key: cord-0290553-l33p5hm1 authors: Momtaz, Samina; Molina, Belen; Mlera, Luwanika; Goodrum, Felicia; Wilson, Jean M. title: Cell type-specific biogenesis of novel vesicles containing viral products in human cytomegalovirus infection date: 2020-12-11 journal: bioRxiv DOI: 10.1101/2020.12.10.420711 sha: d67bbfe1c5a6b3fdad1f85ea33dca9eb29ea3921 doc_id: 290553 cord_uid: l33p5hm1 Human cytomegalovirus (HCMV), while highly restricted for the human species, infects an unlimited array of cell types in the host. Patterns of infection are dictated by the cell type infected, but cell type-specific factors and how they impact tropism for specific cell types is poorly understood. Previous studies in primary endothelial cells showed that HCMV infection induces large multivesicular-like bodies that incorporate viral products including dense bodies and virions. Here we define the nature of these large vesicles using a recombinant virus where UL32, encoding the pp150 tegument protein, is fused in frame with green fluorescent protein (GFP, TB40/E-UL32-GFP). Cells were fixed and labeled with antibodies against subcellular compartment markers and imaged using confocal and super-resolution microscopy. In fibroblasts, UL32-GFP-positive vesicles were marked with classical markers of MVBs, including CD63 and lysobisphosphatidic acid (LBPA), both classical MVB markers, as well as the clathrin and LAMP1. Unexpectedly, UL32-GFP-positive vesicles in endothelial cells were not labeled by CD63, and LBPA was completely lost from infected cells. We defined these UL32-positive vesicles in endothelial cells using markers for the cis-Golgi (GM130), lysosome (LAMP1), and autophagy (LC3B). These findings suggest that virus-containing MVBs in fibroblasts are derived from the canonical endocytic pathway and takeover classical exosomal release pathway. Virus containing MVBs in HMVECs are derived from the early biosynthetic pathway and exploit a less characterized early Golgi-LAMP1-associated non-canonical secretory autophagy pathway. These results reveal striking cell-type specific membrane trafficking differences in host pathways that are exploited by HCMV. Importance Human cytomegalovirus (HCMV) is a herpesvirus that, like all herpesvirus, that establishes a life long infection. HCMV remains a significant cause of morbidity and mortality in the immunocompromised and HCMV seropositivity is associated with increased risk vascular disease. HCMV infects many cells in the human and the biology underlying the different patterns of infection in different cell types is poorly understood. Endothelial cells are important target of infection that contribute to hematogenous spread of the virus to tissues. Here we define striking differences in the biogenesis of large vesicles that incorporate virions in fibroblasts and endothelial cells. In fibroblasts, HCMV is incorporated into canonical MVBs derived from an endocytic pathway, whereas HCMV matures through vesicles derived from the biosynthetic pathway in endothelial cells. This work defines basic biological differences between these cell types that may impact the outcome of infection. were chosen such that infection would be at a similar same stage in each cell type; 209 early in the late phase of infection where the viral assembly compartment (VAC) was 210 apparent, but prior to severe cytopathic effect. 