Intracellular localization of Crimean-Congo Hemorrhagic Fever (CCHF) virus glycoproteins
© Haferkamp et al; licensee BioMed Central Ltd. 2005
Received: 17 February 2005
Accepted: 25 April 2005
Published: 25 April 2005
Crimean-Congo Hemorrhagic Fever virus (CCHFV), a member of the genus Nairovirus, family Bunyaviridae, is a tick-borne pathogen causing severe disease in humans. To better understand the CCHFV life cycle and explore potential intervention strategies, we studied the biosynthesis and intracellular targeting of the glycoproteins, which are encoded by the M genome segment.
Following determination of the complete genome sequence of the CCHFV reference strain IbAr10200, we generated expression plasmids for the individual expression of the glycoproteins GN and GC, using CMV- and chicken β-actin-driven promoters. The cellular localization of recombinantly expressed CCHFV glycoproteins was compared to authentic glycoproteins expressed during virus infection using indirect immunofluorescence assays, subcellular fractionation/western blot assays and confocal microscopy. To further elucidate potential intracellular targeting/retention signals of the two glycoproteins, GFP-fusion proteins containing different parts of the CCHFV glycoprotein were analyzed for their intracellular targeting. The N-terminal glycoprotein GN localized to the Golgi complex, a process mediated by retention/targeting signal(s) in the cytoplasmic domain and ectodomain of this protein. In contrast, the C-terminal glycoprotein GC remained in the endoplasmic reticulum but could be rescued into the Golgi complex by co-expression of GN.
The data are consistent with the intracellular targeting of most bunyavirus glycoproteins and support the general model for assembly and budding of bunyavirus particles in the Golgi compartment.
Crimean-Congo hemorrhagic fever virus (CCHFV) is a member of the genus Nairovirus, one of five genera in the family Bunyaviridae . Bunyaviruses are enveloped particles with a tripartite, single stranded RNA genome of negative polarity [2–4]. The three genome segments encode four structural proteins: the RNA-dependent RNA polymerase (L protein) is encoded by the large (L) segment, the glycoproteins (GN and GC; previously referred to as G1 and G2) are encoded by the medium (M) segment, and the nucleocapsid protein (N) is encoded by the small (S) segment [2–4].
The virus glycoproteins are likely to play an important role in the natural tick-vertebrate cycle of the virus as well as for the high pathogenicity in humans. Indeed, a highly variable mucin-like region at the amino terminus of the CCHFV glycoprotein precursor has recently been identified, a unique feature of nairoviruses within the family Bunyaviridae . A similar serine-threonine-rich domain has been associated with increased vascular permeability and development of hemorrhages in Ebola hemorrhagic fever .
The Nairovirus genus includes 34 described viruses and is divided into seven different serogroups . Only three viruses are known to cause disease: CCHFV, Dugbe virus, and Nairobi sheep disease virus. CCHFV is an arthropod-borne pathogen and the causative agent of a serious form of hemorrhagic fever [7–9] with mortality rates ranging from 15 to 60% [10–17]. The virus is endemic in parts of Africa, Southeastern Europe and Asia as far east as western China [16, 18, 19]. The geographic distribution of CCHFV infections corresponds most closely with the distribution of Hyalomma ticks, suggesting their principal vector role [18, 20, 21]. Hyalomma ticks normally feed on a variety of livestock (sheep, goats, cattle, and ostriches), large wild herbivores, hares, and hedgehogs, which can become infected with CCHFV [13, 18, 22, 23]. In contrast to human infections, infection in these animals generally results in inapparent or subclinical disease but generates viremia levels capable of supporting virus transmission to uninfected ticks [10, 18, 21, 23–25]. Transmission to humans occurs either by bites from infected ticks or direct contact with blood or tissues of infected livestock. Nosocomial infections are common  and represent a major problem in health care institutions .
The widespread geographical distribution of CCHFV, its ability to produce severe human disease with high mortality rates, and fears about its intentional use as a bioterrorism agent http://www.bt.cdc.gov/Agent/Agentlist.asp makes CCHFV an extremely important human pathogen and a worldwide public health concern. Case management and intervention strategies would greatly benefit from knowledge of the biology and pathogenesis of the virus.
Recently, the expression strategy and biosynthesis of the CCHFV glycoproteins have been studied in more detail including the identification of precursor cleavage sites and the determination of the exact N termini of the two major cleavage products, GN and GC [5, 28]. SKI-1, also responsible for the proteolytic processing of the Lassa virus glycoprotein precursor , has been identified as the cellular protease responsible for the processing step that generates the N-terminus of mature GN. Another yet unidentified protease is required for GC processing. However, the exact C-terminus of GN could not yet been determined. Two cleavage sites have been predicted for this processing step, one at amino acid position 808 (RKLL) and the other at 940/944 (KKRKK) favouring the cellular proteases SKI-1 and furin, respectively, as the responsible proteases .
