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. 2019 Apr 26;11(5):390.
doi: 10.3390/v11050390.

Distinct Requirements for HIV-1 Accessory Proteins during Cell Coculture and Cell-Free Infection

Anastasia Zotova  1   2 , Anastasia Atemasova  3 , Alexey Pichugin  4 , Alexander Filatov  5 , Dmitriy Mazurov  6   7
Affiliations

Distinct Requirements for HIV-1 Accessory Proteins during Cell Coculture and Cell-Free Infection

Anastasia Zotova et al. Viruses. .

Abstract

The role of accessory proteins during cell-to-cell transmission of HIV-1 has not been explicitly defined. In part, this is related to difficulties in measuring virus replication in cell cocultures with high accuracy, as cells coexist at different stages of infection and separation of effector cells from target cells is complicated. In this study, we used replication-dependent reporter vectors to determine requirements for Vif, Vpu, Vpr, or Nef during one cycle of HIV-1 cell coculture and cell-free infection in lymphoid and nonlymphoid cells. Comparative analysis of HIV-1 replication in two cell systems showed that, irrespective of transmission way, accessory proteins were generally less required for virus replication in 293T/CD4/X4 cells than in Jurkat-to-Raji/CD4 cell cocultures. This is consistent with a well-established fact that lymphoid cells express a broad spectrum of restriction factors, while nonlymphoid cells are rather limited in this regard. Remarkably, Vpu deletion reduced the level of cell-free infection, but enhanced the level of cell coculture infection and increased the fraction of multiply infected cells. Nef deficiency did not influence or moderately reduced HIV-1 infection in nonlymphoid and lymphoid cell cocultures, respectively, but strongly affected cell-free infection. Knockout of BST2-a Vpu antagonizing restriction factor-in Jurkat producer cells abolished the enhanced replication of HIV-1 ΔVpu in cell coculture and prevented the formation of viral clusters on cell surface. Thus, BST2-tethered viral particles mediated cell coculture infection more efficiently and at a higher level of multiplicity than diffusely distributed virions. In conclusion, our results demonstrate that the mode of transmission may determine the degree of accessory protein requirements during HIV-1 infection.

Keywords: BST2; CRISPR-Cas9 knockout; HIV-1; Nef; Vpu; accessory proteins; cell-to-cell infection; restriction factors.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Characteristics of the generated accessory gene mutants of HIV-1. (A) Western blot analysis of HIV-1 accessory protein expression in 293T cells transiently transfected with the original (wt) pCMV-dR8.2 packaging plasmid or the plasmid containing mutation in one of the accessory genes (indicated as ∆) or mock plasmid. The blots were stained for viral proteins or tubulin as shown on the right. The molecular weights of stained proteins are indicated on the left. The blots with typical staining are presented. (B) The levels of VLP production by 293T cells transfected with wt or mutant HIV-1 packaging plasmid. The supernatants from transfected cells were harvested 48 h post-transfection, filtered, and analyzed using p24 ELISA Kit. The data obtained from the three independent experiments were calculated relative to wt control, and presented as the average values with the standard deviations.
Figure 2
Figure 2
Effects of accessory gene inactivation on HIV-1 replication in HEK 293T/CD4/X4 cells. (A) Schematic representation of experiments designed to quantify and normalize HIV-1 infection in 293T/CD4/X4 cells. The levels of infection estimated by luciferase activity were normalized to the levels of Gag quantified by ELISA. The normalized level of infectivity obtained for wt HIV-1 was set at 1.0, and the infectivity levels for mutants were recalculated relative to that value. The levels of HIV-1 relative infectivity measured in 293T/CD4/X4 cell culture and cell-free infection tests are shown in (B,C), respectively. All data are representative of at least three independent experiments and shown as average values ± standard deviations. *, **, statistically different from the control (wt) by Student’s t-test at p < 0.05 and p < 0.01, respectively.
Figure 3
Figure 3
The requirements for HIV-1 accessory proteins during HIV-1 replication in lymphoid cells. (A) Schematic experimental design for evaluation HIV-1 replication in Jurkat-Raji/CD4 cell cocultures. (B) The results of HIV-1 relative infectivities measured in Jurkat-to-Raji/CD4 cell cocultures. (C) Scheme of experimental set up for detecting infectivity of HIV-1 VLPs derived from Jurkat cells and applied to Raji/CD4 target cells. (D) The levels of infectivity of VLPs purified from Jurkat cells and use to infect Raji/CD4 cells. The levels of replication were calculated as described for Figure 2 and as specified in the Results section. The data shown as average values ± standard deviations are representative of at least three independent experimental repeats. *, **, statistically different from the control (wt) at p < 0.05 and p < 0.01, respectively.
Figure 4
Figure 4
Quantification of cells infected by wt or accessory gene mutant HIV-1 in Jurkat-Raji/CD4 cell coculture using inGFPt and inmCherry vectors. (A) Typical flow cytometry DotPlot graphs showing distribution of single- and double-infected cells at day 3 after cell mixture. Quantitative analysis of single infected (GFP+) and double infected (GFP+ mCherry+) cells (B) and their ratios as a measure of MOI (C). Data are presented as average values ± Std dev from three independent experiments. All values obtained for indicated mutants of HIV-1 in B are statistically different from wt control by Student’s t-test at p < 0.01. **, statistically different from the control (wt) at p < 0.01.
Figure 5
Figure 5
BST2 knockout and replication of Vpu-deficient HIV-1. (A) Flow cytometry analysis of spontaneous and INF-α2b-induced BST2 expression on surfaces of indicated cells. The data are shown as the representative overlaid FACS histograms (A) and as the levels of mean fluorescence intensity (MFI) (B). The color codes in B match those in A. (C) The levels of wt and ∆Vpu HIV-1 replication in Jurkat-Raji/CD4 cell cocultures were comparably examined using parental Jurkat cells (left two bars), BST2 KO Jurkat cells (middle two bars), and BST2 KO Jurkat cells transiently cotransfected with BST2 expression plasmid (right two bars). The infectivity data were normalized to the levels of total Gag and the values obtained for HIV-1 ∆Vpu were calculated relative to the respective wt HIV-1 controls. (D) The levels of MOI detected with wt or KO Jurkat cells and calculated as described for Figure 4C. The results in B–D are representative of at least three independent experiments and shown as averages with standard deviations. *, **, data are statistically different at p < 0.05 and p < 0.01, respectively.
Figure 6
Figure 6
The patterns of HIV-1 and HTLV-1 viral particle expression in BST2-positive and negative T cell lines. (A) Jurkat parental or KO cells were transfected with wt or Vpu(-) HIV-1 or HTLV-1 packaging plasmid and followed fixation/permeabilization stained with the respective anti-Gag Ab. Cells were analyzed using fluorescence deconvolution microscope, and presented as a 0.3-µm optical slices through the middle plane of cells (OptS) or as Z-stack one plane projections obtained by Maximum Likelihood Estimation (MLE) algorithm (scale bar, 5 μm). At least 4-6 cells per sample were analyzed to demonstrate representative cells. The levels of clusterization for HIV-1 ∆Vpu (B) and HTLV-1 (C) were quantified using ImageJ Threshold, Watershed, and Analyze particles software options. A size limit for clusters was set to calculate numbers of clusters per cell. The data obtained from several cells and from different slides were averaged and presented. * and **, the differences between wild type and KO cells are statistically significant at p < 0.05 and p < 0.01, respectively.
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