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Host and Viral Determinants for MxB Restriction of HIV-1 Infection

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Host and Viral Determinants for MxB Restriction of HIV-1 Infection

Kenneth A Matreyek et al. Retrovirology.

Abstract

Background: Interferon-induced cellular proteins play important roles in the host response against viral infection. The Mx family of dynamin-like GTPases, which include MxA and MxB, target a wide variety of viruses. Despite considerable evidence demonstrating the breadth of antiviral activity of MxA, human MxB was only recently discovered to specifically inhibit lentiviruses. Here we assess both host and viral determinants that underlie MxB restriction of HIV-1 infection.

Results: Heterologous expression of MxB in human osteosarcoma cells potently inhibited HIV-1 infection (~12-fold), yet had little to no effect on divergent retroviruses. The anti-HIV effect manifested as a partial block in the formation of 2-long terminal repeat circle DNA and hence nuclear import, and we accordingly found evidence for an additional post-nuclear entry block. A large number of previously characterized capsid mutations, as well as mutations that abrogated integrase activity, counteracted MxB restriction. MxB expression suppressed integration into gene-enriched regions of chromosomes, similar to affects observed previously when cells were depleted for nuclear transport factors such as transportin 3. MxB activity did not require predicted GTPase active site residues or a series of unstructured loops within the stalk domain that confer functional oligomerization to related dynamin family proteins. In contrast, we observed an N-terminal stretch of residues in MxB to harbor key determinants. Protein localization conferred by a nuclear localization signal (NLS) within the N-terminal 25 residues, which was critical, was fully rescuable by a heterologous NLS. Consistent with this observation, a heterologous nuclear export sequence (NES) abolished full-length MxB activity. We additionally mapped sub-regions within amino acids 26-90 that contribute to MxB activity, finding sequences present within residues 27-50 particularly important.

Conclusions: MxB inhibits HIV-1 by interfering with minimally two steps of infection, nuclear entry and post-nuclear trafficking and/or integration, without destabilizing the inherent catalytic activity of viral preintegration complexes. Putative MxB GTPase active site residues and stalk domain Loop 4 -- both previously shown to be necessary for MxA function -- were dispensable for MxB antiviral activity. Instead, we highlight subcellular localization and a yet-determined function(s) present in the unique MxB N-terminal region to be required for HIV-1 restriction.

