title: Second generation imaging of nuclear/cytoplasmic HIV-1 complexes.
abstract: The ability to visualize fluorescent HIV-1 particles within the nuclei of infected cells represents an attractive tool to study the nuclear biology of the virus. To this aim we recently developed a microscopy-based fluorescent system (HIV-IN-EGFP) that has proven valid to efficiently visualize HIV-1 complexes in the nuclear compartment and to examine the nuclear import efficiency of the virus. The power of this method to investigate viral events occurring between the cytoplasmic and the nuclear compartment is further shown in this study through the analysis of HIV-IN-EGFP in cells expressing the TRIMCyp restriction factor. In these cells the HIV-IN-EGFP complexes are not detected in the nuclear compartment, while treatment with MG132 reveals an accumulation of HIV-1 complexes in the cytoplasm. However, the Vpr-mediated transincorporation strategy used to incorporate IN fused to EGFP (IN-EGFP) impaired viral infectivity. To optimize the infectivity of the HIV-IN-EGFP, we used mutated forms of IN (E11K and K186E) known to stabilize the IN complexes and to partially restore viral infectivity in transcomplementation experiments. The fluorescent particles produced with the modified IN [HIV-IN(K)EGFP_IN(E)] show almost 30% infectivity as compared to wild-type NL4.3. Detailed confocal microscopy analysis revealed that the newly generated viral particles resulted in HIV-1 complexes significantly smaller in size, thus requiring the use of brighter fluorophores for nuclear visualization [HIV-IN(K)sfGFP_IN(E)]. The second-generation visualization system HIV-IN(K)sfGFP_IN(E), in addition to allowing direct visualization of HIV-1 nuclear entry and other viral events related to nuclear import, preserves intact viral properties in terms of nuclear entry and improved infectivity.
authors.affiliation: 1 Centre for Integrative Biology (CIBIO), University of Trento , Trento, Italy .
authors.forename: Ashwanth Christopher
authors.initials: AC
authors.lastname: Francis
authors.forename: Cristina
authors.initials: C
authors.lastname: Di Primio
authors.forename: Valentina
authors.initials: V
authors.lastname: Quercioli
authors.forename: Paola
authors.initials: P
authors.lastname: Valentini
authors.forename: Annegret
authors.initials: A
authors.lastname: Boll
authors.forename: Gabriele
authors.initials: G
authors.lastname: Girelli
authors.forename: Francesca
authors.initials: F
authors.lastname: Demichelis
authors.forename: Daniele
authors.initials: D
authors.lastname: Arosio
authors.forename: Anna
authors.initials: A
authors.lastname: Cereseto
mesh_terms: D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000821:virology*; D005456:Fluorescent Dyes / Q000032:analysis; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D057809:HEK293 Cells; D015658:HIV Infections / Q000235:genetics* / Q000821:virology; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D016662:Virus Integration / Q000235:genetics; D053586:Virus Internalization; D014779:Virus Replication / Q000235:genetics
chemical_list: D005456:Fluorescent Dyes; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins