Replication of Primary HIV-1 Isolates Is Inhibited in PM1 Cells Expressing sCD4-KDEL

AbstractExpression of a soluble CD4 molecule (sCD4-KDEL) containing a specific retention signal for the endoplasmic reticulum was shown previously to block propagation of the HIV-1MNprototype strain in a transformed T cell line. However, the virus present in HIV-1-infected individuals is more closely represented by primary HIV-1 isolates which, unlike the HIV-1MNstrain, have not been adapted to growth in cell lines. To determine if sCD4-KDEL could block replication of primary isolates we used the PM1 cell line that has been shown to propagate primary isolates without adaptation. Here we show that the replication of four primary HIV-1 isolates was strongly inhibited in PM1 cells that expressed sCD4-KDEL under control of the HIV-1 LTR. Infection with primary HIV-1 isolates induced sCD4-KDEL expression driven by the LTR, HIV-1 spread was dramatically reduced, and reverse transcriptase activity in the cell culture supernatants was greatly diminished. sCD4-KDEL, therefore, represents a potent inhibitor of HIV-1 replication for gene therapy-based approaches for the treatment of AIDS

Human Immunodeficiency Virus Type 1 (HIV-1)-Specific CD4(+) T Cells That Proliferate In Vitro Detected in Samples from Most Viremic Subjects and Inversely Associated with Plasma HIV-1 Levels

Diminished in vitro proliferation of human immunodeficiency virus type 1 (HIV-1)-specific CD4(+) T cells has been associated with HIV-1 viremia and declining CD4(+) T-cell counts during chronic infection. To better understand this phenomenon, we examined whether HIV-1 Gag p24 antigen-induced CD4(+) T-cell proliferation might recover in vitro in a group of subjects with chronic HIV-1 viremia and no history of antiretroviral therapy (ART). We found that depletion of CD8(+) cells from peripheral blood mononuclear cells (PBMC) before antigen stimulation was associated with a 6.5-fold increase in the median p24-induced CD4(+) T-cell proliferative response and a 57% increase in the number of subjects with positive responses. These p24-induced CD4(+) T-cell proliferative responses from CD8-depleted PBMC were associated with expansion of the numbers of p24-specific, gamma interferon (IFN-γ)-producing CD4(+) T cells. Among the 20 viremic, treatment-naïve subjects studied, the only 5 subjects lacking proliferation-competent, p24-specific CD4(+) T-cell responses from CD8-depleted PBMC showed plasma HIV-1 RNA levels > 100,000 copies/ml. Furthermore, both the magnitude of p24-induced CD4(+) T-cell proliferative responses from CD8-depleted PBMC and the frequency of p24-specific, IFN-γ-producing CD4(+) T cells expanded from CD8-depleted PBMC were associated inversely with plasma HIV-1 RNA levels. Therefore, proliferation-competent, HIV-1-specific CD4(+) T cells that might help control HIV-1 disease may persist during chronic, progressive HIV-1 disease except at very high levels of in vivo HIV-1 replication

Stochastic principles governing alternative splicing of RNA

<div><p>The dominance of the major transcript isoform relative to other isoforms from the same gene generated by alternative splicing (AS) is essential to the maintenance of normal cellular physiology. However, the underlying principles that determine such dominance remain unknown. Here, we analyzed the physical AS process and found that it can be modeled by a stochastic minimization process, which causes the scaled expression levels of all transcript isoforms to follow the same Weibull extreme value distribution. Surprisingly, we also found a simple equation to describe the median frequency of transcript isoforms of different dominance. This two-parameter Weibull model provides the statistical distribution of all isoforms of all transcribed genes, and reveals that previously unexplained observations concerning relative isoform expression derive from these principles.</p></div

Transcript isoform expression pattern of two genes in different conditions.

<p>(A) BRD4. (B) SRSF7. Among 11 transcript isoforms of BRD4 and 12 transcript isoforms of SRSF7, ENST00000371835 and ENST00000409276 are the most dominant isoforms in all four activated conditions, ENST00000263377 and ENST00000477635 are the most dominant isoforms in all four resting conditions, respectively. This result indicates the major transcript isoform can be regulated by single external signal.</p

A model of alternative splicing.

<p>(A) Splicing factor U1 and U2AF search the 5’ GU and 3’ AG splicing sites by 3D and 1D Brownian motion. Multiple candidate splice sites compete for the binding of U1 and U2AF. The binding is ATP-independent and reversible. (B) The binding of U1 and U2AF to the splice sites becomes stable only after the ATP-dependent binding of U2 snRNP. The identification of each intron is equivalent to a minimization process that U1 and U2AF dynamically search their global or local minimal energy sites on the pre-mRNA segment presented for AS. (C) The scaled expression level of transcript isoform follows type III extreme value distribution—a Weibull distribution. The approximate values of parameters <i>a (0</i>.<i>44)</i> and <i>b (0</i>.<i>6)</i> are estimated by curve fitting. Black curve represents the distribution of scaled expression level from experimental data. Red curve represent the Weibull distribution produced by curve fitting.</p

The frequency distribution of the <i>k</i>th dominant transcript isoform.

<p>(A) <i>k</i> = 1. (B) <i>k</i> = 2. <i>k</i> is the rank of a transcript isoform. <i>M</i> is the number of transcript isoforms for a gene. Black curves represent frequency distribution of the experimental RNA-seq data. Red curves represent the frequency distribution of the simulated data from Weibull distribution <i>W(0</i>.<i>39)</i>. KLd is the Kullback-Leibler divergence between the two distributions.</p

Droplet-microfluidics-assisted sequencing of HIV proviruses and their integration sites in cells from people on antiretroviral therapy.

The human immunodeficiency virus (HIV) integrates its genome into that of infected cells and may enter an inactive state of reversible latency that cannot be targeted using antiretroviral therapy. Sequencing such a provirus and the adjacent host junctions in individual cells may elucidate the mechanisms of the persistence of infected cells, but this is difficult owing to the 150-million-fold higher amount of background human DNA. Here we show that full-length proviruses connected to their contiguous HIV-host DNA junctions can be assembled via a high-throughput microfluidic assay where droplet-based whole-genome amplification of HIV DNA in its native context is followed by a polymerase chain reaction (PCR) to tag droplets containing proviruses for sequencing. We assayed infected cells from people with HIV receiving suppressive antiretroviral therapy, resulting in the detection and sequencing of paired proviral genomes and integration sites, 90% of which were not recovered by commonly used nested-PCR methods. The sequencing of individual proviral genomes with their integration sites could improve the genetic analysis of persistent HIV-infected cell reservoirs