Bunyaviruses are known to bud from Golgi membranes and the budding site seems to be defined by an retention of the glycoproteins GN and GC at that particular site [3, 4]. From a number of studies which have addressed the mechanisms of Golgi targeting and retention, one can conclude that the N-terminal located glycoprotein appears to carry the appropriate signal(s) [30–40] So far, no studies have investigated Golgi targeting and retention of nairovirus glycoproteins.
In this study we cloned the complete M segment ORF of CCHFV, strain IbAr10200, into different expression plasmids. Expression and intracellular localization of the glycoproteins GN and GC were studied and compared to glycoproteins generated by virus infection. Using recombinant fusion proteins between the green fluorescence protein (GFP) and CCHFV glycoproteins, the Golgi targeting/retention signal could be mapped to a hydrophobic region within the cytoplasmic domain of the GN protein.
Sequence determination of the full-length CCHFV M segment
Sequence comparison of available CCHFV IbAr10200 M segment sequences
Nucleotide changes compare to U39455 (vRNA position*)
Amino acid changes compare to U39455*
Samples 1 and 2
83: C → T
1671: Gly → Arg
713: T → C
1461: Ser → Gly
1621: T → C
1158: Glu → Gly
2263: G → T
944: Thr → Lys
2926: C → T
723: Arg → Lys
2964: A → G
710: – (silent)
3512: C → T
528: Val → Ile
3550: A → G
515: Phe → Ser
4044: G → T
350: – (silent)
4981: T → G
38: His → Pro
3425: T → C
557: Asn → Asp
3427: C→ A
556: Cys → Phe
3429: G → A
555: – (silent)
3435: T → A
555: – (silent)
3441/42: GT → AG
551: Asp → Ala
3444: A → T
550: – (silent)
4247: G → A
282: – (silent)
4610: T → G
162: Thr → Pro
2760/61: TA → AT
778: Ile → Asn
4684: G → A
137: Ser → Phe
Expression of CCHFV glycoproteins
Intracellular localization of CCHFV glycoproteins
Signals for intracellular targeting of CCHFV glycoproteins
Features and construction details of different GFP-CCHFV G fusion proteins
Glycoprotein fragment [nt] Fragment length
72 bp Oligonucleotide linker
The fusion protein GFP-GNI, containing the TM I of CCHF GN was expressed in the cell cytoplasm in both used cell lines (Figs. 6B and 6C: b) similarly to GFP expressed from the basic vector pHL2823 (Figs. 6B and 6C: a). In case of the signal peptide-containing GFP fusion protein a diffuse staining consistent with the distribution throughout the secretory system was observed (data not shown). Based on this result we conclude that the transmembrane domain TM I does not contain any intracellular targeting signal.
The Golgi localization signal was further analyzed with two more GFP-fusion proteins containing only the 23 amino acids from the predicted TM II directly fused to the C-terminus of GFP. To determine if a specific primary sequence within TM II was recognized as a signal or rather the hydrophobic character of this region was crucial to target GFP to the Golgi complex, the 23 amino acids were fused in two different orientations (Fig. 6A). BHK-21 (Fig. 6B) and 293T (Fig. 6C) cells were transfected with these constructs and GFP expression and intracellular localization were analyzed. Both GFP-fusion proteins showed specific Golgi complex localization demonstrating that TM II contains a Golgi localization signal and that the orientation of the primary amino acid sequence is not important for GFP translocation (Figs. 6B and 6C: k, l). GFP fusion proteins containing either the predicted GC TM (GFP GCA) or cytoplasmic domain (GFP GCB) showed perinuclear staining, suggesting ER localization (Figs. 6B and 6C: m, n).
Enveloped viruses, which do not acquire their lipoprotein coat by budding through the plasma membrane bud at internal membranes, including the inner nuclear membrane (herpesviruses;, the ER (flaviviruses and rotaviruses; [44, 45], the intermediate compartment (ERGIC) (coronaviruses and poxviruses; [46, 47], and the Golgi complex (rubellaviruses, coronaviruses, and bunyaviruses; [4, 46, 48]. Usually, the accumulation of the viral surface proteins in the specific intracellular compartment determines the assembly and budding site of the virus. This intracellular accumulation is dependent on certain compartment-specific retention or retrieval signals.
For almost all bunyaviruses assembly and budding takes place in the Golgi region [4, 46, 48]. However, so far no common motifs could be identified for signals within bunyaviral glycoproteins resulting in Golgi targeting and accumulation. Indeed, even the locations of such signals within bunyaviral glycoproteins are different. For the phlebovirus Uukuniemi (UUK), a Golgi retention signal could be identified within the membrane-proximal half (aa1040) of the 81 aa long cytoplasmic domain of GN [30, 31, 38]. In contrast, for the phlebovirus Punto Toro, such signals were mapped to the transmembrane domain (TM) and the adjacent amino acids of the GN cytoplasmic domain [36, 37]. A similar localization was recently described for the Golgi retention signal in the GN of the phlebovirus Rift Valley Fever (RVF) virus GN . Notable, for the Old World hantavirus Hantaan (HTN) it was reported that the conformation of the GN/GC complex might play a more important role for Golgi accumulation than an actual primary sequence motif .
