___________________________________________________________________________ UTRECHT UNIVERSITY, FACULTY OF VETERINARY MEDICINE DEPARTMENT OF INFECTIOUS DISEASES AND IMMUNOLOGY The role of host-cellular molecules in the coronavirus lifecycle K.G. de Boer Student nr.: 0424374 Master student `Immunity and Infection’ Utrecht University 30 May 2009 Supervisor: Dr. M.H. Verheije ___________________________________________________________________________ ___________________________________________________________________________ The role of host-cellular molecules in the coronavirus lifecycle K.G. de Boer Abstract Coronaviruses are enveloped positive-stranded RNA viruses, which received worldwide attention in 2003 when a coronavirus appeared to be the etiological agent of the severe acute respiratory syndrome (SARS) epidemic in Asia. Such epidemic outbreaks have a great influence on the global population in both a social and an economic perspective. Therefore, development of new intervention strategies in order to reduce the change of a new epidemic outbreak is important. Identification of host-cellular molecules involved in the coronaviral lifecycle could contribute to the development of new therapeutics since host cells are required for coronaviral replication and survival. In this thesis, host-cellular molecules recently found to be involved in the coronaviral lifecycle, are described. ___________________________________________________________________________ 1 ___________________________________________________________________________ Table of Contents Abstract ..................................................................................................................................... 1 Introduction.............................................................................................................................. 3 Entry .......................................................................................................................................... 7 Replication and transcription .............................................................................................. 13 Assembly and budding ........................................................................................................ 17 Maturation and Release ........................................................................................................ 21 Manipulation of the host-cell machinery ........................................................................... 21 Discussion ............................................................................................................................... 22 ___________________________________________________________________________ 2 ___________________________________________________________________________ Introduction Viruses are infectious agents, which need living cells for their reproduction. They contain either RNA or DNA, protected by a protein coat. Although these characteristics are common for all viruses, size, morphology, and complexity are highly variable among different species. For example, some viruses have a lipid envelope in addition to the protein coat, which serves for protection and carry membrane proteins, which are involved in multiple processes of the viral lifecycle. Coronaviruses are enveloped positive-stranded RNA viruses. They belong to the family of Coronaviridea, which is part of the order of Nidovirales. Coronaviruses are currently divided in 3 groups, based on serological and genetic criteria (see table 1). Group Virus Host Associated disease 1 Feline coronavirus (FCoV) Cat Dog Pig Pig Enteritis Pig Respiratory infection Human Human Bat Respiratory infection Respiratory infection Asymptomatic 2a Canine coronavirus (CCoV) Transmissible gastroenteritis virus (TGEV) Porcine epidemic diarrhoea virus (PEDV) Porcine respiratory coronavirus (PRCoV) Human coronavirus (HCoV)-NL63 Human coronavirus (HCoV)-229E Bat coronavirus (bat-CoV)-61, -HKU2, -HKU6, -HKU7, and -HKU8 Murine hepatitis virus (MHV) Respiratory infection / enteritis peritonitis / systemic enteritis Enteritis Enteritis Mouse Respiratory infection/ enteritis / hepatitis / encephalitis Respiratory infection Respiratory infection / enteritis Enteritis / Rat coronavirus (RCoV) Rat Bovine coronavirus (BCoV) Cow Hemagglutinating Pig encephalomyelitis virus (HEV) Human coronavirus (HCoV)-OC43 Human Respiratory infection Human coronavirus (HCoV)-HKU1 Human Respiratory infection Bat coronavirus (bat-CoV)-HKU4, Bat Asymptomatic -HKU5, and bat-SARS-CoV Bat coronavirus (bat-CoV)-DR/2007 Bat Asymptomatic 2b Severe acute respiratory syndrome Human Severe respiratory infection human coronavirus (SARS-CoV) 3 Infectious bronchitis virus (IBV) Chicken Respiratory infection/ enteritis Turkey coronavirus (TCoV) Turkey Enteritis Table 1: Coronavirus groups with main representatives, host and associated diseases. Adapted from de Haan and Rottier, 2005 (3). More recently, HCoV-HKU1 was added to group 2 (4), SARS-CoV was added as a branch of group 2 (5), and nine Bat-CoVs were added to group 1 or 2 (6;7). Coronavirus infections are detected in avian and mammals, including human, mainly causing respiratory and intestinal tract infections. It received worldwide ___________________________________________________________________________ 3 ___________________________________________________________________________ attention in 2003 when it became clear that a coronavirus was the etiological agent of the severe acute respiratory syndrome (SARS). The SARS epidemic started in July 2002 in China. During this epidemic, 8096 people were infected with the SARS virus, which caused mortality in 774 individuals (8). After that, the new coronaviruses HCoV-NL63 in 2004 and HCoV-HKU1 in 2005 were identified, which are less severe but are a cause for common cold in human (4;9). Additionally, the bat-SARS-CoV and bat CoV-61 were identified and closely related to the SARS-CoV, indicating a possible origin of SARS-CoV in bats. Recently, seven new bat-CoV were identified; bat-CoV-HKU2, -HKU6, -HKU7, -HKU8 , -HKU4, -HKU5, and –DR/2007 (6;7). Genome organisation: As reviewed by Pasternak et al. (2006), the genomic RNA size of coronaviruses ranges from 26 to 31 kb, organized in a fixed order: 5’-ORF1a-ORF1bS-E-M-N-3’ (see figure 1) (10). Figure 1: Genomic organization of coronaviruses based on SARS-CoV. Adapted from Pasternak et al. (10). The two open reading frames ORF1a and ORF1b cover two third of the 5’ genome side, encoding the polyproteins pol1a and pol1ab. The polyproteins encode the nonstructural