Second generation imaging of nuclear/cytoplasmic HIV-1 complexes.
AIDS Res Hum Retroviruses
; 30(7): 717-26, 2014 Jul.
Article
en En
| MEDLINE
| ID: mdl-24798748
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Infecciones por VIH
/
Núcleo Celular
/
VIH-1
/
Internalización del Virus
Límite:
Humans
Idioma:
En
Revista:
AIDS Res Hum Retroviruses
Asunto de la revista:
SINDROME DA IMUNODEFICIENCIA ADQUIRIDA (AIDS)
Año:
2014
Tipo del documento:
Article
País de afiliación:
Italia