The Protease Domain in HEV pORF1 Mediates the Replicase’s Localization to Multivesicular Bodies and Its Exosomal Release

Figures

• 

  Figure 1. HEV pORF1 predominantly localizes to CD63-positive vesicular structures. Analyses of subcellular distribution of pORF1 ectopically expressed in A549/D3 cells. (A) Confocal microscopy of double-tagged pORF1; EGFP-mCherry Merge displays the subcellular distribution of EGFP (green)-mCherry (red) fusion protein without insertion of pORF1. C-term, C-terminal mCherry; N-term, N-terminal EGFP. (B) Particle analysis of only C-terminus–containing vesicles (C-term) and N- and C-terminus-containing vesicles (C+N-terminus). ∗∗∗P > .0001; ∼300 particles were analyzed. (C) Confocal microscopy of C-terminally mScarlet-tagged pORF1 (red) and indirect immunofluorescent stain of different organelle marker proteins (green); ERGIC53, ERGIC; GM130, Golgi; EEA1, EEs; Rab11a, REs; LC3B, APs; LAMP2, LYs; CD63, MVBs. (D) Particle analysis of pORF1-containing vesicles (pORF1 alone) and pORF1- and marker protein–containing vesicles (pORF1 + marker); between ∼200 and ∼1000 particles were analyzed for each organelle marker. Single channels depicted as greyscale images with headers indicating the channel coloring; zoom refers to areas magnified from white rectangles in merge; white ellipses indicate nucleus;…

• 

  Figure 2. Generation and characterization of eukaryotic expression constructs encoding different pORF1 domains. (A) Cartoon and surface view of AlphaFold2-based prediction of three-dimensional structure of pORF1 from genotype 3c, isolate 47832c; different coloring represents different domains as stated in the panel; regions colored in magenta represent hinge-like regions between the domains. (B) Representative Western blot of Hek293T cells transfected with the generated constructs (Supplementary Table 1) encoding different pORF1 domains harvested 48 hours after transfection, pORF1 or pORF1ΔPCP as mScarlet fusion protein; scheme below Western blot displays expressed protein from N- to C-terminus including N-terminal StrepTagII. L, ladder; Mock, empty vector transfected cells; tub, detection of tubulin as loading control. Red arrows mark bands reflecting theoretical MW of full-length proteins; numbers in superscript reflect amino acid positions cloned for the respective domain. (C) Representative Western blot of Hek293T cells transfected with StrepII-PCP-mScarlet construct subjected to deglycosylation using PNGase F. neg, cell lysate from mock-transfected cells; pos, cell lysate from…

• 

  Figure 3. pORF1 can be split into different domains, and PCP depicts potential cleavage pattern. (A) Predicted aligned error mapping of the prediction in Figure 2A; green squares indicate certain likelihood of domain formation. (B) Side-by-side comparison of representative Western blots of immunoprecipitation against the N-terminal StrepTagII probed against terminal tags; mScarlet served as StrepTagII negative control. neg, cell lysate from mock-transfected cells; pos, cell lysate from transfections of X-domain in (B). Asterisks mark selected lanes from full Western blot on left side; red lines and numbers represent PCP bands containing the N-terminus; blue lines and numbers represent PCP bands containing only the C-terminus; 1 = higher than expected MW; 2 = expected MW; 3,6 = N-terminal cleavage products; 4,5 = C-terminal cleavage products. Data representative of 3 independent experiments.

• 

  Figure 4. PCP domain in HEV pORF1 localizes to CD63-positive vesicular structures. Analyses of subcellular distribution of all domains contained in pORF1 ectopically expressed in A549/D3 cells. Confocal microscopy of C-terminally mScarlet-tagged pORF1-domains (red) and indirect immunofluorescent stain of CD63 as MVB marker protein (green). Mock refers to empty vector–transfected cells; single channels depicted as greyscale images with headers indicating the channel coloring; zoom refers to areas magnified from white rectangles in merge; white ellipses indicate nucleus; scale bar represents 40 μm. Differences in expression levels between proteins (compare with Figure 2D) were compensated for by thresholding and adjusting red signal intensity. Microscopy performed on Leica TCS SP8 System with 100× objective (numerical arperture 1.4). Images deconvoluted via lightning algorithm using LAS X Control software.

• 

  Figure 5. Deletion of PCP domain in HEV pORF1 leads to loss of localization to CD63-positive vesicular structures. Analyses of subcellular distribution of pORF1, the PCP domain or pORF1 lacking the PCP domain ectopically expressed in A549/D3 cells. (A) Confocal microscopy of C-terminally mScarlet-tagged pORF1 domains/variants (red) and indirect immunofluorescent stain of CD63 as MVB marker protein (green); mScarlet refers to cells transfected with mScarlet-coding vector; single channels depicted as greyscale images with headers indicating the channel coloring; zoom refers to areas magnified from white rectangles in merge; white ellipses indicate nucleus. Scale bar represents 40 μm. Differences in expression levels between proteins (compare with Figure 2D) were compensated for by thresholding and adjusting red signal intensity. Images for pORF1 and PCP were retrieved from Figures 1C and 3 for the sake of visualization alongside pORF1ΔPCP. (B) Three-dimensional reconstruction of z-stacks generated from confocal microscopy in (A); first column displays surface reconstruction; second column displays images from first column clipped in z-pane; third column displays thresholded and processed…

• 

  Figure 6. Both pORF1 and PCP accumulate in exosomes, and genomic RNA is released via exosomes in absence of pORF2/3. (A, C, and E) Normalized mScarlet fluorescence in EVs harvested 72 hours after transfection; ratios were calculated by dividing EV fluorescence by fluorescence in lysates. (B, D, and F) Comparison between fluorescent ratios of pORF1 and pORF1ΔPCP as in (A, C, and E); pORF1 was set as reference, thus displaying 100%. ∗P < .05, ∗∗P < .01, and ∗∗∗∗P < .0001. (G) Relative amount of mScarlet fluorescence in EVs from (A) subjected to isopycnic density gradient centrifugation; ratios were calculated as % of summarized fluorescence in (A). (H) Representative Western blots from samples generated in (A) and (C) probed with antibodies reacting with mScarlet or Alix. L, ladder; neg, cell lysate of untransfected A549/D3 cells; mSC, mScarlet; SN, cell culture supernatant. Red arrows mark pORF1- or pORF1ΔPCP -specific bands and serve purpose to distinguish these from Alix-specific bands visible in these blots. Blue arrows indicate exosomal PCP and pORF1. (I) Ratio of genomic HEV RNA in cell lysates versus in EV pellets originating from A549/D3 cells stably electroporated with the…

• 

  Figure 7. Hypothetical model integrating our findings in the context of the HEV life cycle. (A) HEV pORF1 is recruited to MVBs in PCP-dependent manner. (B) Genomic replication of HEV takes place at surface of MVBs, which is mediated via the pORF1 pool facing the cytosol. (C) pORF1 along with viral RNA is incorporated into ILVs of MVBs and forms the intralumenal pool of pORF1. (D) Viral proteins pORF1-3 are translated. (E) RNA-loaded virions are incorporated into ILVs of MVBs as well. (F) Both pORF1 and virions are released via exosomes. This leads to release of exosomes representing classic eHEV, pORF1-loaded exosomes, and pORF1- and capsid-loaded exosomes. In target cells, these facilitate initial genomic replication, because pORF1 is readily available without a first step of translation. In addition, exosomes loaded with genomic RNA being in complex with pORF1 mediate a capsid-independent spread. eHEV, quasi-enveloped HEV.