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A Role for the Host Coatomer and KDEL Receptor in Early Vaccinia Biogenesis

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A Role for the Host Coatomer and KDEL Receptor in Early Vaccinia Biogenesis A role for the host coatomer and KDEL receptor in early vaccinia biogenesis Leiliang Zhanga,1, Stella Y. Leea,1, Galina V. Beznoussenkob, Peter J. Petersc, Jia-Shu Yanga, Hui-ya Gilberta, Abraham L. Brassd,e, Stephen J. Elledged, Stuart N. Isaacsf, Bernard Mossg, Alexander Mironovb, and Victor W. Hsua,2 aDivision of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115; bDepartment of Cell Biology and Oncology, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (Chieti), Italy; cDivision of Cell Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands; dDepartment of Genetics, Center for Genetics and Genomics, Brigham and Women’s Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115; eGastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114; fDepartment of Medicine, Division of Infectious Diseases, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; and gLaboratory of Viral Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 Communicated by Michael B. Brenner, Harvard Medical School, Boston, MA, November 20, 2008 (received for review August 14, 2008) Members of the poxvirus family have been investigated for their coatomer (4), was on the virus (data not shown). Because this applications as vaccines and expression vectors and, more recently, initial detection occurred in the context of viral-mediated over- because of concern for their potential as biological weapons. expression of a host protein, we first sought to ascertain the Vaccinia virus, the prototypic member, evolves through multiple presence of coatomer on wild-type virus that did not encode for forms during its replication. Here, we show a surprising way by any foreign protein. By ImmunoGold EM, we detected ␤-COP which vaccinia hijacks coatomer for early viral biogenesis. Whereas on intracellular infectious forms of the WR strain, MV and WV coatomer forms COPI vesicles in the host early secretory system, (Fig. 1A). Subsequently, EM morphometry revealed that ␤-COP vaccinia formation bypasses this role of coatomer, but instead, on intracellular viruses was enriched by Ϸ4-fold over that on the depends on coatomer interacting with the host KDEL receptor. To host Golgi, where coatomer is normally concentrated (Table 1). gain insight into the viral roles of these two host proteins, we have Thus, because a subunit of coatomer was being specifically detected them on the earliest recognized viral forms. These find- enriched on the virus, we next sought to determine functionally ings not only suggest insights into early vaccinia biogenesis but whether it had a role in vaccinia replication. also reveal an alternate mechanism by which coatomer acts. An initial key hurdle was that coatomer had been shown to be COPI ͉ morphogenesis essential for eukaryotic cell viability from mammals to yeast (5, 6). Thus, we first sought to define a condition of partially perturbing coatomer that would allow us to detect a potential accinia virus initiates replication in discrete regions of the role for coatomer in vaccinia replication and yet not result in Vhost cytoplasm, known as viral factories. Here, viral DNA global cellular dysfunction. When HeLa cells were treated with and proteins first assemble into a core particle, which then siRNA against ␤-COP for 48 h, we observed partial reduction in undergoes membrane wrapping to become an immature virus endogenous ␤-COP (Fig. 1B). When cells were subsequently (IV). The IV then undergoes extensive membrane transforma- infected with WR virus for 24 h in this condition, we found that tion to become a mature virus (MV), which then acquires Ϸ additional layers of membrane from the host trans-Golgi net- viral replication was reduced by 4-fold (Fig. 1B). work to become a wrapped virion (WV). Upon transport to the To show that this reduction upon the partial crippling of host periphery, the WV fuses with the plasma membrane to coatomer was not caused by generalized cellular dysfunction, we become extracellular viruses. Events that lead to MV formation assessed two distinct parameters. With respect to a host process, have been intensely investigated, because the MV is the first, most we examined clathrin-mediated endocytosis, which did not in- CELL BIOLOGY abundant infectious form generated. MV formation also entails a volve coatomer (4). Tracking the internalization of transferrin complex process of membrane morphogenesis, which has intrigued (Tf) receptor (TfR) through fluorescently-labeled Tf bound to not only virologists but cell biologists over the years (1–3). surface TfR, we found that TfR internalization was not signif- As viruses often hijack host factors, an intriguing possibility is icantly affected (Fig. 1C). We also examined the