While extensive studies have been performed regarding intracellular budding sites and glycoprotein accumulation for members of the genera Orthobunyavirus, Phlebovirus, Hantavirus and Tospovirus [30, 32, 34–36, 39, 40], nothing is known for members of the genus Nairovirus. Here we demonstrated, for the first time, that the CCHFV GN protein is membrane associated and contains a Golgi localization motif. In addition we have mapped this signal to a hydrophobic region (TM II) within the predicted cytoplasmic tail . Co-expressed GN and GC result in a specific Golgi accumulation and co-localization using specific Golgi markers, whereas individual expressed GC is retained in the ER. These results imply that the two CCHFV glycoproteins have to interact and form hetero-oligomers for a proper Golgi transport of GC.
GFP-fusion proteins containing different portions of the CCHF GN glycoprotein allowed mapping of the Golgi targeting sequence within the cytoplasmic domain. Interestingly, we located the signal downstream of the predicted protease cleavage site RKLL at position 808 of the CCHFV precursor GPC, responsible for generating the C-terminus of the mature GN protein [5, 28], implying that this cleavage site might not be used during the maturation process of GN. Furthermore, we could demonstrate that the addition of only the hydrophobic region from the predicted TM II within the GN cytoplasmic domain targeted a GFP-fusion protein to the Golgi complex. This shows that the 23 amino acids of TM II are sufficient and necessary for targeting GFP to the Golgi region, whereas the first 99 amino acids from the cytoplasmic domain and the TM I domain do not contribute to Golgi targeting.
The results obtained from the GFP-GN fusion proteins seem contradictory to the studies with the GN expression plasmid. IFA data combined with confocal microscopy co-localization studies of cells transfected with GNs expression plasmids demonstrated a clear Golgi complex staining (Fig. 3A: b, g; Fig. 4e). Since GNs contains only the first 87 amino acids of the predicted cytoplasmic domain without the predicted TM II sequence, we expected that the corresponding GFP-fusion protein GFP-GNA would show similar intracellular localization. However, the diffuse staining throughout the cytoplasm of transfected cells demonstrates that the first 87 amino acids are not sufficient to target the GFP to the Golgi complex (GFP-GNA; Figs. 6B and 6C: c). A possible explanation for this discrepancy is the existence of a second Golgi localization signal located within the GN ectodomain. Such a signal would be the reason for the Golgi localization pattern of GNs, whereas GFP-GNC and fusion proteins containing longer fragments of the predicted GN cytoplasmic domain localize to the Golgi region because of a Golgi localization signal located in TM II.
CCHFV GC protein expressed by its own retained in the ER and did not relocate into the Golgi complex. Interestingly, similar to all described GC proteins of phleboviruses CCHFV GC proteins also contain a lysine-based ER retrieval signal (KKXX;  within the predicted cytoplasmic domain. In case of single expressed GC protein this signal is most likely responsible for the ER localization of the protein, even though GFP-CCHF GCA fusion proteins containing only the predicted TM showed perinuclear staining pattern (Figs. 6B and 6C: m). However, co-expression with GN protein leads to interaction between these two proteins most likely resulting in masking of the ER retrieval signal and an accumulation of the heterodimer in the Golgi complex, due to the Golgi retention signal(s) located on GN (Fig. 3A: d, e, i, j; Fig. 4g). A similar phenomenon with conflicting transport/targeting signals was previously described for the rubellavirus E1 and E2 proteins [48, 50, 51].
In conclusion, we were able to express CCHF GN and GC glycoproteins individually as well as from the precursor GPC. GN could be localized to the Golgi compartment, whereas GC was found in the ER. Co-expression of both proteins resulted in Golgi rescue of GC, indicating that proper interaction between GN and GC is important for transportation of the heterodimer out of the ER. The potential Golgi targeting signal could be localized to a hydrophobic region within the cytoplasmic domain in the GN protein. Furthermore, our results suggest that additional signals could be localized within the GN ectodomain.
Further characterization of the CCHFV GN Golgi retention signals could provide helpful information to understand the proteolytic cleavage event(s) of the GPC and the glycoprotein maturation process. The different CCHFV G expression plasmids might show also useful for the generation of virus-like particles (VLPs) as well as for identification of interaction sites between the viral glycoproteins and the ribonucleoproteins.
The identification of the potential budding site(s) of nairoviruses and the detailed analysis of the Golgi localization signal of the CCHFV GN protein will allow subsequent studies for targeting the glycoprotein accumulation during the development of antiviral strategies or even for rational vaccine design.