proteins (nsps) and are translated directly after nucleocapsid disassembly and release of the genomic RNA into the host cytoplasma (11). The ORF1b is translated using a ribosomal frameshift mechanism (12). As reviewed by Pasternak et al. (2006), the structural and accessory proteins are encoded on a nested set of subgenomic (sg) mRNA’s (10). For transcription of these sg mRNA’s, first sg-length minus strands are transcribed from the full-length genome. A 3’ transcription regulatory sequence (TRS) and leader sequence (LS) are present on all sg minus strands. The currently accepted model of transcription is the ‘discontinuous minus-strand RNA synthesis’ model. The transcription is assumed to be discontinuous, because the LS encoded on the 5’ end of the positive-stranded RNA genome is common in all sg minus strands. To complete transcription of a minus stranded sg RNA containing the body coding sequence with the body TRS, the common LS is added by jumping to the leader TRS (see figure 2). To enable a jump of the minus strand from the body TRS to the leader TRS, RNA and protein interactions bring the body and leader region in close contact. Eventually, the leader sequence is added and this sg minus-strand RNA is transcribed to positive-strand sg mRNA, which is used for translation (10;13;14). ___________________________________________________________________________ 4 ___________________________________________________________________________ Figure 2: The currently accepted mechanism of coronavirus sg mRNA transcription; discontinuous minusstrand RNA synthesis. Adapted from Pasternak et al. (10). Non-structural proteins: The pol1a and pol1ab polyproteins are cleaved by the viral main protease (Mpro) and papain-like protease (PLpro) resulting in 16 nsps: nsp1 to nsp16 (see figure 3). As reviewed by Snijder et al. 2006, several functional domains on the nsps have been identified; nsp7, nsp8, and nsp9 contain RNA-binding domains (RBD), suggesting involvement in viral replication (11). Nsp3, nsp4 and nsp6 contain a transmembrane domain, indicating replication to occur at cellular membranes. Nsp3 contains an ADP-ribose-1’-monophosphatase (ADRP) domain and nsp14 contains an exonuclease (exo) domain. Nsp13 contains both a Zinc-binding (Z) domain and a helicase (Hel) domain. Finally, nsp16 contains a ribose-2’-Omethyltransferase (MT) domain. Figure 3: Polyprotein organization of coronaviruses based on SARS-CoV. Cleavage sites are indicated with grey and black arrowheads, respectively (11). Structural proteins: As reviewed by de Haan and Rottier (2005), the structural proteins are the building blocks of the virus (3). The N protein encapsidates the viral genomic RNA and thereby forms the nucleocapsid. Additionally, the N protein is involved in regulation of viral replication (15). The lipid envelope contains 3 or 4 different viral structural proteins (3). The M protein is the most prominent envelope protein and undergoes interactions with most other viral components. The small and low abundant E protein does probably have a morphogenetic function by taking strategic positions to generate the required membrane curvature during budding. The S protein forms non-covalently linked homotrimers, which are responsible for attachment and fusion with the host cell. Additionally to the M, E, and S proteins, some coronaviruses carry hemaglutinin (HE) at the envelope membrane. The function of HE protein is not totally clear; implications of involvement in virus motility by binding to mucopolysaccharides on the intestinal wall were found. Additionally, the HE protein can possibly play a role in infections of other organs besides the respiratory or intestinal tract. ___________________________________________________________________________ 5 ___________________________________________________________________________ Accessory proteins: Accessory proteins are together with the structural proteins encoded on the nested set of sg mRNA strands. The accessory proteins are nonessential in vitro, however the function of these proteins in vivo is unknown. They may contribute to the variability in pathogenicity and infectivity among coronaviral species. Coronavirus lifecycle The viral lifecycle of coronaviruses is divided in four steps, as also reviewed by de Haan and Rottier (3). An overview of the coronaviral lifecycle is summarized below (see also figure 4), although each step is described in more detail in the sub-sections of this thesis. (1A) Attachment: Prior to entry, the globular head of the S protein (the S1 domain) on the viral envelope binds to the receptor expressed on the target cell. Next to binding of the S protein to its receptor, several strain-specific supplementary attachment molecules are involved (16). (1B) Entry: It is not clearly established for all coronaviruses if entry occurs via either the endosomal route or occurs directly at the plasma membrane (16). After attachment, the stalk-like region of the S protein (the S2 domain) is responsible for fusion with the membrane (3). In case of endosomal entry, fusion of the viral envelope with the endosome is realized by either cleavage or conformational changes of the S protein. (2) Replication/transcription: After disassembly, the genomic RNA is released into the cytoplasm of the target cell. The replication and transcription complex, consisting of coronaviral nsps and the genomic RNA, were found to co-localize with intracytoplasmatic double membrane vesicles (DMVs) (11;13). Localization of replication and transcription complexes to DMVs could be beneficial for coronaviral replication efficiency, in facilitating organization of the replication complex, recruitment of hostcellular molecules, and protection against host-defence mechanisms. (3) Assembly/budding: After translation, the structural proteins S, M, and E are inserted into the rough endoplasmic reticulum (ER) and transported to the ER-toGolgi intermediate compartment (ERGIC), where budding of helical nucleocapsid particles takes place (13). (4) Maturation and release: The constitutive secretory pathway is used for viral release. During transport, some coronavirus strains undergo maturation already, by sugar modification and cleavage of the S protein into two subunits. After cleavage the subunits remain non-covalently linked. After maturation and release newly formed viral particles can start a new lifecycle after finding a permissive target cell. The coronavirus rely on host-cellular molecules to fulfil their lifecycle. However, many involved proteins and mechanisms are not identified yet. This thesis covers