Identification of alternatively spliced mRNAs encoding potential new regulatory proteins in cattle infected with bovine leukemia virus

Alexandersen, Søren; Carpenter, Susan; Christensen, Jesper; Storgaard, Torben; Viuff, Birgitte; Wannemuehler, Yvonne; Belousov, J; Roth, James A.

doi:10.1128/jvi.67.1.39-52.1993

Cited by 52 publications

(26 citation statements)

References 109 publications

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“…BLV shares many properties of genomic structure, viral replication, and pathology with HTLV. The R3 and G4 proteins share some homologies with regulator proteins of HTLV (p12I, pl3", and p30" or Rof and Tof) and human immunodeficiency virus (Vpr) (3). This suggests that all these proteins belong to a common family of complex retrovirus regulatory proteins.…”

Section: Discussionmentioning

confidence: 98%

“…Our data show that the G4 ORF is important in vivo although its product is dispensable to establish an infection. Since G4 mRNAs are detected preferentially at the beginning of lymphocytosis (3), the behavior of pBLVIG4 will be particularly interesting to analyze for pathogenesis. In contrast to cattle, all BLV-infected sheep die as a consequence of leukemia.…”

Section: Discussionmentioning

confidence: 99%

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Attenuation of bovine leukemia virus by deletion of R3 and G4 open reading frames.

Willems¹,

Kerkhofs²,

Dequiedt³

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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Complex oncoviruses contain, in addition to the classical retroviral genes (gag, pol, and env), a region (X) located between the envelope sequences and the 3' long terminal repeat. The X region contains two genes, tax and rex, whose protein products are involved in transcriptional and posttranscriptional regulation of viral expression. In addition to these activators, the bovine leukemia virus (BLV) and the human T-cell leukemia virus (HTLV) contain alternative open reading frames (R3 and G4 for BLV; p30, p13, and p12 for HTLV). As a virus/animal model for HTLV-induced leukemogenesis, BLV provirus can be injected intradermally into sheep, where it induced B-lymphocyte transformation. Deletion of the R3 and G4 sequences from an infectious and tumorigenic BLV provirus greatly impaired the in vivo propagation of the viruses as demonstrated by DNA polymerase chain reaction, RNA blots, structural-protein ELISA, and immunofluorescence analysis. Our results show that the alternative open reading frames are required for maintaining high virus loads during the course of persistent infection in vivo. Thus, R3 and G4 are candidates for antiviral drug development. Furthermore, viruses with a deletion in these sequences should be tested as live attenuated vaccines.

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Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Attenuation of bovine leukemia virus by deletion of R3 and G4 open reading frames.

Willems¹,

Kerkhofs²,

Dequiedt³

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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“…Expression of the BLV genome is repressed in vivo, and even sensitive methods, such as reverse transcription-PCR (RT-PCR) and in situ hybridization, have detected only limited viral RNA expression (2,26,30,37,42,63). However, the virus can be reactivated ex vivo, by growing untransformed BLVinfected lymphocytes from aleukemic sheep (31,39,42,50).…”

mentioning

confidence: 99%

In Vivo Rescue of a Silent tax -Deficient Bovine Leukemia Virus from a Tumor-Derived Ovine B-Cell Line by Recombination with a Retrovirally Transduced Wild-Type tax Gene

Broeke

Bagnis

Ciesiolka

et al. 1999

J Virol

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The lack of bovine leukemia virus (BLV) expression is a consistent finding in freshly isolated ovine tumor cells and in the B-cell lines derived from these tumors. In order to gain further insight into the mechanisms of BLV silencing in these tumors, we have used the YR2 B-cell line, which was derived from the leukemic cells of a BLV-infected sheep. This cell line contains a single, monoclonally integrated, silent provirus, which cannot be reactivated either by stimulation in vitro or by in vivo injection of the tumor cells or cloned proviral DNA in sheep. Sequence analysis of the tax gene from the YR2 cell line identified two G-to-A transitions (G 7924 to A 7924 and G 8149 to A 8149 ) that result in E-to-K amino acid changes at positions 228 and 303 in the Tax protein.Following retroviral vector-mediated transfer of a wild-type tax gene into YR2 cells, we showed that BLV mRNA, viral proteins, and virions were produced, demonstrating that the cellular factors required for virus expression were present in the original YR2 cell line. Injection of this transduced YR2 cell line in sheep led to the rescue of replication-competent BLV proviruses. The integrated competent proviruses exhibited unique chimeric tax genes, which arose from homologous recombination between the transduced wild-type tax and the YR2-derived tax sequences. Furthermore, in one of these functional recombinant proviruses, only the A 8149 -to-G 8149 reversion was present, providing clear evidence that the defect underlying the silent phenotype in YR2 cells results from a single C-terminal E 303 -to-K 303 amino acid substitution in the BLV Tax protein. Our observations suggest that a single strategically located mutation in tax provides a mechanism for BLV inactivation in B-cell tumors.

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“…The Tax proteins have an immortalization potential in cell culture and collaborate with the Ha-ras oncogene to transform primary cells (16,30,43,50,51,55). At least two other mRNAs encoding putative proteins are transcribed from alternative open reading frames located in the region between the env and the tax/rex genes (R3 and G4 for BLV; p30, p13, and p12 for HTLV) (1,2,5,20). HTLV-1 p12 has been shown to cooperate with bovine papillomavirus E5 in cell transformation and to bind to the ␤ and ␥ chains of the interleukin-2 receptor (13,26).…”

mentioning

confidence: 99%

Both wild-type and strongly attenuated bovine leukemia viruses protect peripheral blood mononuclear cells from apoptosis

Dequiedt

Hanon

Kerkhofs

et al. 1997

J Virol

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Bovine leukemia virus (BLV) and the human T-cell leukemia viruses belong to the same subfamily of oncoviruses. Although much attention has focused on the mechanisms of cell proliferation and transformation by these viruses, experiments on the apoptotic process have yielded conflicting data in in vitro cell culture. Experimental infection of sheep with BLV proviruses offers the opportunity to analyze apoptosis in vivo.Here, we show that BLV-infected peripheral mononuclear cells, cultivated ex vivo, are protected from spontaneous programmed cell death. Moreover, the virus is able to specifically interfere with the apoptotic program of infected B lymphocytes. Strongly attenuated mutant proviruses that harbor deletions in the G4 and/or R3 genes also decrease the global susceptibility to apoptosis at levels similar to those obtained with the wild-type virus. In addition, cell culture supernatants from wild-type and mutant viruses can prevent uninfected cells from undergoing programmed cell death. These observations demonstrate that the R3 and G4 genes are not required to maintain both direct and indirect protection against apoptosis. They also imply that the level of programmed cell death observed ex vivo is independent of the amounts of proviruses in the animals. The failure of these cells to undergo apoptosis might be related to the pathogenesis induced by BLV.

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Identification of alternatively spliced mRNAs encoding potential new regulatory proteins in cattle infected with bovine leukemia virus

Cited by 52 publications

References 109 publications

Attenuation of bovine leukemia virus by deletion of R3 and G4 open reading frames.

Attenuation of bovine leukemia virus by deletion of R3 and G4 open reading frames.

In Vivo Rescue of a Silent tax -Deficient Bovine Leukemia Virus from a Tumor-Derived Ovine B-Cell Line by Recombination with a Retrovirally Transduced Wild-Type tax Gene

Both wild-type and strongly attenuated bovine leukemia viruses protect peripheral blood mononuclear cells from apoptosis

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