Cells and virus
BHK-21 (baby hamster kidney), 293T (human embryonic kidney), VeroE6 (African green monkey kidney) and SW13 cells (human adenocarcinoma cells)(American Type Culture Collection) were grown on plastic dishes in Glasgow (BHK-21), Eagle's minimal essential (293T, VeroE6), or Leibovitz L15 (SW13) medium, respectively, supplemented with 5 to 10% fetal calf serum, 2 mM L-glutamine, 100 IU of penicillin/ml, and 100 μg of streptomycin/ml (Invitrogen). The CCHFV, strain IbAr10200, isolated in 1970 from ticks (Hyalomma excavatum) in Nigeria (Sokoto), kindly provided by Special Pathogens Branch, Centers for Disease Control and Prevention, Atlanta (T. G. Ksiazek), was used for all experiments. The CCHFV stocks were prepared on SW13 cells by infection of T162 cell culture flasks with a 1:100 dilution. Supernatant was collected three days post infection (p.i.), clarified from cell debris by low speed centrifugation (3,000 × g, 10 min, 4°C), and aliquots were stored in liquid nitrogen. Virus titers were determined either by plaque assay or 50% tissue culture infectious dose assay (TCID50).
Sequence determination of the full-length CCHFV M segment
Total RNA was isolated 7 days post infection from VeroE6 cells infected with CCHFV (1:1000 dilution of virus stock; 10-3 pfu, RNA sample #1). Additional CCHFV RNA was kindly provided by J. Smith, USAMRIID, Alphavax, Durham, N.C. (RNA sample #2). CCHFV specific M segment vRNA or cRNA molecules were reverse transcribed using the primers CCHF M1 (TCTCAAAGAAATAGTGGCGGCACGCAGTC) or CCHF M2 (TCTCAAAGAAATACTTGCGGCACGTCAGT) for the reverse transcription reaction, respectively. The resulting cDNA molecules were used as templates for subsequent PCR reactions producing overlapping PCR fragments covering the entire CCHFV M segment. PCR products were inserted into pCR4 using the TOPO TA cloning kit (Invitrogen). Prior to sequence determination, positive clones were screened by PCR technology (primer TOPO F: AGCTCGGATCCACTAGTAACG and TOPO R: ATGCTCGAGCGGCCGCCAGTG) and restriction enzyme digest (Eco RI). For vRNA and cRNA-based constructs three of the cloning plasmids were sequenced using primers specific for the M segment ORF. The sequence results were aligned to the genebank sequence U39455 using the Align Plus 5 program of the Clone Manager Professional Suite 6 (Scientific & Educational Software). Determined nucleotide exchanges and the corresponding amino acid differences are listed in Table 1. For sequence determination of the M segment ends CCHFV specific vRNA and cRNA molecules were ligated using T4 RNA ligase (Roche) prior to the reverse transcription reaction (vRNA: M32: AGAACCAGAGGCCTGTTCAA, cRNA: M33: AAGGTGTCTGTGCCGGTTGT). Subsequent PCR amplification with primers CCHF M34 (AATACTAGTCTAAT CCACTGGCTGGTGTT) and M35 (AATGAATTCTGCCGAACTGTTCTCTAC) generated fragments containing both segment ends. PCR products were inserted into pCR4 (Invitrogen) for direct sequence determination as described above. In total, 12 cRNA/mRNA and 37 vRNA clones were analyzed using T7 and T3 promoter-specific primers.
CCHFV glycoprotein expression plasmids
Based on the recently published N-terminal sequence determination of mature CCHFV glycoproteins , expression plasmids for both glycoproteins were generated. In case of the CCHFV GN two constructs were generated since the C terminus of the mature GN is not yet experimentally determined: pCMV CCHF GN "short" (GNs) contains the GN-ORF from pos. 519 to 807, preceding the predicted C-terminal cleavage site RKLL at position 808 (44). pCMV CCHF GN "long" (GNl) consists of pos. 519 to 1040 extending the GN-ORF to the experimentally determined N-terminal end of GC. The PCR fragments (GNs: RF346/352, GNl: RF353/352) were inserted after Bsm BI endonuclease treatment into pDisplay (Invitrogen) previously digested with Bgl II/Xma I digest, resulting in CMV-driven (human cytomegalovirus immediate early promoter and enhancer) expression plasmids for CCHFV GN. The Ig κ-chain signal peptide sequence and the hemagglutinin A (HA) epitope of the pDisplay vector were used for correct intracellular processing and detection, respectively. The CCHFV GC was PCR-amplified using primers CCHF M9 (AGTTGGTCTAGCCAATGTGTG) and RF351 (AATCGTCTCAAATTCATGGAGAC AGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACTTCCTAGATAGTACAGCTAAAGGCATG) (pos. 1041 to 1684).