an update about host-cellular molecules which are found to contribute to the coronaviral lifecycle in literature published from 2006 till 2009. Each step of the viral lifecycle is discussed separately. ___________________________________________________________________________ 6 ___________________________________________________________________________ Figure 4: Viral lifecycle of coronaviruses based on SARS-CoV (16). Entry As reviewed by de Haan and Rottier (2006), the S protein is responsible for attachment and entry of the coronavirus in the host-cell and eventually cell-cell fusion by expression of S on the cell surface (16). The interactions between the S protein and host-cellular receptors during coronaviral entry are the most intensivelystudied parts of the coronaviral lifecycle. However, findings are highly variable due to differences in tropism of coronavirus strains (17). Additionally, viruses behave differently in different cell types and environmental circumstances, and cell culture adaptation can lead to variability within strains (18;19). Differences between the mechanisms of virus-cell fusion and cell-cell fusion make it even more complicated (20). Therefore, the findings concerning host-virus interactions for certain coronaviruses are difficult to extrapolate for coronaviruses in general. In this section the most prominent host interactions concerning coronaviral attachment and entry have been summarized. ___________________________________________________________________________ 7 ___________________________________________________________________________ Attachment Host receptors As reviewed by de Haan and Rottier (2006), the amino-terminal S1 subunit of the coronaviral S protein is responsible for attachment to the host receptor (16). The receptor-binding domain of the S1 subunit is different among coronavirus strains, resulting in variability of host receptors for different virus-strains. For numerous coronaviral species a major host receptor has been identified, which plays an important role in infection. ACE2 for SARS-CoV: The main receptor required for SARS-CoV infection is the angiotensin-converting enzyme 2 (ACE-2) (21). Different SARS-CoV strains were found to have a variable affinity to this receptor. Remarkably, the function of ACE2 as the host receptor essential for SARS-CoV infection have been questioned recently, as will be discussed in the section ‘co-factors’ (1). ACE2 for HCoV-NL63: Next to SARS-CoV, ACE2 was also found for HCoV-NL63 to function as an important receptor for infection (22). Although FACS analysis found a lower affinity of the HCoV-NL63 S protein to the ACE2 receptor compared with the S protein of SARS-CoV, antibody blocking of the ACE2 receptor on host cells resulted in inhibition of infection in a rate comparable with inhibition of the ACE2 receptor in SARS-CoV infection. hAPN for HCoV-229E: For HCoV-229E, human aminopeptidase N (hAPN or CD13) was found to be an important receptor needed for infection. Although it is not sure whether hAPN is the main receptor, antibody blocking of hAPN results in strong inhibition of HCoV-229E entry (22). pAPN for TGEV: Porcine aminopeptidase N (pAPN) was found to serve as a receptor for transmissible gastroenteritis virus (TGEV) (23). fAPN for FCoV: Feline aminopeptidase N (fAPN) was found to serve as a receptor for feline coronaviruses (24). Additionally, it was found that fAPN could serve as a receptor for other members of serogroup I coronaviruses like canine coronavirus, porcine coronavirus TGEV, and human coronavirus HCoV-229E. CEACAM1 receptor for MHV: For MHV the main receptor is found to be carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1 or MHVR) (25). Sialic acids: For the group 2 coronaviruses HcoV-OC43 and BCoV, the main receptors were found to be O-acetylated sialic acids (26). Although controversial findings were observed, sialic acids were also found to be important in group 3 coronavirus infections (27). HLA class 1 and HCoV-OC43: In addition to sialic acids, HLA class 1 antigens have been reported to serve as one of the host receptors for HCoV-OC43 (28). Co-factors Co-factors have been found to augment coronaviral attachment in addition to the host receptor. Although it is difficult to distinguish between host receptors and co___________________________________________________________________________ 8 ___________________________________________________________________________ factors sometimes, co-factors are aimed to contribute to the infection efficiency in addition to the host receptor. Lectins: As reviewed by de Haan and Rottier (2006), opposite roles for lectins concerning SARS-CoV infections were found (16). The lectins DC-SIGN, DCSIGNR/L-SIGNL/CD209L, and LSECtin were found to augment binding to the ACE2 receptor and thereby coronaviral entry (29;30). Additionally, cell-mediated transfer was promoted by binding to DC-SIGN and L-SIGN (30;31). On the contrary, other findings suggested that expression of the lectin L-SIGN inhibited cell-mediated transfer and promoted proteasome-dependent viral degradation (32). Recently, Han et al. (2007) found that DC-SIGN and L-SIGN were able to serve as alternative receptors for infection with pseudotyped murine leukemia virus bearing the SARS-CoV S protein (1). DC-SIGN and L-SIGN transfected cells were infected by the SARS-CoV S containing pseudotype independently of ACE2 expression (see figure 5). Additionally, co-transfection of ACE2 with DC/L-SIGN provided evidence that ACE2-mediated infections were augmented minimally. These findings are Figure 5: ACE2, DC-SIGN, and L-SIGN are contradictive to its role as a co-factor as able to serve as a SARS-CoV receptor. HeLa described previously (30). Recently, the cells were transfected with ACE2, DC-SIGN, role of DC-SIGN as a co-factor was also L-SIGN or pcDNA empty vector, which served as the negative control (1). found for feline CoV FIPV (33). In this study, expression of hDC-SIGN in non-permissive cells rescued FIPV infection. Expression of fAPN was required for this augmentation, indicating that hDC-SIGN does not serve as an alternative receptor but as a co-factor in FIPV infections. These results indicate that even recent publications can give contradictive conclusions. HLA-C: Although the host receptor responsible for hCoV-HKU1 infection is not elucidated yet, the host protein HLA-C was identified as a co-factor using transduction of a cDNA library in cells, which eventually were exposed to the S protein S1 subunit (27). The interaction of HLA-C with the S protein of hCoV-HKU1 was confirmed in a