Figures

Figure 1
Figure 1
MxB antiviral activities. (A) Western blot of HOS cells expressing MxB-HA. β-actin was monitored to control for sample loading. (B) Western blot of untransduced or MxB-HA expressing HOS cell lysates extracted after treatment for 24 h with 1,000 U/ml of interferon α (IFN) as indicated. (C) Immunofluorescence microscopy of HOS cells stably expressing MxB-HA or mock-transduced cells. Blue-tinted ovals demarcate cell nuclei due to Hoescht 33342 staining of DNA. White horizontal bar, 10 μm. (D) Infection of MxB-HA expressing cells with various retroviral vectors plotted as percent infection of mock-transduced cells. Results are a summary of 6 independent experiments with error bars denoting 95% confidence intervals.
Figure 2
Figure 2
HIV-1 CA and sensitivity to MxB restriction. (A) Infectivities of CA mutant viruses normalized to the WT by input level of exogenous RT activity and expressed as percent of WT HIV-1 infection. (B) Sensitivity of the 17 CA single mutant viruses to MxB-HA, expressed as percent infection versus mock-transduced cells. The single letter codes along the bottom of the graph represent the WT residue at that position. (C) Normalized infectivities of 10 CA double mutant viruses relative to the WT. (D) Sensitivity of HIV-1 CA double missense mutant viruses (half black, half white filled circles) as compared to the parental single mutant viruses. Q63A alone was not tested. (E) Scatterplot comparing the sensitivity of CA missense mutant viruses to MxB-HA restriction with sensitivity to the rhesus TRIM5α TFP allele. (F) Pairwise comparison summary of MxB sensitivity with various CA-related manipulations of host cells, with Spearman rank correlation and P values denoted; see Additional file 1: Figure S1 for the corresponding scatterplots. Panel A-D results are averages of at least 6 independent experiments, with error bars denoting 95% confidence intervals.
Figure 3
Figure 3
Partial resistance of D64N/D116N IN active site mutant virus to MxB restriction. (A) Levels of NC mutant H23C, RT mutant V148I, and IN mutant D167K and D64N/D116N infectivities, normalized to the WT based on input levels of exogenous RT activity, in control (dark grey) versus MxB-expressing (light grey) cells. Results are plotted relative to the WT virus in control cells (set at 100%). (B) The re-plot of panel A results highlights the infectivities of the indicated viruses in MxB-expressing cells relative to control cells. (C) RT-normalized levels of D64N/D116N IN mutant infectivities with versus without additional CA mutations in control (dark grey) versus MxB-expressing (light grey) cells. The infectivity of the WT CA, D64N/D116N IN mutant virus in control cells was set to 100%. (D) Extent of MxB restriction of WT and CA mutant viruses that carry WT (dark grey) or D64N/D116N (NN) mutant IN (white bars). Results are an average of 5 independent experiments, with error bars denoting 95% confidence intervals.
Figure 4
Figure 4
WT and D64N/D116N IN mutant DNA metabolism in control and MxB-expressing cells. (A) Levels of WT and D64N/D116N infectivities in control (dark grey) and MxB-expressing (light grey) HOS cells normalized for input RT cpm; the level of WT HIV-1 infection in control cells was set to 100% (left panel). Right panel, re-plot to highlight levels of WT and D64N/D116N IN mutant restriction by MxB. (B) Upper panel, WT and D64N/D116N (NN) late reverse transcription (LRT) products in control and MxB-expressing cells. WT and NN curves are black and red, respectively. Dashed lines, values from MxB-expressing cells. Lower panel, integration as assessed by Alu-R qPCR. (C) Levels of 2-LTR circles using conventional qPCR conditions. (D and E) Levels of 2-LTR circles using Jxn2 and Jxn1 qPCR conditions, respectively. The 4-member graphs to the left are split into two panels on the right in panels C-E to highlight the responses of WT (black lines) and NN mutant (red lines) viruses to MxB restriction (dashed lines). Results are an average of 2 independent experiments, with error bars denoting standard deviation (downward bars omitted to ease interpretation of coincident time points).
Figure 5
Figure 5
PIC activity and integration site sequencing strategy. (A) PICs extracted from the cytoplasm (left panel) or nucleus (center panel) of mock-transduced (dark grey bars) or MxB-expressing cells (light grey bars) were assessed for in vitro integration activity. Percent infectivity, determined 48 h after infection (right panel), revealed the level of MxB restriction under these infection conditions. Results are the average of 4 independent experiments, with error bars denoting standard error of the mean. (B) Integration site sequencing strategy. In the representative HIV-1 provirus the viral DNA internal to the LTRs is a single bold line and the abutting cellular DNA is two thin lines (the region to be sequenced is in red). Thin blue lines, asymmetric DNA linker. The bold extensions of PCR primers denote elements required for Illumina sequencing. HIV-1 DNA harbors numerous MseI sites; only the relevant site downstream from the upstream U5 sequence is shown.
