The apparent interferon resistance of transmitted HIV-1 is possibly a consequence of enhanced replicative fitness

HIV-1 transmission via sexual exposure is an inefficient process. When transmission does occur, newly infected individuals are colonized by the descendants of either a single virion or a very small number of establishing virions. These transmitted founder (TF) viruses are more interferon (IFN)-resistant than chronic control (CC) viruses present 6 months after transmission. To identify the specific molecular defences that make CC viruses more susceptible to the IFN-induced ‘antiviral state’, we established a single pair of fluorescent TF and CC viruses and used arrayed interferon-stimulated gene (ISG) expression screening to identify candidate antiviral effectors. However, we observed a relatively uniform ISG resistance of transmitted HIV-1, and this directed us to investigate possible underlying mechanisms. Simple simulations, where we varied a single parameter, illustrated that reduced growth rate could possibly underly apparent interferon sensitivity. To examine this possibility, we closely monitored in vitro propagation of a model TF/CC pair (closely matched in replicative fitness) over a targeted range of IFN concentrations. Fitting standard four-parameter logistic growth models, in which experimental variables were regressed against growth rate and carrying capacity, to our in vitro growth curves, further highlighted that small differences in replicative growth rates could recapitulate our in vitro observations. We reasoned that if growth rate underlies apparent interferon resistance, transmitted HIV-1 would be similarly resistant to any growth rate inhibitor. Accordingly, we show that two transmitted founder HIV-1 viruses are relatively resistant to antiretroviral drugs, while their matched chronic control viruses were more sensitive. We propose that, when present, the apparent IFN resistance of transmitted HIV-1 could possibly be explained by enhanced replicative fitness, as opposed to specific resistance to individual IFN-induced defences. However, further work is required to establish how generalisable this mechanism of relative IFN resistance might be

The Interferon Resistance of Transmitted HIV-1 is Possibly a Consequence of Enhanced Replicative Fitness

Underlying data for pape

Anti-HIV-1 ISGs inhibit the CC virus more potently than transmitted HIV-1.

(A) Candidate anti-HIV-1 effectors that were more inhibitory than the known anti-HIV-1 effector IFITM3 were tested against CH058 GIN GFP-IRES-Nef) TF and CC viruses on MT4-R5 cells, as in 2F-G, normalised to the level of infection observed in the presence of an empty vector control. (B-C) ISGs in Fig 3A were tested for ability to induce cell death or ISRE (IFN-stimulated response element). In B, MT4 and TMZR5 cells were tested for cell death when expressing ISGs by flow cytometry using the LIVE/DEAD fixable dead cell stain kit (Invitrogen) and MT4 cell viability was additionally tested using the luminescence based CytoTox-Glo Cytotoxicity assay (Promega). In C, IFN induction by candidate ISGs was measured by flow cytometry using MT4 cells expressing an ISRE-GFP construct. (D) Candidate ISGs from Fig 3A were checked for their changes in expression upon type I IFN stimulation using the Interferome database to determine their ‘ISG-ness’. (E-F) Validation of the 8 most potent anti-HIV-1 effector ISGs, along with IFITM1 and IFITM2 controls, against CH058 TF and CC viruses, In E, TMZR5 cells transduced with pLV constructs containing the indicated ISGs or RFP as a control, were challenged with CH058 TF or CC and sampled daily to monitor virus spread. GFP-positive cells were enumerated using flow cytometry. Viral spreading replication experiments took place on two occasions, a typical result with contemporaneous controls is shown. In F, data from panel E represented as area under the curve (AUC).</p

Viruses used in this study.

Viruses used in this study.</p

Because of the proapoptotic effect of IFNs, the viability of IFN-treated TMZR5 cells was also assessed in parallel cultures to those used in Fig 1.

Viability was tested using flow cytometry using the LIVE/DEAD fixable dead cell stain kit (Invitrogen). (PDF)</p

S2 Fig -

Western blot gels assessing protein expression levels and IFN induction of expression of (A) CD80, (B) FNDC3B, (C) MICB (D) TMEM140, (E) CD38, (F) SCARB2. (G) TMZR5 cells (modified to express CD38 and SCARB2) were challenged with NHG and sampled daily to monitor virus spread. GFP-positive cells were enumerated via flow cytometry. (H) Western blots of the seven CRISPR guides and non-targeting control guide cell lines for CD38 in PM1 cells. (I) PM1 cell lines were pre-treated for 24 hours with the IFNα14 doses indicated and were subsequently challenged with NHG and sampled daily to monitor virus spread. (J) Western blots of the seven CRISPR guides and non-targeting control guide cell lines for SCARB2 in TMZR5 cells. (K) TMZR5 cell lines were pre-treated for 24 hours with the IFNα14 doses indicated and were subsequently challenged with NHG and sampled daily to monitor virus spread. Viral spreading replication experiments took place on two occasions, a typical result is shown. The white line in A indicates that the CD80 image was flipped for this blot to correct sample order, but the blot is the same for both portions of this image. Raw western blot images can be viewed in S7 Fig. (PDF)</p

Transmitted HIV-1 of the CH040 virus pair is more resistant to IFN and AZT than the matched chronic virus.

(A-B) TMZR5 cells were treated with indicated doses of IFNα14 for 24 hours before being challenged with the CH040 TF and CC viruses. Cells were sampled daily to monitor virus spread and GFP-positive cells were enumerated via flow cytometry. (C-D) TMZR5 cells were pre-treated with a range of azidothymidine (AZT) doses for 2 hours prior to infection with the CH040 TF or CC viruses. Cells were sampled daily to monitor virus spread and GFP-positive cells were quantified via flow cytometry.</p

Amino acid frequencies at sites that exhibit amino acid changes between sequenced TF/CC pairs are shown and compared to HxB2 (reference sequence).

(PDF)</p

The CH058 TF virus is more resistant to IFN than its matched CC virus.

(A-B) TMZR5 GFP-reporter cells were challenged with matched TF and CC VSV-G pseudotyped IMC pairs and sampled daily to monitor virus spread. GFP-positive cells were enumerated via flow cytometry. (C) To investigate the effect of IFN early in the viral life cycle (incoming infection), TMZR5 cells were pre-stimulated with different doses of IFNα14 for 24 hours prior to infection with serially diluted CH058 TF and CC viruses. To limit replication to a single cycle, infected cells were treated with dextran sulphate 17–18 hours post infection. The percentage of GFP-positive cells was determined at 48 hours post-infection using flow cytometry. To investigate the effect of IFN late in the viral life cycle (production effect), TMZR5 cells were pre-treated with IFNα14, and after 24 hours cells were challenged with CH058 TF and CC viruses at an MOI of 0.5 for 6 hours, before the inoculum was removed. At 46–48 hours post-infection, cell-free, filtered virus containing supernatants were titrated on TMZR5 cells. ND indicates not detected. (D-E) TMZR5s were treated with the indicated dose of IFNα14 for 24 hours before being challenged with the CH058 TF and CC virus pair. Cells were sampled daily to monitor virus spread and GFP-positive cells were enumerated via flow cytometry. Annotated fold change values refer to the maximum difference in (%) infection out of the timepoints tested. Viral spreading replication experiments took place on two occasions and a typical result is shown.</p

Raw western blot images.

(PDF)</p