co-immunoprecipitation experiment. Overexpression of HLA-C in two different cell lines increased attachment of hCoV-HKU1 S protein 100/200 fold, and down regulation by RNAi and antibody-mediated blocking of HLA-c resulted in inhibition of entry up to around 60% (see figure 6). The fact that the amount of HLAC-expression on host cells did not correlate with infection efficiency and not all HLAC expressing cells were permissive for infection indicated that HLA-C is not the only receptor involved in infection. ___________________________________________________________________________ 9 ___________________________________________________________________________ Figure 6: Entry inhibition of CoV-HKU1 by HLA-c RNAi and HLA-C antibody blocking. A549 cells were exposed to CoV-HKU1 pseudotyped virus, VSV-G plasmid served as the positive control (27). Fusion As reviewed by de Haan and Rottier (2006), the carboxyterminal S2 subunit of the S protein is responsible for virus-cell fusion (16). The S protein is cleaved into the S1 and S2 subunits to enable the S2 fusion peptide to come in close-contact with the membrane. Known cleavage mechanisms of the coronaviral S protein occur either during maturation by furin-like enzymes (34), or after attachment in the endosomal vesicle of the target cell by cathepsins (35). Coronaviral entry occurs either directly via the plasma membrane or using the endosomal route. Furin-like enzymes: As reviewed by de Haan and Rottier (2006), the S protein of group 2 coronaviruses – but not SARS-CoV - are cleaved into S1 and S2 subunits during maturation by furin-like enzymes (16). The S protein of HCoV-HKU1 was also found to be cleaved by furin or furin-like enzymes (34). Using HIV-pseudotyped virus bearing the HCoV-HKU1 S protein, cleavage of the S protein and thereby viral particle release was inhibited by addition of furin or furin-like enzyme inhibitors. Another study performed by de Haan et al. (2008) showed that cleavage of the S protein is not restricted to group 2 coronaviruses; several members of group 1 contain a furin cleavage sequence in their S protein, which could be cleaved by furin-like enzymes (18). However, implications were found that furin-mediated cleavage does not affect virion infectivity, but does influence cell-cell fusion (36). Cathepsin L: For the SARS-CoV, cathepsin L was found to serve as an alternative protease capable to cleave the S protein and thereby mediate membrane fusion (37;38). However, also in vitro trypsin treatment is capable to cleave the S protein and thereby promote infection of SARS-CoV, indicating that other proteases could be involved in cleavage of the S protein in addition. Although to a lower extend compared than for SARS-CoV, reduced levels of hCoV229E were observed after the addition of cathepsin L inhibitors compared with the untreated control, indicating that hCoV-229E is also capable to use these proteases during endosomal entry (35). For the feline enteric coronavirus (FECV), entry was ___________________________________________________________________________ 10 ___________________________________________________________________________ found to be both cathepsin L- and cathepsin B-dependent (17). In particular, cells were treated with specific cathepsin B- and L-inhibitors pre-infection resulting in inhibition of coronaviral entry (see figure 7). On the contrary, feline infectious peritonitis virus (FIPV) was shown to be dependent on cathepsin B but not cathepsin L. These results indicate that within feline coronaviruses different mechanisms are used for entry. Treatment of cathepsin inhibitors after infection did not affect infectivity, indicating that cathepsins are involved in the coronaviral entry process. Control Cathepsin L-inhibition Cathepsin B-inhibition Figure 7: The effect of Cathepsin L and B inhibition pre-infection on FIPV and FECV entry. The fCoV N protein is stained with a fluorescent antibody (green) (17). Endosomal acidification: For SARS-CoV it was thought that the acidification of the endosomal vesicles is required for coronaviral entry, as Control it caused activation of host proteases, which eventually NH4Cl enabled cleavage of the S protein. Wang et al. (2008) Chloroquine found that SARS-CoV makes use of the endosomal Bafilomycin A1 route for infection (2). By fluorospectrometric monitoring, the GFP-labelled ACE2 receptor was found to follow the endosomal route after exposure to retroviral pseudoviruses bearing the SARS-CoV S protein. Co-localization of SARS-CoV S protein with the ACE2 receptor, and SARS-CoV S protein with the early endosome antigen 1 (EEA1) were confirmed by dual labelling. Figure 8: Inhibition of viral infectivity (GFP expression) in cells treated with lysosomotropic agents prior to infection with pseudovirus bearing the SARS-CoV S protein (2). ___________________________________________________________________________ 11 ___________________________________________________________________________ The use of the endosomal route was found to be pH dependent, because different lysosomotropic agents inhibited infection of spike-bearing pseudovirus (see figure 8). In addition to SARS-CoV, FECV infection was also found to be highly pH dependent (17). In contrast to FECV, the coronavirus FIPV resulted to be pH independent, again indicating different mechanisms of entry within FCoV strains. Cholesterol: As earlier indications already suggested, cholesterol is found to be important as a co-factor in coronaviral fusion with the cellular membrane. The effect was confirmed recently by Lu et al. (2008), who studied the depletion of cholesterol on SARS-CoV entry (39). The role of cholesterol was found to be included in the role of lipid rafts; depletion of cholesterol resulted in disruption of lipid rafts. Lipid rafts are cholesterol and sphingolipids enriched regions of the plasma membrane, which serves as a platform for host-cellular proteins involved in signal transduction. Next to its role in signal transduction, lipid rafts were found to serve as an entry, assembly and budding site for several microbial pathogens, as reviewed by Campbell et al. (2001) (40). In Vero E6 cells, approximately 70% of the ACE2 receptors co-localized with the lipid-raft marker caveolin-1, indicating a role in coronaviral attachment and entry (39). Co-localization was determined by sucrose gradient fractionation followed by western blot, and fluorescent antibody staining followed by confocal microscopy. Cholesterol depletion was found to play a role in the entry process and not attachment as such; cholesterol depletion did not affect binding of SARS-CoV S to the ACE2 receptor, but using a single cycle infectivity assay, SARS-CoV infectivity was found to be inhibited for about 90% in the cholesterol depleted cells (39). In line with the above described findings, involvement of cholesterol in SARS-CoV infection was also found by Wang et al. 2008 (2). The SARS-CoV entry-route did make use of lipid rafts, because cholesterol depletion by methyl-β-cyclodextrin (MβCD) did affect infectivity significantly (see figure 9a). The addition of lysomotropic agents inhibited pseudotyped viral infectivity, indicating that the endocytotic pathway was pH dependent. However, in contrast with Lu et al. (2008) (39), SARS-CoV infection was found to be caveolae-independent (2). Inhibition of caveolae-dependent endocytosis by addition of filipin and nystatin did not affect infectivity of retroviral pseudoviruses bearing the SARS-CoV S protein (see figure 9b). Additionally, fluorescent antibody staining of the pseudovirus bearing SARS-CoV S protein and caveolin-1 did not reveal co-localization. Next to caveolin, the endocytotic-route taken by SARS-CoV was also found to be clathrin-independent, as inhibition of clathrin-mediated endocytosis by both the drug cholopromazine and clathrin heavy chain siRNA did not reduce SARS-CoV S protein pseudovirus infectivity significantly (see figure 9c). Altogether, cholesterol is involved, but in which manner remains to be elucidated. ___________________________________________________________________________ 12 ___________________________________________________________________________ A B Control Control Chlorpromazine Control siRNA Clathrin HC siRNA MβCD C Control Control Filipin Control Nystatin Figure 9: Infection of SARS-CoV S protein bearing pseudoviruses involves cholesterol but is not clathrin- or caveolae-mediated. A: Influence of cholesterol depletion by MβCD on infectivity of SARS-CoV S protein bearing pseudoviruses in Vero E6 cells B: Influence of clathrin-mediated endocytosis inhibition by the drug chlorpromazine and clathrin siRNA on infectivity of SARS-CoV S protein bearing pseudoviruses in Vero E6 cells. C: Influence of caveolae-mediated endocytosis inhibition by the drugs nystatin and filipin on infectivity of SARS-CoV S protein bearing pseudoviruses in Vero E6 cells (2). Replication and transcription As described in the introduction, coronaviral replication and transcription occurs at DMVs. Influence of host-cellular molecules on both formation of DMVs and the process of replication and transcription will be discussed in this section. Origin of the coronaviral replication-transcription sites Host-cellular molecules are involved in DMV formation, however generally accepted mechanisms have changed over time. Previously, it was thought that DMVs originated from autophagosomes, which also exhibit vesicles with double membranes like DMVs. Autophagy is a host-cellular survival system, which breaks down cytoplasmic components by fusion of autophagosomes with lysosomes during starvation (41). As reviewed by Mizushima et al. (2002), autophagy requires two Atg5-dependent protein conjugation systems; Atg5-Atg12 conjugate and the ___________________________________________________________________________ 13 ___________________________________________________________________________ Atg8/LC3 conjugate (42). The Atg8/LC3 conjugate requires modification of the free Cterminal glycine of LC3, which is Atg5 dependent (see figure 10) (43). Figure 10: Schematic overview of the autophagy pathway: LC3 recruitment at the double-membraned autophagosomes is Apg5 (=Atg5) dependent (42). Previous findings indicated that DMVs needed for coronaviral replication were supplied using the autophagy pathway; Atg5 knockout of mouse embryonic stem cells impaired MHV replication (44). However, several recent findings argue against involvement of the autophagy pathway. Co-staining of LC3 and the N protein after SARS-CoV infection did only partially show co-localization (13). Additionally, knockdown of Atg5 in mice-derived macrophages did not affect MHV replication (43). More recently, the membrane tubuli, which co-localized with SARS-CoV nsp3, were frequently found in close proximity to ER cisternae and therefore implicate ERorigin (13). The membrane tubules were in close proximity of DMVs, suggesting that DMVs are derived from these tubular ER-protrusions. Immuno-labeling of ER (antiPDI) and nsp3 with specific antibodies found clustering at sites probably corresponding to DMVs. Oostra et al. (2007) confirmed that the early secretory pathway plays an important role in formation of coronaviral replication sites (45). In particular, inhibition of ER protein export by H89 affected viral replication both early (1-4 h.p.i.) and late (4-7 h.p.i.) in infection. Verheije et al. (2008) also found that the MHV replication complex formation was closely connected to the early secretory pathway (see figure 11) using the drug brefeldin A (BFA), which induces redistribution of Golgi proteins into the ER and thereby blocks transport by the secretory pathway (46). BFA targets several guanine nucleotide exchange factors (GEFs); the golgi-specific resistance factor 1 (GBF1), the BFA-inhibited GEF 1 (BIG1) and 2 (BIG-2). GEFs were found to be involved in the formation of MHV replication complexes (RC), as BFA reduces the production of DMVs. By depletion of each GEF separately using siRNA, GBF1 was found to be essential for MHV replication. GBF1 activates ADP-ribosylation factor 1 (ARF1), which regulates the budding of vesicles from the ER in the secretory pathway, confirming the role of the secretory pathway. ___________________________________________________________________________ 14 ___________________________________________________________________________ The late transport secretory pathway was found to be less involved, as blocking of the transport from ERGIC (or VTCs) to cis-Golgi did not impair MHV replication. Figure 11: Schematic representation of the link between RCs and the early secretory pathway, and the inhibitors used for inhibition of indicated steps (46). Role of host proteins in viral replication and transcription As reviewed by de Haan and Rottier (2005), three different virus-host interactions concerning coronaviral replication and transcription have been found previously (16). Firstly, the heterologous nuclear ribonucleoprotein (hnRNP) family members (hnRNPA1, PTB and SYNCRYP) were found to affect coronavirus replication and transcription by binding to different regions of the genomic RNA. Secondly, mitochondrial-aconitase