Bsm BI- and Xho I-restricted PCR fragments were inserted into the plasmid pCAGGS/MCS  prior digested with Eco RI/Xho I digest, resulting in a chicken β-actin-driven expression plasmid for CCHFV GC. For correct intracellular processing of the CCHFV GC we inserted the Ig κ-chain signal peptide of the pDisplay vector via forward oligonucleotide primer RF351.
Different expression strategies (CMV-, chicken β-actin-driven) were used for the different CCHFV glycoproteins to yield maximum expression levels.
CCHFV glycoprotein expression plasmid DNA was transfected into subconfluent BHK-21 or 293T cells (3 × 106) using 2 to 4 μg of the respective plasmid and 8 μl of liposome plus buffer (LipofectAMINE PLUS; Life Technologies, Invitrogen) mixed in serum-free MEM and incubated for 15 min at room temperature. After addition of 12 μl of liposome reagent, incubation was continued for a further 15 min. The cells were incubated at 37°C with the DNA-Lipofectamine mixture for 3 h. To determine the efficiency of transfection, plasmid pHL2823, expressing enhanced GFP (EGFP) under the CMV immediate early promoter and enhancer (R. Flick and G. Hobom, unpublished), was transfected similarly. After further incubation for 20–24 h in MEM containing 2% FCS, the transfected cells were fixed and CCHFV glycoprotein expression levels determined using indirect immunofluorescence assays (IFA).
Indirect immunofluorescence assay
293T or BHK-21 cells grown on coverslips within a 6-well dish were transfected as described above. After 20 to 44 h, cycloheximide (final concentration of 0.18 mM) was added when indicated to inhibit further protein synthesis. The cells were incubated for an additional 2 to 5 h and then washed with phosphate buffer saline (PBS, pH 7.5) and fixed in methanol:acetone (50:50, V/V) for 20 min at -20°C. Permeabilization was omitted by fixation with paraformaldehyde when surface-expressed proteins were to be detected. After fixation, cells were washed with PBS and blocked for at least 30 min with PBS containing 5 % bovine serum albumin (BSA). Poly- or monoclonal antisera were diluted in PBS containing 1 % BSA and incubated for 1 h at room temperature. After several washes with PBS, goat anti-rabbit or mouse immunoglobulin secondary antibodies conjugated to fluorescein isothiocyanate (FITC) or tetramethyl rhodamin isothiocyanate (TRITC) were incubated with the cells for 45 to 60 min at room temperature. Procedures were repeated for double labeling with a different antiserum and fluorescent probe, and at the end of the procedure the slides were washed with PBS overnight.
Intracellular localization of the glycoproteins GN was determined by co-localization with commercially available organelle-specific fluorescent dyes (Molecular Probe Inc., Oregon, USA): BODIPY-TR C5 ceramide was selected as an indicator of the Golgi region. In addition Golgi (mannosidase II; Sigma) and ER-specific (Calreticulin; Sigma) monoclonal or polyclonal antibodies were used.
Sample preparation and immunocytochemical staining were the same as for wide-field fluorescence microscopy. The fluorescence staining patterns were analysed with a ZEISS LSM 510 UV META laser scanning confocal microscope (Jena, Germany) equipped with a Coherent Enterprise II 81 mW Argon UV laser, a Lasos 30 mW Argon laser, and 5 mW HeNe laser. Images were acquired with a C-apochromat 63/1.2 corr. water-immersion lens. FITC-stained proteins were imaged with excitation at 488 nm and with a 505 to 530 nm bandpass emission filter. Golgi marker BODIPY-TR C5 ceramide were imaged with excitation at 543 nm and with a 570 to 655 nm bandpass emission. DAPI-stained DNA was imaged with excitation at 364 nm and emission through a 385 to 470 bandpass filter. Merged pictures for analysis of intracellular co-localization were generated using Zeiss LSM Image Brower 3.2 software.
Alkaline carbonate extraction was performed on BHK-21 cells 24–48 h post transfection. The protocol described in Current Protocols in Cell Biology Online, John Wiley & Sons, Inc. was followed. Briefly, BHK-21 cells were transfected with individual constructs as described before. At 24 to 48 h post transfection, supernatant was removed and cells were washed three times with PBS followed by an additional washing step with 100 ml NaCl. Occasionally, the transfected cells would detach from the plate thus, the non-adherent cells were isolated between washes by microcentrifugation (2 min at 1000 × g). Remaining cells were scraped (adherent) or resuspended (non-adherent) into 1 ml of ice-cold 100 mM sodium carbonate, pH 11.5 and homogenized (five strokes) in a 2 ml Dounce homogenizer. The homogenate was then incubated for 30 min on ice and 1 ml of sodium carbonate was added to attain the necessary volume for subsequent ultracentrifugation (2 ml). The homogenate was then centrifuged for 60 min at 50.000 rpm using a TLS-55 rotor (Beckman) at 4°C. Following centrifugation, the supernatant was transferred to a fresh tube and concentrated three to five times. The pellet was resuspended in 250 μl of sodium carbonate. Pellet and supernatant fractions were then mixed with 4× SDS-PAGE sample buffer containing β-mercaptoethanol and run on SDS-PAGE. Protein gels were then transferred to PVDF transfer membrane (Amersham Biosciences) using a Trans-blot SD semi-dry transfer apparatus (Bio-Rad). Proteins were subsequently visualized by immunoblot.
