Entry and Transcription as Key Determinants of Differences in CD4 T-Cell Permissiveness to Human Immunodeficiency Virus Type 1 Infection

Abstract

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.

Differential Pathogenesis of Primary CCR5-Using Human Immunodeficiency Virus Type 1 Isolates in Ex Vivo Human Lymphoid Tissue

“…However, in our ex vivo tissues, no infection with any of the primary R5 HIV-1 isolates affected the levels of the 16 measured cy- tokines/chemokines. Earlier, similar results were reported for an R5 laboratory strain and for recombinant viruses carrying R5 Envs, whereas an X4 strain significantly changed chemokine release (9,27). However, one recent study (8) has suggested that R5 HIV-1 infection of fetal thymic organ cultures induces IL-10 and transforming growth factor ␤, cytokines not studied here, and thereby upregulates the expression of CCR5.…”

Stable gene expression occurs from a minority of integrated HIV-1-based vectors: transcriptional silencing is present in the majority

“…VSV-Gmediated entry is predominantly endocytic rather than the virus cell fusion mechanism of the CD4 route. 31 This may have some influence on subsequent transcriptional activity, 32 as we have previously shown that blocking downstream receptor signaling may affect infectivity. 33 However, recent data suggest that entry using VSV-G does not influence integration site selection compared with entry via CCR5 receptor-mediated fusion (F Bushman, personal communication).…”

Characterization of the Influence of Mediator Complex in HIV-1 Transcription

“…We also quantified the unspliced tat/rev/nef mRNA transcripts, transcribed during the elongation process that follows the initial transcription process (26). A reduction in viral elongated transcripts was observed after silencing of all MED subunits (Fig.…”

“…Gene expression assays for all analyzed human genes were purchased from Invitrogen. For viral RNA quantification, the following primers and probe amplifying tat/rev/nef mRNA were used: forward 5Ј-GGATCTGTCTCTGTCTCTCTCTCCACC-3Ј, reverse 5Ј-ACAGTCAGACTCATCAAGTTTCTCTATCAAAGCA-3Ј, and the dual-labeled fluorescent probe FAM 5Ј-TTCCTTCG-GGCCTGTCGGGTCCC-3Ј TAMRA (26). TAR transcripts were quantified with the following primers and probe: forward 5Ј-GGGTCTCTCTGGTTAGA-3Ј, reverse 5Ј-GGGTTCCCT-AGTTAG-3Ј, and the probe that is complementary to the TAR loop region of the HIV-1 LTR and also dual-labeled, FAM 5Ј-GCCTGGGAGCTCTCTGG-3Ј TAMRA (27,28).…”