binding-interaction with coronaviral RNA was found to be involved in viral replication (47). Thirdly, coronaviral RNA interactions with poly-Abinding proteins were found (48). Deletion of parts of the poly-A tail reduced replication, indicating, but not confirming, a role of these proteins in replication efficiency. Although interactions of host-cellular molecules with coronaviral RNA were found to be involved in the replication and transcription process, for all these interactions complete molecular mechanisms have not been elucidated yet. Since 2006 two new host-cellular molecules have been identified, as described below. The N protein and GSK-3: N protein phosphorylation was suggested to play a role in viral replication (reviewed by de Haan and Rottier, 2005) (16). The major phosphorylation sites of the SARS-CoV N protein were found between amino acids 179-210; also called the central SR-rich region. Using NetPhos software, glycogen synthase kinase-3 (GSK-3) was predicted to function as a potential host-derived kinase for this SR-rich region of the SARS-CoV N protein (49). Indeed, GSK-3-specific inhibition resulted in defective N protein phosphorylation, assessed as differences in migration rate in NuPAGE gels. An antibody against phosphorylated N-ser-177 ___________________________________________________________________________ 15 ___________________________________________________________________________ showed specific inhibition of N-ser-177 phosphorylation by GSK-3 inhibition. Further evidence was provided by the fact that a ‘gain of function’ approach by constitutive active GSK-3 expression restored GSK-3 specific inhibited N protein phosphorylation. Using an in vitro kinase assay, mutation of Ser-189 and Ser-207 to both alanine prevented N protein phosphorylation by GKS-3. Inhibition of GSK-3 activity resulted in a reduction of viral titer in vitro in SARS-CoV and MHV infections. The fact that N proteins are important in coronaviral replication suggests that phosphorylation of the SR-rich region could be important in this process, although the role of phosphorylation in other steps during the lifecycle cannot be excluded. Helicase and Ddx5: Using a yeast two-hybrid system interactions between SARS-CoV helicase (located at Nsp13) and Asp-Glu-Ala-Asp box polypeptide 5 (Ddx5) were observed (50). Eventually, interaction was verified with a mammalian two-hybrid assay and co-immunoprecipitation (see figure 12). Figure 12: Co-immunoprecipitation of HA-Helicase and c-myc-Ddx5 using HA- and myc-antibodies (IP:immunoprecipitation, ID: immunodetection) (50). Ddx5 is a multifunctional host-cellular protein involved in transcription regulation. Therefore, this interaction might play a role in coronaviral transcription efficiency. The effect of siRNA knockdown of Ddx5 in fetal rhesus kidney cells on viral load was assessed by real-time RT-PCR and viral titers were calculated by back-titration. Ddx5 knockdown resulted in decrease of both viral load and titer of SARS-CoV (see figure 13). In conclusion, Ddx5 is involved in SARS-CoV replication in vitro. However, the exact role of Ddx5 in the transcription process has to be investigated. Figure 13: Effect of Ddx5 siRNA on viral replication, measured in viral RNA copies and viral titers (50). ___________________________________________________________________________ 16 ___________________________________________________________________________ Assembly and budding During coronavirion assembly, the viral components are combined in such a way that complete viral particles are formed. The assembly process can be subdivided in nucleocapsid and envelope assembly. During nucleocapsid assembly the genomic RNA is packaged by N proteins into a helical capsid structure. Next, the envelope is assembled by the packaging of nucleocapsid particles into an ERGIC-derived lipid membrane. During this process, the membrane curves around the nucleocapsid particle, a process called budding. Prior to budding, virus-derived structural proteins are transported and directed to the ERGIC membrane using both viral and cellular factors. Interactions between the different components of the coronavirus itself are studied intensively. On the contrary, the role of host-cellular proteins in coronavirus assembly and budding has hardly been explored. In this section most recent findings (> 2006) concerning host-coronavirus interactions during the assembly process are summarized. Nucleocapsid assembly Nucleocapsid assembly is mainly regulated by the interactions between N proteins and the genomic RNA, as reviewed by de Haan and Rottier (2005) (3). To our knowledge, no host-cellular protein interactions with coronaviral proteins contributing to nucleocapsid assembly are found. The N protein and APOBEC3G: In contrast to contribute to the coronaviral assembly process, a virus-host interaction which might exhibit anti-retroviral properties was found. The host-cellular protein human APOBEC3G (hA3G) was found to interact with SARS-CoV N proteins (51). More specific, the nucleocapsid domain of the HIV1 Gag protein, known to be responsible for hA3G incorporation in HIV-1 virion formation, was replaced by coronavirus derived N protein, forming a chimeric construct. Chimeric constructs containing different regions of the SARS-CoV N protein were used to determine the region of the coronaviral N protein able to incorporate hA3G in VLPs. By western immunoblotting of VLPs, hA3G incorporation was found in the constructs containing the amino acid 86-302 region of the SARS-CoV N protein. Incorporation of hA3G was found to be partially RNA dependent, as RNAse treatment prior to analysis reduced purification of constructhA3g complexes in a GST pull-down assay considerably. The interaction between the SARS-CoV N protein and hA3G was confirmed by co-immunoprecipitation after cotransfection of SARS-CoV N and M proteins with hA3G in 293T cells. Furthermore, incorporation of hA3G in M and N containing virus-like particles (VLPs) was found by sucrose density-gradient fractionation. The binding domain of hA3G with SARS-CoV N protein was investigated by cotransfection of the M and N protein with different hA3G mutants. The hA3G interaction-domain with SARS-CoV N protein appeared to be localized between residues 104-156. This hA3G binding-domain is comparable with the previous found ___________________________________________________________________________ 17 ___________________________________________________________________________ interaction with HIV-1 nucleocapsid, indicating that a similar mechanism for