Figure 6
Figure 6
Integration patterns in control versus MxB-expressing cells. (A) Number of integration sites counted in 1.25 kb bins (x-axis ticks) were plotted as percent of total from 30 kb upstream (negative x-axis value) to 30 kb downstream of TSSs. Blue and red lines, data from control and MxB-expressing HOS cells, respectively; The green plot, MRC values. The sites ~4 kb to 12 kb upstream of TSSs with significantly greater levels of integration than random in WT HOS cells in the vast majority of cases also mapped within RefSeq genes, which was attributed to the presence of internal promoters in the human genome [44]. (B) Frequency of HIV-1 integration sites surrounding CpG islands. Line colorings are same as in panel A. See Additional file 3: Figure S3 for statistical analysis of panel A and B results. (C) Percent integration sites (y-axis) plotted against number of genes per Mb (x-axis) for infections conducted using control (blue line) and MxB-expressing (red line) HOS cells. Wilcox Rank-Sum test analysis of gene density targeting values (Table 1) yielded P values < 2.2 × 10−302 for all comparisons (WT versus MRC, MxB versus MRC, and WT versus MxB). Fisher’s exact test was therefore conducted on the subset of integration sites that fell between 8 and 19 genes per Mb (bracket). Herein, WT and MxB-expressing cells harbored 34.8% and 35.4% of all integrations, respectively; the MRC value was 30.2%. The WT versus MRC comparison yielded P <2.2 × 10−308. MxB-expressing cells versus MRC yielded P =2.8 × 10−124 whereas P =0.01 was determined for control versus MxB-expressing cells. (D) Same as in panel C, except the following data from Ocwieja et al. [45] was analyzed: control siGL2, 7,140 integration sites; TNPO3 si4, 3,923 sites; RANBP2 si6, 2,114 sites. TNPO3, transportin 3. The panel D graph was smoothed using kernal estimation [45] due to relatively fewer numbers of integration sites.
Figure 7
Figure 7
Activities of MxB GTPase active site mutant proteins. (A) X-ray crystal structure of dynamin active site with bound phosphomethylphosphonic acid guanylate ester (GCP) (pdb code 3zyc). Dynamin side chain contacts with GCP (cyan backbone) are labeled, with the corresponding conserved MxB residue labeled in parenthesis. Dotted black lines denote hydrogen bonds. Remaining colors: red, oxygen atoms; blue, nitrogen; orange, phosphorus. (B) (top) WT or MxB mutant protein activities against HIV-1 (dark grey), EIAV (light grey) or FIV infection (striped). Results are an average of at least 6 independent experiments, with error bars denoting 95% confidence intervals. (bottom) Representative western blots from at least 3 independent experiments. (C) Immunofluorescent microscopy of HOS cells expressing WT MxB or the indicated mutant protein. Each bar represents a distance of 10 μm.
Figure 8
Figure 8
MxB stalk mutants and antiviral activity. (A) The MxB protein schematic highlights regions of sequence diversity from the human MxA protein; each red dot denotes an MxB position where 5 consecutive residues differed from MxA. (B) Antiviral activities of MxB stalk mutant proteins. Results are an average of at least 5 independent experiments, with error bars denoting 95% confidence intervals. (C) Representative immunofluorescent microscopy results; white bar, 10 μm. See Additional file 4: Figure S4 for results of quantitative western blotting.
Figure 9
Figure 9
Functional heterologous NLS and NES sequences. (A) Schematic of MxB nuclear localization mutants. The location of heterologous NLS and NES sequences are denoted in blue and red, respectively; the common C-terminal HA tag is in green. Western blotting (B), antiviral activity (C), and immunofluorescent microscopy (D) of WT MxB and mutant proteins. Results in panel C represent the geometric mean of at least 8 independent experiments, with error bars denoting 95% confidence intervals. The differences in HIV-1 restriction by MxB Δ1-25 or MxB + NES with WT MxB was highly significant (P <0.0001). The horizontal bars in panel D represent the distance of 10 μm.
Figure 10
Figure 10
Activity and localization of N-terminal deletion mutants. Schematic (A), western blotting (B), antiviral activity (C), and immunofluorescence microscopy (D) of N-terminal deletion mutants. Panel C results represent the geometric mean of at least 6 independent experiments, with error bars denoting 95% confidence intervals. All mutant proteins restricted HIV-1 infection to significantly lower levels than WT MxB (P <0.002). The bars in panel D represent 10 μm.

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