Following transfer, the blot was blocked overnight in 5 % skim milk + 0.1 % Tween. The following morning, the blot was washed three times with PBS/0.1 % Tween then incubated with the primary antibody, e.g. anti-GFP (Oncogene) at a 1:2000 dilution in PBS for 1 h at room temperature with rocking. The blot was then washed three times with PBS/0.1 % Tween and incubated with the secondary antibody, goat anti rabbit HRP (Sigma) at a 1:30.000 dilution in PBS for 1 h at room temperature with rocking. The blot was then washed with PBS/0.1 %Tween three times, followed by three washes with PBS. Proteins were visualized using the ECL+plus Western Blotting Detection system (Amersham Biosciences).
We are grateful to Stuart T. Nichol (Special Pathogens Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, GA) for providing antibodies against the CCHF virus glycoproteins GC and GN. We also thank Sherif R. Zaki (Molecular Pathology and Ultrastructure Activity, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, GA) for providing anti-CCHFV IbAr10200 immune serum. CCHF viral stocks and viral RNA were kindly provided by Special Pathogens Branch, Centers for Disease Control and Prevention, Atlanta (T. G. Ksiazek) and by J. Smith, USAMRIID, Alphavax, Durham, N.C.
Images were captured using the laser confocal scanning microscope at the Infectious Disease and Toxicology Optical Imaging Core (OIC) facility with the assistance of Eugene Knutson, manager, and supervision of Thomas Albrecht, director, at the Department of Microbiology and Immunology, University of Texas Medical Branch (UTMB). We also like to thank Thomas Bednarek at the UTMB, Department of Pathology Imaging Laboratory, for his assistance with the illustrations.
S.H. was supported by the Boehringer Ingelheim Foundation, and performed this work in partial fulfillment of the requirements for a Ph.D. degree from the Julius-Maximilians University Wuerzburg.
- van Regenmortel MHV, Fauquet CM, Bishop DML, Carstens EB, Estes MK, Lemon SM, Maniloff J, Mago MA, McGeoch DJ, Pringle CR, Wicknen RB: 7th report of the International Committee of Taxonomy of Viruses. Virus Taxonomy 2000, 599-621.Google Scholar
- Elliott RM, Schmaljohn CS, Collett MS: Bunyaviridae genome structure and gene expression. Curr Top Microbiol Immunol 1991, 169: 91-141.PubMedGoogle Scholar
- Schmaljohn CS, Le Duc JW: Bunyaviridae. In Topley and Wilson's Microbiology and Microbial Infections. 9th edition. Edited by: Collier LH. London , Edward Arnold; 1998:601-628.Google Scholar
- Schmaljohn CS, Hooper JW: Bunyaviridae: the viruses and their replication. In Fields virology. Edited by: Knipe DMPMH. Philadelphia , Lippincott Williams and Wilkins; 2001:1581-1602.Google Scholar
- Sanchez AJ, Vincent MJ, Nichol ST: Characterization of the glycoproteins of Crimean-Congo hemorrhagic fever virus. J Virol 2002,76(14):7263-7275. 10.1128/JVI.76.14.7263-7275.2002PubMed CentralView ArticlePubMedGoogle Scholar
- Yang ZY, Duckers HJ, Sullivan NJ, Sanchez A, Nabel EG, Nabel GJ: Identification of the Ebola virus glycoprotein as the main viral determinant of vascular cell cytotoxicity and injury. Nat Med 2000,6(8):886-889. 10.1038/78645View ArticlePubMedGoogle Scholar
- Casals J: Antigenic similarity between the virus causing Crimean hemorrhagic fever and Congo virus. Proc Soc Exp Biol Med 1969,131(1):233-236.View ArticlePubMedGoogle Scholar
- Chumakov MP: A new tick-borne disease-Crimean hemorrhagic fever. In Crimean Hemorrhagic Fever (Acute Infectious Capillary Toxicosis). Edited by: Sokolov AACMPKAA. Simferopol, Russia , Izd. Otd. Primorskoi Armii; 1945.Google Scholar
- Chumakov MP: Contribution to 30 years of investigation of Crimean haemorrhagic fever. Med Virol 1974, 22: 5-18.Google Scholar
