Primary T-cells from human CD4/CCR5-transgenic rats support all early steps of HIV-1 replication including integration, but display impaired viral gene expression.
BACKGROUND
In vivo studies on HIV-1 pathogenesis and testing of antiviral strategies have been hampered by the lack of an immunocompetent small animal model that is highly susceptible to HIV-1 infection. Since native rodents are non-permissive, we developed transgenic rats that selectively express the HIV-1 receptor complex, hCD4 and hCCR5, on relevant target cells. These animals display a transient low-level plasma viremia after HIV-1YU-2 infection, demonstrating HIV-1 susceptibility in vivo. However, unlike macrophages, primary CD4 T-cells from double-transgenic animals fail to support viral spread ex vivo. To identify quantitative limitations or absolute blocks in this rodent species, we quantitatively assessed the efficiency of key steps in the early phase of the viral replication cycle in a side-by-side comparison in infected cell lines and primary T-cells from hCD4/hCCR5-transgenic rats and human donors.


RESULTS
Levels of virus entry, HIV-1 cDNA synthesis, nuclear import, and integration into the host genome were shown to be remarkably similar in cell lines and, where technically accessible, in primary T-cells from both species. In contrast, a profound impairment at the level of early HIV gene expression was disclosed at the single-cell level in primary rat T-cells and most other rat-derived cells. Macrophages were a notable exception, possibly reflecting the unique transcriptional milieu in this evolutionarily conserved target cell of all lentiviruses. Importantly, transient trans-complementation by ex vivo nucleofection with the Tat-interacting protein Cyclin T1 of human origin markedly elevated HIV gene expression in primary rat T-cells.


CONCLUSIONS
This is the first study that has quantitatively determined the efficiency of consecutive steps in the HIV-1 replication cycle in infected primary HIV target cells from a candidate transgenic small animal and compared it to human cells. Unlike cells derived from mice or rabbits, rat cells complete all of the early steps in the HIV-1 replication cycle, including provirus integration in vivo, with high efficiency. A deficiency in gene expression was disclosed at the single cell level and could be counteracted by the human pTEFb transcription complex factor Cyclin T1. Collectively, these results provide the basis for the advancement of this transgenic rat model through strategies aimed at boosting HIV-1 gene expression in primary rat CD4 T-cells, including human Cyclin T1 transgenesis.
Goffinet
Christine
C
Department of Virology, University of Heidelberg, Heidelberg, Germany. Christine.goffinet@med.uni-heidelberg.de
Michel
Nico
N
Allespach
Ina
I
Tervo
Hanna-Mari
HM
Hermann
Volker
V
Kräusslich
Hans-Georg
HG
Greene
Warner C
WC
Keppler
Oliver T
OT
D000818:Animals; D030801:Animals, Genetically Modified; D015704:CD4 Antigens / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D056906:Cyclin T; D016213:Cyclins / Q000378:metabolism; D004195:Disease Models, Animal; D005260:Female; D015967:Gene Expression Regulation, Viral; D015658:HIV Infections / Q000276:immunology / Q000503:physiopathology / Q000821:virology*; D015497:HIV-1 / Q000472:pathogenicity / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats; D019713:Receptors, CCR5 / Q000235:genetics / Q000378:metabolism*; D013601:T-Lymphocytes / Q000821:virology*; D053586:Virus Internalization; D014779:Virus Replication
C503684:CCNT1 protein, human; D015704:CD4 Antigens; D056906:Cyclin T; D016213:Cyclins; D019713:Receptors, CCR5