant virions were constructed in NIH 3T3 and rat2 cells, and the viral particles released by these cells were characterized for protein and RNA content. The results indicated that most mutations block replication and specifically inhibit the packaging of the MuLV genomic RNA. In some of the mutants, the packaging of the endogenous rat VL30 RNA was not affected as profoundly as was MuLV RNA. NC also seems to have another function distinct from dimer formation and packaging: one mutation reduced viral RNA packaging by only fivefold but completely abolished viral cDNA synthesis, suggesting a defect in reverse transcription.

Section: Most Of the Mutations That We Have Constructed In Thementioning

confidence: 99%

Characterization of Moloney murine leukemia virus mutants with single-amino-acid substitutions in the Cys-His box of the nucleocapsid protein

Meric

Goff

1989

248

149

“…In two of these studies, a third RNA component, slightly smaller than 32S, was reported and considered an additional genomic RNA of SFFV (36) or of an endogenous virus (15). It has not been shown that the presence of 32S RNA in SFFV(Fr-MLV) is necessary to induce spleen foci in mice, nor was it ruled out that the 32S RNA associated with SFFV(Fr-MLV) was a viral contaminant such as the 30S defective retrovirus-like RNAs present in some stocks of MLVs (17,26,44,45). To test directly whether the 32S RNA associated with SFFV(Fr-MLV) is correlated with spleen focus formation, we have examined the spleen focus-forming activity and the RNA components of viruses released from FRE cells and FRE cells nonproductively infected with SFFV before and after superinfection with Fr-MLV.…”

Section: Resultsmentioning

confidence: 99%

“…This could arise by deletion of a recombinant virus carrying Fr-MLV and amphotropic MLV sequences or by unequal crossing-over between two viruses with such sequences, resulting in specific deletions. Alternatively, SFFV could be the product of recombination between Fr-MLV and amphotropic MLV and an unknown defective prototype of SFFV possibly analogous to the 30S defective retrovirus-like RNA found in mouse and rat cells (17,26,44,45). This hypothesis would explain the defectiveness of SFFV because one of the parents of SFFV would itself be defective.…”

Section: Discussionmentioning

confidence: 99%

Spleen focus-forming Friend virus: identification of genomic RNA and its relationship to helper virus RNA

Evans¹,

Duesberg²,

Troxler³

et al. 1979

Self Cite

The genome of the defective, murine spleen focus-forming Friend virus (SFFV) was identified as a 50S RNA complex consisting of 32S RNA monomers. Electrophoretic mobility and the molecular weights of unique RNase T1-resistant oligonucleotides (T1-oligonucleotides) indicated that the 32S RNA had a complexity of about 7.4 kilobases. Hybridization with DNA complementary to Friend murine leukemia virus (Fr-MLV) has distinguished two sets of nucleotide sequences in 32S SFFV RNA, 74% which were Fr-MLV related and 26% which were SFFV specific. By the same method, SFFV RNA was 48% related to Moloney MLV. We have resolved 23 large Ti-oligonucleotides of SFFV RNA and 43 of Fr-MLV RNA. On the basis of the relationship between SFFV and Fr-MLV RNAs, the 23 SFFV oligonucleotides fell into four classes: (i) seven which had homologous equivalents in Fr-MLV RNA; (ii) six more which could be isolated from SFFV RNA-Fr-MLV cDNA hybrids treated with RNases A and TI; (iii) eight more which were isolated from hybrids treated with RNase TI; and (iv) two which did not have Fr-MLV-related counterparts. Surprisingly, the two class iv oligonucleotides had homologous counterparts in the RNA of six amphotropic MLVs including mink cell focus-forming and HIX-MLVs analyzed previously. The map locations of the 23 SFFV T1-oligonucleotides relative to the 3' polyadenylic acid

“…A characteristic feature of VL30 retrotransposons is that they can be packaged into MLV virions, leading to their horizontal spread during retrovirus dissemination (17,19,33,34,67,78,79). Based on this observation, we asked whether the 5Ј region of VL30m RNA could replace the MLV Eϩ sequence functionally in a recombinant viral RNA.…”

Section: Discussionmentioning

confidence: 99%

“…VL30m elements do not encode virus-like proteins, have little sequence homology to Moloney murine leukemia virus (MoMLV) and have not been implicated in retroviral carcinogenesis (16,27). However, VL30 RNA possesses the unusual property of being packaged into MLV particles that are propagated in cell culture (17,19,33,34,67,78,79). As a consequence, VL30 elements are able to retrotranspose from cell to cell at high frequency via MLV particles (17,27).…”

mentioning

confidence: 99%

Identification of an Internal Ribosome Entry Segment in the 5′ Region of the Mouse VL30 Retrotransposon and Its Use in the Development of Retroviral Vectors

López-Lastra

Ulrici

Gabus

et al. 1999

Mouse virus-like 30S RNAs (VL30m) constitute a family of retrotransposons, present at 100 to 200 copies, dispersed in the mouse genome. They display little sequence homology to Moloney murine leukemia virus (MoMLV), do not encode virus-like proteins, and have not been implicated in retroviral carcinogenesis. However, VL30 RNAs are efficiently packaged into MLV particles that are propagated in cell culture. In this study, we addressed whether the 5′ region of VL30m could replace the 5′ leader of MoMLV functionally in a recombinant vector construct. Our data confirm that the putative packaging sequence of VL30 is located within the 5′ region (nucleotides 362 to 1149 with respect to the cap structure) and that it can replace the packaging sequence of MoMLV. We also show that VL30m contains an internal ribosome entry segment (IRES) in the 5′ region, as do MoMLV, Friend murine leukemia virus, Harvey murine sarcoma virus, and avian reticuloendotheliosis virus type A. Our data show that both the packaging and IRES functions of the 5′ region of VL30m RNA can be efficiently used to develop retrotransposon-based vectors.