Vpr14-88-Apobec3G fusion protein is efficiently incorporated into Vif-positive HIV-1 particles and inhibits viral infection.
BACKGROUND
APOBEC3G (A3G), a deoxycytidine deaminase, is a potent host antiviral factor that can restrict HIV-1 infection. During Vif-negative HIV-1 replication, A3G is incorporated into HIV-1 particles, induces mutations in reverse transcribed viral DNA and inhibits reverse transcription. However, HIV-1 Vif counteracts A3G's activities by inducing its degradation and by blocking its incorporation into HIV-1 particles. Thus, it is interesting to elucidate a mechanism that would allow A3G to escape the effects of Vif in order to rescue its potent antiviral activity and to provide a possible novel therapeutic strategy for treating HIV-1 infection.


RESULTS
In this study, we generated an R88-A3G fusion protein by fusing A3G to a virion-targeting polypeptide (R14-88) derived from HIV-1 Vpr protein and compared its antiviral effects relative to those of HA-tagged native A3G (HA-A3G). Our study showed that transient expression of the R88-A3G fusion protein in both Vif(-) and Vif(+) HIV-1 producing cells drastically inhibited viral infection in HeLa-CD4-CCR5-cells, CD4(+) C8166 T cells and human primary PBMCs. Moreover, we established CD4(+) C8166 T cell lines that stably express either R88-A3G or HA-A3G by transduction with VSV-G-pseudotyped lentiviral vector that harbor expression cassettes for R88-A3G or HA-A3G, respectively, and tested their susceptibility to Vif(+) HIV-1 infection. Our results clearly reveal that expression of R88-A3G in transduced CD4(+) C8166 cells significantly blocked Vif(+) HIV-1 infection. In an attempt to understand the mechanism underlying the antiviral activity of R88-A3G, we demonstrated that R88-A3G was efficiently incorporated into viral particles in the presence of Vif. Moreover, PCR analysis revealed that R88-A3G significantly inhibited viral cDNA synthesis during the early stage of Vif(+) virus infection.


CONCLUSIONS
Our results clearly indicate that R88 delivers A3G into Vif(+) HIV-1 particles and inhibits infectivity and spread of the virions among CD4(+) T cells. This study provides evidence for an effective strategy to modify a host protein with innate anti-HIV-1 activity and rescue its potent anti-HIV potential in the presence of Vif. Further characterization and optimization of this system may lead to the development of an effective therapeutic approach against HIV-1 infection.
Ao
Zhujun
Z
Laboratory of Molecular Human Retrovirology, Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada.
Yu
Zhe
Z
Wang
Lina
L
Zheng
Yingfeng
Y
Yao
Xiaojian
X
D000071480:APOBEC-3G Deaminase; D015496:CD4-Positive T-Lymphocytes / Q000821:virology; D002460:Cell Line; D003564:Cytidine Deaminase / Q000737:chemistry / Q000235:genetics*; D016346:Gene Products, vif / Q000737:chemistry / Q000235:genetics*; D015658:HIV Infections / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D014158:Transcription, Genetic; D054325:vpr Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000378:metabolism*
D016346:Gene Products, vif; D011993:Recombinant Fusion Proteins; D054325:vpr Gene Products, Human Immunodeficiency Virus; C514912:vpr protein, Human immunodeficiency virus 1; D000071480:APOBEC-3G Deaminase; C464924:APOBEC3G protein, human; D003564:Cytidine Deaminase