Modeling of the viral life cycle.

<p>(<b>A</b>) Raw data of measured viral replication intermediates (mean [dots] with one standard error) and curves of fitted progression model (solid lines). The temporal dynamics of each step in the viral life cycle was generated individually by modeling the net effect of production, decay, initial viral input, and experimental noise of the corresponding marker intermediate (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003161#ppat.1003161.s001" target="_blank">Text S1</a></b> and <b>Figure S4</b> and <b>S5 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003161#ppat.1003161.s001" target="_blank">Text S1</a></b>). (<b>B</b>) Activity profile of individual steps of the viral life cycle estimated from the progression model. Each violin spans the 98% quantile of the viral step with width proportional to activity level at each given point in time. The plus symbol (‘+’) denotes the peak of the activity and the inner white violin its 95% bootstrap confidence interval. In the shaded area, expected values extrapolated beyond the last observed time point (24 h, dashed line) are shown.</p

Transcriptome changes upon exposure to infectious and non-infectious viral particles.

<p>Principal component analysis is used to explore the overall variance structure of the transcriptome datasets. With each point representing a whole transcriptome sample, the figure presents the transcriptome of cells that were universally infected (HIV), cells exposed to heat-inactivated virus (Heat-inactivated), cells exposed to a mixture of 1∶10 infectious/heat-inactivated virus (HIV[1/10]), and non-infected cells (Mock). One mock sample failed and is not plotted. The transcriptome of mock cells and that of cells exposed to heat-inactivated viruses clustered together across the top principal components. Infected cells, on the other hand, spread away from the mock space as infection progressed, with the most distant dot corresponding to the latest time point (24 h). The mixture 1/10 infectious/noninfectious material occupies the intermediate space. Clustering of the two hours samples corresponds to end of cell exposure to the virus or control materials.</p

Core gene validation.

<p>RT-qPCR was used to validate key patterns of expression using heat-inactivated virus, primary cells, and natural viral envelope. (<b>A</b>) Analysis of 14 representative genes using competent or heat-inactivated HIV-based vector. The graphs depict the 24 dynamics of expression (log₂ fold change of VSV.G pseudotyped HIV-infected over mock) of eight upregulated genes (red lines), five downregulated genes (blue), and one control (<i>RPL31</i>, black line) in SupT1 cells exposed to similar amount of viral particles, only competent HIV (top panel), 1∶10 competent HIV∶heat-inactivated HIV (middle panel), and only heat-inactivated HIV (bottom panel). (<b>B</b>) Analysis in primary CD4+ T cells isolated from two healthy blood donors. Depicted are the 24 dynamics of expression (log₂ fold change of VSV.G pseudotyped HIV-infected over mock) of the upregulated (red), downregulated (blue), and control (black) genes. (<b>C</b>) Correlation analysis of RT-qPCR for the 14 representative genes at all time points in primary cells (donor 1) infected by VSV.G or CXCR4 pseudotyped HIV. Log₂ fold change linear regression yielded <i>r²</i> = 0.22, <i>p</i><10⁻⁴.</p

Clusters of host genes correlated with viral progression.

<p>Temporal expression patterns of 7,991 genes modulated in concordance with key steps of viral replication (panel <b>A</b>) were grouped into 18 clusters with differential expression profiles at three phases of the viral life cycle, namely reverse transcription, integration, and late phase. The cluster code characters ‘+’ and ‘−’ mark significant (<i>p</i><10⁻²) upregulation and downregulation, respectively, while ‘o’ indicates no significant deviation from zero. For example, the cluster ‘−+o’ contains 373 genes downregulated during reverse transcription, upregulated during integration, and unregulated during the late phase. In total, six upregulated clusters (<b>B</b>), four clusters with mixed patterns of regulation (<b>C</b>), and eight downregulated clusters (<b>D</b>) were found. Details of clusters are available at the dedicated web resource <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003161#ppat.1003161-Bartha1" target="_blank">[6]</a>.</p