Entry and transcription as key determinants of differences in CD4 T-cell permissiveness to human immunodeficiency virus type 1 infection

Isolated primary human cells from different donors vary in their permissiveness-the ability of cells to be infected and sustain the replication of human immunodeficiency virus type 1 (HIV-1). We used replicating HIV-1 and single-cycle lentivirus vectors in a population approach to identify polymorphic steps during viral replication. We found that phytohemagglutinin-stimulated CD4(+) CD45RO(+) CD57(-) T cells from healthy blood donors (n = 128) exhibited a 5.2-log-unit range in virus production. For 20 selected donors representing the spectrum of CD4 T-cell permissiveness, we could attribute up to 42% of the total variance in virus production to entry factors and 48% to postentry steps. Efficacy at key intracellular steps of the replicative cycle (reverse transcription, integration, transcription and splicing, translation, and budding and release) varied from 0.71 to 1.45 log units among donors. However, interindividual differences in transcription efficiency alone accounted for 64 to 83% of the total variance in virus production that was attributable to postentry factors. While vesicular stomatitis virus G protein-mediated fusion was more efficacious than CCR5/CD4 entry, the latter resulted in greater transcriptional activity per proviral copy. The phenotype of provirus transcription was stable over time, indicating that it represents a genetic trait.

SV40 activates transcription from the transferrin receptor promoter by inducing a factor which binds to the CRE/AP-1 recognition sequence

Peter Martin Beard, Nicole Paduwat

During the course of lytic infection by simian virus 40 (SV40), expression of both the viral late genes and certain host cellular genes is induced. The promoter of the cellular transferrin receptor (TR) gene contains a DNA sequence which is similar to the AP-1- and AP-4-binding region in SV40 which has been implicated in the control of the viral late promoter. Expression of TR is needed for cells to enter S-phase and is therefore expected to be important for the SV40 lytic cycle. Here we show that the level of TR mRNA in vivo was increased by SV40 infection. A factor which activates transcription from the TR promoter in vitro was specifically induced in SV40-infected cells. Gel mobility shift assays with an oligonucleotide comprising this part of the TR promoter showed three nucleoprotein complexes to be formed with proteins from CV-1 cells. Following SV40 infection, one of the complexes was increased ten-fold. Formation of this complex was specifically reduced by competition with the phorbol ester-responsive element of the collagenase gene, implying that the factor is a member of the AP-1/Jun/Fos family. Cross-linking of the complex by ultraviolet light showed major DNA-binding components to be proteins of about 55 kD and 47 kD. Removal of this factor by adding the oligonucleotide to in vitro transcription reactions with the TR promoter, abolished the activation of TR transcription. The factor which binds to the TR promoter co-sedimented with SV40 chromosomes extracted late in infection. This suggests that similar transcriptional regulatory proteins are involved in controlling transcription from both the SV40 and the TR promoters, and that the virus can use a common mechanism to induce viral and host cellular transcription.

1991

Efficient gene transfer into human primary blood lymphocytes by surface-engineered lentiviral vectors that display a T cell-activating polypeptide

Didier Trono

In contrast to oncoretroviruses, lentiviruses such as human immunodeficiency virus 1 (HIV-1) are able to integrate their genetic material into the genome of nonproliferating cells that are metabolically active. Likewise, vectors derived from HIV-1 can transduce many types of nonproliferating cells, with the exception of some particular quiescent cell types such as resting T cells. Completion of reverse transcription, nuclear import, and subsequent integration of the lentivirus genome do not occur in these cells unless they are activated via the T-cell receptor (TCR) or by cytokines or both. However, to preserve the functional properties of these important gene therapy target cells, only minimal activation with cytokines or TCR-specific antibodies should be performed during gene transfer. Here we report the characterization of HIV-1-derived lentiviral vectors whose virion surface was genetically engineered to display a T cell-activating single-chain antibody polypeptide derived from the anti-CD3 OKT3 monoclonal antibody. Interaction of OKT3 IgGs with the TCR can activate resting peripheral blood lymphocytes (PBLs) by promoting the transition from G(0) to G(1) phases of the cell cycle. Compared to unmodified HIV-1-based vectors, OKT3-displaying lentiviral vectors strongly increased gene delivery in freshly isolated PBLs by up to 100-fold. Up to 48% transduction could be obtained without addition of PBL activation stimuli during infection. Taken together, these results show that surface-engineered lentiviral vectors significantly improve transduction of primary lymphocytes by activating the target cells. Moreover these results provide a proof of concept for an approach that may have utility in various gene transfer applications, including in vivo gene delivery.

2002

Adeno-Associated Virus Activates an Innate Immune Response in Normal Human Cells but Not in Osteosarcoma Cells

Peter Martin Beard

Adeno-associated virus (AAV) is a small, DNA-containing dependovirus with promising potential as a gene delivery vehicle. Given the variety of applications of AAV-based vectors in the treatment of genetic disorders, numerous studies have focused on the immunogenicity of recombinant AAV. In general, AAV vectors appear not to induce strong inflammatory responses. We have found that AAV2, when it infects the osteosarcoma cells U2OS, can initiate part of its replicative cycle in the absence of helper virus. This does not occur in untransformed cells. We set out to test whether the cellular innate antiviral defenses control this susceptibility and found that, in nonimmune normal human fibroblasts, AAV2 induces type I interferon production and release and the accumulation of nuclear promyelocytic leukemia bodies. AAV fails to mobilize this defense pathway in the U2OS cells. This permissiveness is in large part due to impairment of the viral sensing machinery in these cells. Our investigations point to Toll-like receptor 9 as a potential intracellular sensor that detects AAV2 and triggers the antiviral state in AAV-infected untransformed cells. Efficient sensing of the AAV genome and the ensuing activation of an innate antiviral response are thus crucial cellular events dictating the parvovirus infectivity in host cells.

2011