Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-07
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • The mammalian two hybrid results described above were all

    2020-05-28

    The mammalian two-hybrid results described above were all obtained in 293T cells, and thus the higher RLU observed specific to the hCrm1 fusion could be consistent with human Oleandrin synthesis having an unknown activator that interacts with hCrm1 HEAT repeat 9A and somehow facilitates Crm1-Rev/RRE function. Although this scenario is unlikely based upon the biochemical results of Fig. 1, the mammalian two hybrid experiments were repeated in murine B78 cells using the same Gal4DBD and VP16AD fusion constructs. To achieve adequate transfection efficiency and consequent protein expression levels, B78 cells were transfected using Amaxa nucleofector system with various fusion constructs along with HIV vector FG12, which encodes both an RRE and an eGFP reporter (Qin et al., 2003). Transfection efficiency was ~ 10%, as judged by epifluorescence microcopy of eGFP expressed from FG12 vector. Cells were allowed to recover and proliferate for 4 d and then lysed for FFLUC RLU. B78 cells mock- or transfected with only Crm1 or Rev fusion constructs exhibited low RLU, whereas there was a significant increase in RLU in the presence of both the mCrm1 and Rev fusions (Fig. 7). There was a significantly higher amount of RLU in the presence of both the hCrm1 and Rev fusions compared to each individually or in the presence of mCrm1 and Rev fusions, whereas the highest levels of RLU were observed for the positive control interaction (p53 and SV40 Tag), as expected. Similar results were observed when B78 cells were transfected with cationic lipids (not shown). These results suggest that the more favorable genetic interaction observed between hCrm1 and Rev compared to mCrm1 and Rev is cell-type independent, since it is observed in both human and murine cells.
    Discussion We and others have shown that a major defect to infectious HIV production in murine cells is due to a block at nuclear export of unspliced and partially spliced viral mRNAs (Coskun et al., 2006; Sherer et al., 2011; Swanson et al., 2010). Not only does this result in reduced viral structural protein expression but also decreased levels of cytoplasmic genomic viral mRNA for packaging. This defect was corrected in part by provision of human chromosome 2 (Coskun et al., 2007; Coskun et al., 2006), and more recently it has been demonstrated that at least one of the responsible gene products is Crm1 (Elinav et al., 2012; Nagai-Fukataki et al., 2011; Okada et al., 2009; Sherer et al., 2011), which was known to associate with HIV Rev/RRE complex and facilitate nuclear export of intron-containing viral mRNAs. Murine (m) Crm1, which is precisely the same length at 1071 aa as hCrm1, appears to be a loss-of-function gene, and the important functional differences for HIV infectivity mapped to a cluster or patch of amino acid residues in HEAT repeat 9A, which is distinct and physically distant from the known NES binding domain of Crm1 (Elinav et al., 2012; Sherer et al., 2011). Previously, in vitro experiments had shown that in the presence of HIV Rev two regions of Crm1 were protected against endoprotease treatment—a region encompassing amino acids 800–820 and also an Aspartate at 716, although the functional importance of these regions of Crm1 were not tested (Askjaer et al., 1998). Rev itself has a single canonical NLS and a leucine rich NES, the latter of which termed the activation domain and is thought to bind to Crm1, but no other Rev sequences or domains have been implicated in Rev binding to Crm1. Rev with two NLS sequences does not have enhanced function in murine cells, whereas Rev with two separated NES sequences (Rev ×2NES) appears to restore function in terms of CA production and infectious virus release in murine cells (Aligeti et al., 2014), such that addition of hCrm1 is unnecessary, which we have confirmed here. This finding is consistent with a fundamental defect in mouse cells being the inability of viral intron-containing mRNAs to be exported from the nucleus. However, the result with Rev ×2NES does not directly address the question as to why mechanistically mCrm1 is less active than hCrm1.