211 To define the composition of MVBs that incorporate viral cargo, we labeled cells with 212 the classic MVB markers CD63 and LBPA. CD63 is a Tetraspanin-group protein that is 213 present in MVBs or late endosomes (LEs) and at the cell surface 33 . Based on these results, we next tested if the UL32-GFP-positive vesicles in 280 HMVECs could be derived from the biosynthetic pathway. We labeled infected HMVECs 281 with the trans-Golgi marker, p230. A tubular network of p230 was observed in the 282 perinuclear region of uninfected HMVECs, but this TGN marker did not colocalize with 283 UL32-GFP-positive vesicles (Fig. 4E) , suggesting that the UL32-GFP-positive vesicles 284 do not derive from the trans-Golgi network. Next, we tested the small GTPase, Rab6, which regulates retrograde transport from 286 the endosomal compartment via the trans-Golgi to the endoplasmic reticulum 48 . A 287 previous study found that Rab6 recruits UL32 to the viral assembly compartment by 288 binding to dynein, a microtubule motor protein 49 . Rab6 was scattered through the 289 cytoplasm in uninfected cells and did not colocalize with UL32-GFP vesicles in infected 290 HMVECs (Fig. 4F ). This data suggests that endosomal and trans-Golgi membrane 291 traffic is not involved in the biogenesis of these vesicles in HMVECs. 292 A previous study showed that HCMV assembly compartment formation alters the 293 recycling endosomal Rab cascade marked by the small GTPase Rab11 50 . Rab11 was 294 distributed as small punctate structures in the cytoplasm of uninfected cells and did not 295 colocalize with UL32-GFP-positive vesicles in infected HMVECs (Fig. 4G ). The small 296 GTPase, Rab14, which is involved in the biosynthetic trafficking between Golgi and 297 endosomes and the plasma membrane 51 , also did not colocalize with UL32-GFP-298 positive vesicles in HMVECs (Fig. 4H ). These data suggest that UL32-positive vesicles 299 are not derived from endosomal recycling compartments. 300 Due to our observation of the presence of clathrin on the membrane of UL32-GFP-301 positive vesicles in HMVECs, we next asked if UL32-GFP positive vesicles contained 302 clathrin-associated adaptor proteins: AP-2, AP-1, or AP-3. AP-2 binds to the 303 phosphatidylinositol 2-phosphate (PIP2) in the plasma membrane and the cargo in 304 clathrin-mediated endocytosis 35 . AP-2 labeled small puncta in uninfected HMVECs, and 305 this distribution did not change with infection in infected HMVECs (Fig. 4I) . However, 306 AP-2 was somewhat less intense and more diffuse, possibly due to infection-induced 307 increase in cell size. AP-2 did not colocalize with UL32-GFP vesicles (Fig. 4I) . AP-1 recruits clathrin to the TGN and contributes to the biogenesis of vesicles from the TGN 309 52 . Uninfected HMVECs showed the small punctate structure of AP-1 in the perinuclear 310 region (Fig. 4J) , similar in appearance to p230 (see Fig. 4E ). AP-1, like AP-2, also did 311 not colocalize with UL32-GFP vesicles (Fig. 4J ). AP-3, mediates the transport from TGN 312 to LE or lysosome/Lysosome Related Organelles 53 . As with all other adaptor proteins, 313 AP-3 also did not colocalize with UL32-GFP-positive vesicles in infected HMVECs (Fig. 314 4K ). Together, these findings demonstrate a striking lack of colocalization of UL32-GFP 315 with common endosomal and biosynthetic markers and support the idea that these 316 vesicles in HMVECs originate from a non-classical membrane trafficking pathway. HMVECs showed elongated vesicular labeling of LAMP1 throughout the cytoplasm (Fig. 326 5A). In infected HMVECs, LAMP1 labeling was not substantially altered. However, the 327 UL32-GFP containing vesicles colocalized with LAMP1 (Fig. 5A) . HMVECs could be related to these structures, we next examined if the UL32-GFP 341 vesicles contained GM130. In infected HMVECs, GM130 labeling was detected as a 342 well-defined and characteristic ring-structure around the VAC (Fig. 5C) . Further, GM130 343 colocalizes with the UL32-GFP vesicles in HMVECs (Fig. 5C ). These finding indicate 344 that the UL32-GFP vesicles may derive from an early biosynthetic, Golgi-mediated 345 pathway. 346 347 UL32-GFP containing vesicles are marked by LAMP1, but not LC3B and GM130 in 348 fibroblasts. We next sought to analyze the association of LAMP1, LC3B, and GM130 349 with UL32-GFP-positive vesicles in infected fibroblasts. LAMP1 labelling in uninfected 350 fibroblasts appeared as elongated vesicular structures in the cytoplasm (Fig. 6A) . 351 Infected fibroblasts demonstrated a scattered and fine granule labelling of LAMP1 352 around the VAC, consistent with previous observations (Fig. 6A ) 43 . However, UL32-353 GFP containing vesicles also colocalize with LAMP1 (Fig. 6A ). LAMP1 has been reported to localize in the late endosomes (LEs), apart from their primary localization at 355 the lysosomal compartments 58-60 . This finding indicates that colocalization of UL32-356 GFP-positive vesicles with LAMP1 in infected fibroblasts may derive from either LEs or 357 from the lysosomes. 358 LC3B was present on vesicle structures throughout the cytoplasm in uninfected 359 fibroblasts (Fig. 6B) . However, LC3B localized predominantly to the VAC and no 360 colocalization was detected with UL32-GFP vesicles in infected fibroblasts (Fig. 6B) . In 361 uninfected fibroblasts, GM130 localized in a tubular, perinuclear region typical of the cis-362 medial Golgi (Fig. 6C) . In infected fibroblasts, GM130 was localized to the classic ring-363 like structure of the VAC and showed no colocalization with UL32-GFP vesicles (Fig. 364 6C) . 365 We quantified the colocalization of LAMP1, LC3B and GM130 with UL32-GFP in 366 both HMVECs and fibroblasts (Fig. 7) . In HMVEC and fibroblasts, 92% and 99% of the 367 UL32-GFP vesicles colocalized with LAMP1, respectively (Fig. 7A) . However, LC3B and 368 GM130 showed strong discordance between HMVECs and fibroblasts; 87 % of UL32-369 GFP-positive vesicles colocalized with LC3B in infected HMVECs, compared to none in 370 infected fibroblasts (Fig. 7B) . Similarly, 88% of UL32-GFP vesicles localized with 371 GM130 in HMVECs, compared to none in infected fibroblasts (Fig. 7C) . The absence of 372 classic MVB markers on UL32-GFP vesicles in HCMV-infected HMVECs coupled with 373 the presence of LC3B and GM130 suggest that these vesicles are derived from a 374 distinct pathway from that associated with infection in fibroblasts. indicating their biogenesis from a pathway distinct from endocytic trafficking pathways. 407 The loss of LBPA in the infected HMVECs also implies the generation of altered ILVs or 408 altered lipid metabolism. This may be due to viral interference with LBPA synthesizing 409 enzymes or their precursors, e.g. phosphatidyl glycerol 68 . Altered ILV generation is 410 supported by the lack of colocalization of these vesicles with the exosomal marker ALIX. 411 A previous report showed ALIX-independent ILVs with altered biogenesis and shape, Based on our findings, we propose a model in which HCMV controls maturation and 447 egress from distinct host pathways in infected HMVECs and fibroblasts (Fig. 8) Human Cytomegalovirus (HCMV) -Revised Review of cytomegalovirus seroprevalence and 471 demographic characteristics associated with infection Severe Symptomatic Primary Human Cytomegalovirus Infection despite Effective 473 Innate and Adaptive Immune Responses Overview of human cytomegalovirus 475 pathogenesis Maternal Immunity and the Natural History of Congenital Human Cytomegalovirus 477 Infection Advancing Our Understanding of Protective Maternal 479 Immunity as a Guide for Development of Vaccines To Reduce Congenital Cytomegalovirus 480 Infections Aging, sex, inflammation, frailty, and CMV and HIV infections Chronic Low-grade Inflammatory Phenotype (CLIP) and Senescent 486 Immune Dysregulation New advances in CMV and immunosenescence Known unknowns: how might the 490 persistent herpesvirome shape immunity and aging? 