- Gonzalez JP, Camicas JL, Cornet JP, Wilson ML: Biological and clinical responses of west African sheep to Crimean-Congo haemorrhagic fever virus experimental infection. Res Virol 1998,149(6):445-455. 10.1016/S0923-2516(99)80013-2View ArticlePubMedGoogle Scholar
- Leshchinskaya EV: Clinical picture of Crimean hemorrhagic fever (in Russian). (in English: NAMRU3-1856) 1965, 7: 226-236 (in English: NAMRU3-1856).Google Scholar
- Oldfield EC, Wallace MR, Hyams KC, Yousif AA, Lewis DE, Bourgeois AL: Endemic infectious diseases of the Middle East. Rev Infect Dis 1991, 13 Suppl 3: S199-217.View ArticlePubMedGoogle Scholar
- Shepherd AJ, Swanepoel R, Cornel AJ, Mathee O: Experimental studies on the replication and transmission of Crimean-Congo hemorrhagic fever virus in some African tick species. Am J Trop Med Hyg 1989,40(3):326-331.PubMedGoogle Scholar
- Swanepoel R: Nairovirus Infections. In Exotic Viral Infections. Edited by: Porterfield JS. London ; 1995.Google Scholar
- Swanepoel R, Gill DE, Shepherd AJ, Leman PA, Mynhardt JH, Harvey S: The clinical pathology of Crimean-Congo hemorrhagic fever. Rev Infect Dis 1989, 11 Suppl 4: S794-800.View ArticlePubMedGoogle Scholar
- Watts DMTGKKJLHH: Crimean-Congo hemorrhagic fever. In The arboviruses: epidemiology and ecology. Edited by: Monath TP. Boca Raton, Florida , CRC Press Inc.; 1988:177-222.Google Scholar
- Yen YC, Kong LX, Lee L, Zhang YQ, Li F, Cai BJ, Gao SY: Characteristics of Crimean-Congo hemorrhagic fever virus (Xinjiang strain) in China. Am J Trop Med Hyg 1985,34(6):1179-1182.PubMedGoogle Scholar
- Hoogstral H: The epidemiology of tickborne Crimean-Congo hemorrhagic fever in Asia, Europe and Africa (Review). J Med Entomol 1979, 15: 307-417.View ArticleGoogle Scholar
- Schwarz TF, Nitschko H, Jager G, Nsanze H, Longson M, Pugh RN, Abraham AK: Crimean-Congo haemorrhagic fever in Oman. Lancet 1995,346(8984):1230. 10.1016/S0140-6736(95)92936-3View ArticlePubMedGoogle Scholar
- Logan TM, Linthicum KJ, Bailey CL, Watts DM, Moulton JR: Experimental transmission of Crimean-Congo hemorrhagic fever virus by Hyalomma truncatum Koch. Am J Trop Med Hyg 1989,40(2):207-212.PubMedGoogle Scholar
- Sparagano OA, Allsopp MT, Mank RA, Rijpkema SG, Figueroa JV, Jongejan F: Molecular detection of pathogen DNA in ticks (Acari: Ixodidae): a review. Exp Appl Acarol 1999,23(12):929-960. 10.1023/A:1006313803979View ArticlePubMedGoogle Scholar
- Zeller HG, Cornet JP, Camicas JL: Experimental transmission of Crimean-Congo hemorrhagic fever virus by west African wild ground-feeding birds to Hyalomma marginatum rufipes ticks. Am J Trop Med Hyg 1994,50(6):676-681.PubMedGoogle Scholar
- Fagbami AH, Tomori O, Fabiyi A, Isoun TT: Experimantal Congo virus (Ib -AN 7620) infection in primates. Virologie 1975,26(1):33-37.PubMedGoogle Scholar
- Swanepoel R, Leman PA, Burt FJ, Jardine J, Verwoerd DJ, Capua I, Bruckner GK, Burger WP: Experimental infection of ostriches with Crimean-Congo haemorrhagic fever virus. Epidemiol Infect 1998,121(2):427-432. 10.1017/S0950268898001344PubMed CentralView ArticlePubMedGoogle Scholar
- Fisher-Hoch SP, McCormick JB, Swanepoel R, Van Middlekoop A, Harvey S, Kustner HG: Risk of human infections with Crimean-Congo hemorrhagic fever virus in a South African rural community. Am J Trop Med Hyg 1992,47(3):337-345.PubMedGoogle Scholar
- Mayers DL: Exotic virus infections of military significance. Hemorrhagic fever viruses and pox virus infections. Dermatol Clin 1999,17(1):29-40, vii-viii. 10.1016/S0738-081X(98)00065-0View ArticlePubMedGoogle Scholar
- Vincent MJ, Sanchez AJ, Erickson BR, Basak A, Chretien M, Seidah NG, Nichol ST: Crimean-Congo hemorrhagic fever virus glycoprotein proteolytic processing by subtilase SKI-1. J Virol 2003,77(16):8640-8649. 10.1128/JVI.77.16.8640-8649.2003PubMed CentralView ArticlePubMedGoogle Scholar