replication of that host coat proteins, which are well known to act in membrane HIV and found that this viral process was not significantly morphogenesis to form intracellular transport vesicles from host affected (Fig. 1D). Thus, we concluded that we had achieved a compartmental membrane (4), participate in early viral biogen- condition of partially perturbing coatomer without leading to esis. However, none has been identified thus far. Yet, another generalized cellular dysfunction. issue relates to the origin of the earliest viral membrane, which We next considered that the standard vaccinia replication is needed for the acquisition of viral infectivity. A compelling assay measured both viral formation and viral infectivity (7). way to demonstrate a host origin for this viral membrane would Thus, we sought to assess viral formation more directly by be the detection of host integral membrane proteins on early purifying intracellular viruses from infected cells, and then viral forms, but none has been detected thus far. quantifying for intact viruses by EM examination. Partial silenc- We became interested in these key issues of vaccinia biogen- esis through an initial serendipitous detection of coatomer on vaccinia virus. Pursuing this finding, we find that coatomer is Author contributions: L.Z., S.Y.L., A.L.B., S.J.E., S.N.I., B.M., A.M., and V.W.H. designed important for early viral formation, and this role is linked to research; L.Z., S.Y.L., G.V.B., P.J.P., J.-S.Y., H.-y.G., A.L.B., and A.M. performed research; L.Z., another host protein, the KDEL receptor (KDELR). Further S.Y.L., G.V.B., P.J.P., J.-S.Y., H.-y.G., A.L.B., S.J.E., S.N.I., B.M., A.M., and V.W.H. analyzed elucidation of how these two proteins play interlinking roles in data; and L.Z., S.Y.L., S.N.I., B.M., and V.W.H. wrote the paper. vaccinia formation suggests insights into viral biogenesis and The authors declare no conflict of interest. how coatomer can be regulated. 1L.Z. and S.Y.L. contributed equally to this work. 2To whom correspondence should be addressed. E-mail: [email protected]. Results This article contains supporting information online at www.pnas.org/cgi/content/full/ In the course of using vaccinia virus as an expression vector, we 0811631106/DCSupplemental. observed by ImmunoGold EM that ␤-COP, a subunit of © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0811631106 PNAS ͉ January 6, 2009 ͉ vol. 106 ͉ no. 1 ͉ 163–168 Downloaded by guest on September 29, 2021 Fig. 1. Coatomer acts in vaccinia formation. (A) HeLa cells were infected with the WR strain of vaccinia virus for 16 h and then examined by EM with ImmunoGold labeling for ␤-COP. (Scale bar: 50 nm.) (B) HeLa cells, either treated with siRNA against ␤-COP for 48 h or mock-treated, were then infected with WR virus for 24 h. The vaccinia replication assay was then performed, with the mean and standard error from 3 experiments shown (P Ͻ 0.01). Gel shows the level of ␤-COP comparing siRNA-treated versus mock-treated HeLa cells. (C) The surface pool of TfR on HeLa cells, either treated with siRNA or mock-treated, was labeled with fluorescently tagged Tf, and then tracked for internalization into early endosomes. (Scale bar: 10 ␮m.) (D) TZM-bl cells were transfected with siRNAs against different targets, ␤-COP, CXCR4, Tat, or Luciferase as control, or mock-treated. After infection with HIV-IIIB, ␤-galactosidase activity was measured, which was expressed as relative light units (RLU), and then normalized to the condition of mock treatment. The mean with standard deviation from 3 experiments is shown. (E) HeLa cells, either treated with siRNA against ␤-COP for 48 h or mock-treated, were then infected with WR virus for 24 h. Intracellular viral forms were then purified from infected cells, followed by EM examination for intact viruses. Quantitation was performed from 10 randomly selected images, and then expressed as mean with standard error (P Ͻ 0.01). ing of ␤-COP resulted in a Ϸ5-fold reduction in intracellular As the results thus far allowed us to conclude that coatomer viruses detected (Fig. 1E). participated in vaccinia formation, we next sought to gain further As ␤-COP is a subunit of coatomer (4), a prediction was that insight into its role. In this respect, whereas ARF1 is a key coatomer as an entire complex participated in viral formation. regulator of coatomer for host COPI vesicle formation (9), an To confirm this idea, we also examined a mutant CHO cell line earlier vaccinia study had shown that MV formation was not that had been characterized to express a temperature-sensitive affected by treating cells with brefeldin A (BFA) (10), which form of ␧-COP, which became misfolded at the nonpermissive would have perturbed ARF1-regulated COPI transport (9). temperature, resulting in the degradation of the entire coatomer Thus, these observations suggested the possibility that viral complex (6).
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