incorporation is involved. In addition to SARS-CoV, a HIV-1 chimeric construct containing the HCoV-229E N protein was also able to incorporate hA3G in VLPs. In addition, this reaction was found to be RNA-dependent using a GST pull-down assay. These results indicate that incorporation of hA3G in coronaviral VLPs is not SARS-CoV specific. The hA3G protein might function as an inhibitor of viral replication by C-to-U editing of minus strands during reverse transcription, as APOBEC3G is a member of cytidine deaminases. However, as the coronavirus does not exhibit a reverse transcription reaction of the genome, the role of hA3G in coronaviral lifecycle remains unclear. Envelope assembly The M protein and β-actin: the M and E protein together, but not alone, can induce the formation of virus-like particles (52), implicating that these proteins have an essential role in coronaviral budding. Therefore, to study the role of host-cellular proteins in the assembly process, the interaction between coronaviral M protein and hostcellular proteins was investigated. A two-hybrid system identified an interaction between IBV M protein and host-cellular β-actin (53). Co-transfection followed by coimmunoprecipitation confirmed this interaction. Furthermore, the M protein and βactin were found to co-localise in the Golgi area. Specifically, the interaction-region of the M protein is localized on the C-terminal cytoplasmic portion. By testing multiple mutation constructs, the amino acids P159 and E160 of M protein were found to be responsible for the interaction with β-actin. Viral replication is not regulated by this interaction, as the amounts of full-length negative-stranded RNA PCR fragments of wild type and Δ159/160 M IBV mutants increased equally. Although less mRNA expression in the Δ159/160 M IBV mutants was detected, disruption of actin filaments and inhibition of actin polymerization by cytochalasin D did not lead to a reduction in the amount of N protein present in IBV infected cell lysates (see figure 14). On the contrary, the amount of N protein in the medium of cytochalasin-treated IBV-infected cells was decreased. These results suggest that the interaction between the IBV M protein and β-actin does not influence viral replication and transcription, but moreover, that viral assembly and release is affected. Of note, the role of β-actin in the lifecycle of other coronaviral species is questionable, because amino acids 159 and 160 of the IBV M protein are not conserved in all coronaviruses. Figure 14: Disruption of actin filaments by cytochalasin D inhibits IBV replication but not replication and transcription. Cytochalasin D or the negative control DMSO was added to IBV infected Vero cells at indicated timepoints post infection (53). ___________________________________________________________________________ 18 ___________________________________________________________________________ Accessory protein 7a and hSGT: coronaviral accessory proteins are non-essential for in vitro replication and are not conserved between different virus groups. For the majority of these proteins, their function in the coronaviral lifecycle is still unknown. Recent investigations showed that SARS-CoV accessory protein 7a (S7a) might be involved in the coronaviral assembly process, because interactions of S7a with both M and E proteins were found by co-immunoprecipitations (54). Furthermore, an interaction was revealed between S7a and the human small glutamine-rich tetratricopeptide repeat-containing protein (hSGT). The interaction was confirmed by co-immunoprecipitation and immunofluorescent staining (see figure 15). In particular, hSGT and S7a were found to co-localize around the perinuclear area. The interaction of hSGT was localized within amino acids 125-158; the tetratricopeptide repeat (TPR) 2 region. Previously, diverse functions of SGT TPR regions were found, like binding to viral and non-viral proteins (55-57). hSGT was found to interact with the HIV-I accessory protein Vpu and the structural protein Gag, which are both involved in the viral particle release (56). This similarity to the found interaction between SARS-CoV structural proteins (M protein and E protein), an accessory protein (S7a), and hSGT implicate that a similar mechanism may regulate viral packaging and release. Figure 15: Immunofluorescent microscopy of hSGT and S7a co-localization (54). The S protein and COPI: M and E proteins together are capable to generate empty enveloped vesicles, which at a microscopic view resemble viral particles (52). However, to produce infectious viruses all required components need to be incorporated, including viral RNA. Viral particles lacking the S protein can not be infectious, because the S protein is essential for host attachment and entry. McBride et al. (2007) investigated the mechanism of S protein incorporation and found that cycling of SARS-CoV S protein between the ER and Golgi compartment may be required (58). SARS-CoV S proteins have a dibasic motif (KxHxx) on its cytoplasmic tail, which is found to function as an ER retrieval signal. An in vitro binding’s assay with S fusion proteins showed that the KxHxx-motif of the S protein binds to the host-derived coatomer complex I (COPI). COPI-coated vesicles are responsible for retrograde transport of proteins from the Golgi to the ER (see figure 16), as reviewed by Lee et al. (2004) (59). Although differences were small, KxHxx mutants of the S ___________________________________________________________________________ 19 ___________________________________________________________________________ protein were processed in the Golgi compartment slight more rapidly. To validate the small differences the effect of the KxHxx-motif on S protein cycling was studied using chimeric proteins as a reporter. In this way, lower expression levels could be measured and thereby earlier timepoints post-transfection. Using this chimeric mutant, more S protein expression was present at the plasma membrane indicating that less S proteins were retained in the ER-Golgi area. These results indicate that a reduction of S expression at the cell surface and a delay in migration towards the cell surface may indeed be caused by the KxHxx- motif. Thereby, the cycling of the S protein in the secretory pathway might facilitate S incorporation in the virion particle (58). The interaction between COPI and the S protein might be a way to retrieve S from other compartments to the side of budding. This mechanism could also be used by other viral proteins, although more investigations are needed to verify