SIVmac239 Vpx Nuclear Import
Location
CSU Ballroom
Start Date
12-4-2022 2:00 PM
End Date
12-4-2022 3:30 PM
Student's Major
Biological Sciences
Student's College
Science, Engineering and Technology
Mentor's Name
Allison Land
Mentor's Department
Biological Sciences
Mentor's College
Science, Engineering and Technology
Description
Vpx is a Human Immunodeficiency Virus type 2 (HIV-2)/Simian Immunodeficiency Virus (SIV) protein whose activity permits successful viral infection of non-dividing cells. The two principal functions of Vpx are mediating nuclear import of the HIV-2/SIV reverse transcribed genome and antagonizing SAMHD1, a host-cell innate immune protein primarily localized in the nucleus. Previous studies have identified multiple mechanisms of Vpx nuclear translocation, including Importin-α/β dependent and independent pathways that rely on multiple non-canonical nuclear localization signals (ncNLS) and phosphorylation by MAPK/ERK-2. Although predominantly found in the nucleus, concurrent or exclusive Vpx localization in the cytoplasm has been observed in specific cell types and a subset of HIV-2/SIV strains. We observe that HIV-2 ROD9 Vpx demonstrates significantly higher nuclear localization in HeLa cells compared to Vpx proteins from HIV-2 7312a and SIVmac239. This discrepancy is likely due to key amino acid differences that impact nuclear import and/or export pathways exploited by Vpx. To investigate amino acids relevant for nuclear import, site-directed mutagenesis was executed on SIVmac239 Vpx to increase its homology to HIV-2 ROD9 Vpx. HeLa cells were transfected with various Cherry-tagged SIVmac239 Vpx constructs followed by treatment with leptomycin B (inhibits nuclear export). Representative cells were then imaged with confocal microscopy and used to quantify localization patterns displayed by each Vpx construct. Interestingly, our data from the SIVmac239 V67T Vpx construct suggests that other residues outside the C-terminal 65-SYTKYRYL-72 minimal ncNLS must contribute to efficient Vpx nuclear uptake. Collectively, these various constructs help narrow down the particularly relevant nuclear localization mechanisms for this virus in HeLa cells and serve as a platform for additional study in other biologically relevant cell types, such as macrophages. By providing greater insight into viral nuclear import, these data may galvanize the development of effective HIV-2/SIV therapeutics that target commonly exploited host-cell nuclear entry pathways.
SIVmac239 Vpx Nuclear Import
CSU Ballroom
Vpx is a Human Immunodeficiency Virus type 2 (HIV-2)/Simian Immunodeficiency Virus (SIV) protein whose activity permits successful viral infection of non-dividing cells. The two principal functions of Vpx are mediating nuclear import of the HIV-2/SIV reverse transcribed genome and antagonizing SAMHD1, a host-cell innate immune protein primarily localized in the nucleus. Previous studies have identified multiple mechanisms of Vpx nuclear translocation, including Importin-α/β dependent and independent pathways that rely on multiple non-canonical nuclear localization signals (ncNLS) and phosphorylation by MAPK/ERK-2. Although predominantly found in the nucleus, concurrent or exclusive Vpx localization in the cytoplasm has been observed in specific cell types and a subset of HIV-2/SIV strains. We observe that HIV-2 ROD9 Vpx demonstrates significantly higher nuclear localization in HeLa cells compared to Vpx proteins from HIV-2 7312a and SIVmac239. This discrepancy is likely due to key amino acid differences that impact nuclear import and/or export pathways exploited by Vpx. To investigate amino acids relevant for nuclear import, site-directed mutagenesis was executed on SIVmac239 Vpx to increase its homology to HIV-2 ROD9 Vpx. HeLa cells were transfected with various Cherry-tagged SIVmac239 Vpx constructs followed by treatment with leptomycin B (inhibits nuclear export). Representative cells were then imaged with confocal microscopy and used to quantify localization patterns displayed by each Vpx construct. Interestingly, our data from the SIVmac239 V67T Vpx construct suggests that other residues outside the C-terminal 65-SYTKYRYL-72 minimal ncNLS must contribute to efficient Vpx nuclear uptake. Collectively, these various constructs help narrow down the particularly relevant nuclear localization mechanisms for this virus in HeLa cells and serve as a platform for additional study in other biologically relevant cell types, such as macrophages. By providing greater insight into viral nuclear import, these data may galvanize the development of effective HIV-2/SIV therapeutics that target commonly exploited host-cell nuclear entry pathways.
Recommended Citation
Mayhew, Hunter. "SIVmac239 Vpx Nuclear Import." Undergraduate Research Symposium, Mankato, MN, April 12, 2022.
https://cornerstone.lib.mnsu.edu/urs/2022/poster-session-02/25