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Proceedings of the National Academy of Sciences of the United States of 618 America The enigmatic endosome -sorting the ins and outs of endocytic 620 trafficking Herpes simplex virus type 1 production requires a functional ESCRT-622 III complex but is independent of TSG101 and ALIX expression Host factors involved in retroviral budding and release Microbiol 9 Lysobisphosphatidic acid controls endosomal cholesterol levels Late endosomal cholesterol accumulation leads to impaired intra-endosomal 628 trafficking Autophagy, EVs, and Infections: A Perfect Question for a Perfect Time The Autophagic Machinery in Viral Exocytosis Enterovirus Transmission by Secretory Autophagy The Role of Secretory Autophagy in Zika Virus Transfer through 635 the Placental Barrier Nonlytic viral spread enhanced by 637 autophagy components Diverting autophagic membranes for exocytosis Phosphatidylserine vesicles enable efficient en bloc transmission of 641 enteroviruses The LC3-conjugation machinery specifies the loading of RNA-binding proteins 643 into extracellular vesicles β-Coronaviruses Use Lysosomes for Egress Instead of the Biosynthetic Secretory 645 Pathway Human herpesvirus-6 induces MVB formation, and virus egress occurs by an 647 exosomal release pathway CD63 and LBPA show differential association with UL32-GFP-positive 663 vesicles in HMVECs and fibroblasts HMVECs (MOI, 4) or fibroblasts (MOI,1) were fixed at 96 and 48 hpi, respectively Cells were labelled with mouse anti-CD63 (red) and imaged by confocal microscopy B) Quantification of the percentage of vesicles that contain the CD63 669 that are also positive for UL32-GFP, ** p< 0.01. (C) Uninfected and infected cells were 670 labeled with anti-LBPA (red) and imaged by confocal microscopy. Infection results in 671 loss of LBPA from infected HMVECs, and the infected HMVEC is outlined due to lack of 672 LBPA staining. UL32-GFP positive vesicles are indicated by the arrowhead (inset Quantification of the percentage of vesicles that contain the LBPA that are also positive 675 for UL32-GFP, **p< 0.01. For each quantification, 700 to 900 vesicles were counted for 676 each marker. E. UL32-GFP accumulates in the lumen of the large vesicles in both 677 HMVEC and fibroblasts (arrows) Clathrin heavy chain colocalizes with UL32-GFP-positive vesicles in In uninfected cells, clathrin is 684 distributed in diffuse puncta throughout the cell. There is substantial colocalization of 685 clathrin heavy chain and UL-32-GFP vesicles (arrows, insets) in both HMVECs and 686 fibroblasts Quantification of clathrin-positive UL32-GFP-positive vesicles do not contain typical endocytic and 691 biosynthetic trafficking markers in HMVECs Cells were labelled with (A) anti-EEA1 (early endosomes), (B) anti-Rab5 (early endosomes), (C) anti-ALIX (late endosomes), (D) anti-Rab7 (late 694 endosomes, (E) anti-p230 (trans-Golgi), (F) anti-Rab6 (trans-Golgi), (G) anti-Rab11 695 (recycling endosomes), (H) anti-Rab14 (endosomes), (I) anti-AP trans-Golgi) antibodies and secondary 697 antibodies conjugated to Alexa-fluor 647. The UL-32-GFP-positive vesicles (arrows) do 698 not colocalize with any of these markers Fig. 5. LAMP1, GM130, and LC3B localize to UL32-GFP-positive vesicles in Uninfected or TB40/E-UL32-GFP-infected HMVECs were labelled with (A) 703 anti-LAMP1 (late endosomes-lysosomes), (B) anti-LC3B (autophagosomes), and (C) Nuclei, blue. In uninfected cells, LAMP1 and LC3B are distributed throughout 706 the cytoplasm. GM130 is localized to the perinuclear region LC3B, and GM130 all colocalize on UL32-GFP-positive vesicles (arrows). Scale bars Uninfected or TB40/E-UL32-GFP-infected fibroblasts were 713 labelled with (A) anti-LAMP1 (late endosomes-lysosomes), (B) anti-LC3B 714 (autophagosomes), (C) anti-GM130 (cis-Golgi) antibodies and secondary antibodies 715 conjugated to Alexa-fluor 647 (red). Nuclei, blue. In uninfected cells, LAMP1 and LC3B 716 are distributed in the cytoplasm. GM130 localizes to the perinuclear region Quantification of LAMP1, LC3B, and GM130 positive UL32-GFP vesicles LC3B (B), and GM130 (C) positive UL32-GFP vesicles 723 on both cell types were illustrated. Two hundred to four hundred vesicles were counted 724 for each marker