this hypothesis. Figure 16: ER-Golgi transport: retrograde transport is regulated by the coatomer complex I (COPI) (59). Palmitoylation of the E protein: Boscarino et al. (2008) investigated the role of E protein palmitoylations during coronaviral assembly (60). Palmitoylation is the posttranslational lipidation of cysteine residues, which could be important for proteins in their interactions with host membranes during assembly. Bio-informatical software predicted which cysteines of the E protein were likely to be post-translational palmitoylated. The E protein of MHV was found to have several candidates, from which mutations were tested on the ability of VLP formation after co-transfection with S, M, and N. Mutant constructs of 3 cysteine residues on the MHV E protein resulted in incompetent VLP production. Two mutations did already affect, but not block, VLP formation. These results indicated that at least 2 cysteine residues need to be palmitoylated to formulate proper VLPs. The exact role of palmitoylation in the assembly process is not clear, although it does not seem to play a role in localization ___________________________________________________________________________ 20 ___________________________________________________________________________ to the assembly site, because both mutants and wild-type E proteins were localized to Golgi vesicles. In conclusion, in recent years substantial progress in the understanding of hostcellular mechanisms involving coronaviral assembly was made. However, underlying molecular mechanisms and functions of interactions are still hard to identify. Maturation and Release Probably multiple host-cellular molecules are involved in the maturation and release of coronaviruses. However, the only known cellular-molecules are furin-like enzymes which cleave the S-protein of most group 2 coronaviruses, as described in the section entry and attachment. The virus is released using the conventional secretory pathway, however no host-cellular factors are recently found to be involved in coronaviral release. Manipulation of the host-cell machinery As most host-virus interactions described in this thesis contribute to coronaviral lifecycle efficiency directly, host-viral interactions could also be induced to affect the host-cell machinery, which could lead to increased coronaviral replication indirectly. The N protein and B23: a co-localization of host-cellular B23 with the SARS-CoV N protein was observed, which probably affects the host-cell machinery (61). The interaction between SARS-CoV N protein and B23 was confirmed by a protein binding assay and co-immunoprecipitation, and resulted to be RNA independent. HeLa cells were transfected with the SARS-CoV N protein and immunofluorescent staining followed by confocal microscopy showed co-localization in the perinuclear area (see figure 17). SARS-CoV N protein B23 MERGE Figure 17: Co-localization of transfected SARS-CoV N protein with host-cellular B23 (61). The B23 protein is known to be involved in cell-cycle progression (62). After phosphorylation of B23 at Thr199 it serves as a substrate for CDK2/Cyclin E, ___________________________________________________________________________ 21 ___________________________________________________________________________ regulating centrosome duplication. The binding of SARS-CoV M inhibits the Thr199 phosphorylation of B23 and thereby probably cell-cycle progression (61). It remains to be identified which step of the coronaviral lifecycle benefits from this interaction. Discussion Host-cellular molecules are involved in virtually each step of the coronaviral lifecycle. Unravelling interactions between coronaviruses and host-cellular molecules could help to understand the pathogenic differences among species and contribute to the development of new therapeutics. The coronaviral lifecycle is not elucidated completely yet, which is partially due to several complicating factors involving coronaviral research. Firstly, findings could not be extrapolated to coronaviruses in general, unless interactions are confirmed with other strains. Another complicating factor is that some viruses, like HCoVHKU1, can not be passaged in culture yet. In addiction, it is dangerous to work with human coronaviruses like SARS-CoV, as it is highly infectious. To make the research more applicable, the use of pseudotyped viruses is introduced. A drawback of this method is the uncertainty if the pseudotyped virus reflects the mechanisms of the wild-type coronavirus correctly. Furthermore, viruses are evolving in a relatively high rate, which, in theory, makes viral research non-ending. Although coronaviral research is mainly focussed on the virus itself, the improvements on the field of functional genomics and proteomics and the increased use of genomic libraries and two-hybrid systems have contributed to identification of multiple new host-cellular molecules over the past years. Identification of important host-cellular molecules involved in the coronaviral lifecycle could be used as targets for the development of new intervention-strategies. Host receptor identification is the easiest and fastest way of finding new therapeutic targets. The knowledge about the main receptor could be used for neutralizingantibody treatment of infected individuals. As recently was found that multiple hostmolecules could serve as the receptor required for infection, combination therapy including multiple targets could possibly improve drug efficacy. New interventionstrategies are important to dampen a new upcoming epidemic as soon as it is noticed. Another epidemic can have a great impact on social and economic activities, as currently proven with the H1N1 influenza outbreak, which originated from Mexico in April 2009 and disrupted the economy on a large scale. Next to the re-emergence of a coronaviral epidemic, it is important to take into account that coronaviruses could theoretically be used as a biological weapon. Altogether, the investigations on the contribution of the host-cellular machinery to coronaviral infection efficiency are ongoing and will keep attention in order to search for new targets, which are needed to intervene with infection and thereby prevent spreading. ___________________________________________________________________________ 22 ___________________________________________________________________________ References 1. Han,D.P., Lohani,M., and Cho,M.W. (2007) J.Virol. 81, 12029-12039. 2. Wang,H., Yang,P., Liu,K., Guo,F., Zhang,Y., Zhang,G., and Jiang,C. 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