- Lenz O, ter Meulen J, Klenk HD, Seidah NG, Garten W: The Lassa virus glycoprotein precursor GP-C is proteolytically processed by subtilase SKI-1/S1P. Proc Natl Acad Sci U S A 2001,98(22):12701-12705. 10.1073/pnas.221447598PubMed CentralView ArticlePubMedGoogle Scholar
- Andersson AM, Melin L, Bean A, Pettersson RF: A retention signal necessary and sufficient for Golgi localization maps to the cytoplasmic tail of a Bunyaviridae (Uukuniemi virus) membrane glycoprotein. J Virol 1997,71(6):4717-4727.PubMed CentralPubMedGoogle Scholar
- Andersson AM, Pettersson RF: Targeting of a short peptide derived from the cytoplasmic tail of the G1 membrane glycoprotein of Uukuniemi virus (Bunyaviridae) to the Golgi complex. J Virol 1998,72(12):9585-9596.PubMed CentralPubMedGoogle Scholar
- Chen SY, Compans RW: Oligomerization, transport, and Golgi retention of Punta Toro virus glycoproteins. J Virol 1991,65(11):5902-5909.PubMed CentralPubMedGoogle Scholar
- Chen SY, Matsuoka Y, Compans RW: Golgi complex localization of the Punta Toro virus G2 protein requires its association with the G1 protein. Virology 1991,183(1):351-365. 10.1016/0042-6822(91)90148-5View ArticlePubMedGoogle Scholar
- Gerrard SR, Nichol ST: Characterization of the Golgi retention motif of Rift Valley fever virus G(N) glycoprotein. J Virol 2002,76(23):12200-12210. 10.1128/JVI.76.23.12200-12210.2002PubMed CentralView ArticlePubMedGoogle Scholar
- Lappin DF, Nakitare GW, Palfreyman JW, Elliott RM: Localization of Bunyamwera bunyavirus G1 glycoprotein to the Golgi requires association with G2 but not with NSm. J Gen Virol 1994, 75 ( Pt 12): 3441-3451.View ArticleGoogle Scholar
- Matsuoka Y, Chen SY, Compans RW: A signal for Golgi retention in the bunyavirus G1 glycoprotein. J Biol Chem 1994,269(36):22565-22573.PubMedGoogle Scholar
- Matsuoka Y, Chen SY, Holland CE, Compans RW: Molecular determinants of Golgi retention in the Punta Toro virus G1 protein. Arch Biochem Biophys 1996,336(1):184-189. 10.1006/abbi.1996.0547View ArticlePubMedGoogle Scholar
- Pettersson RF, Andersson A, Melin L: Mapping a retention signal for Golgi localization of a viral spike protein complex. Cold Spring Harb Symp Quant Biol 1995, 60: 147-155.View ArticlePubMedGoogle Scholar
- Shi X, Lappin DF, Elliott RM: Mapping the Golgi targeting and retention signal of Bunyamwera virus glycoproteins. J Virol 2004,78(19):10793-10802. 10.1128/JVI.78.19.10793-10802.2004PubMed CentralView ArticlePubMedGoogle Scholar
- Spiropoulou CF, Goldsmith CS, Shoemaker TR, Peters CJ, Compans RW: Sin Nombre virus glycoprotein trafficking. Virology 2003,308(1):48-63. 10.1016/S0042-6822(02)00092-2View ArticlePubMedGoogle Scholar
- Flick R, Elgh F, Pettersson RF: Mutational analysis of the Uukuniemi virus (Bunyaviridae family) promoter reveals two elements of functional importance. J Virol 2002,76(21):10849-10860. 10.1128/JVI.76.21.10849-10860.2002PubMed CentralView ArticlePubMedGoogle Scholar
- Flick R, Flick K, Feldmann H, Elgh F: Reverse genetics for crimean-congo hemorrhagic fever virus. J Virol 2003,77(10):5997-6006. 10.1128/JVI.77.10.5997-6006.2003PubMed CentralView ArticlePubMedGoogle Scholar
- Mettenleiter TC: Herpesvirus assembly and egress. J Virol 2002,76(4):1537-1547. 10.1128/JVI.76.4.1537-1547.2002PubMed CentralView ArticlePubMedGoogle Scholar
- Mackenzie JM, Jones MK, Westaway EG: Markers for trans-Golgi membranes and the intermediate compartment localize to induced membranes with distinct replication functions in flavivirus-infected cells. J Virol 1999,73(11):9555-9567.PubMed CentralPubMedGoogle Scholar
- Suzuki H: A hypothesis about the mechanism of assembly of double-shelled rotavirus particles. Arch Virol Suppl 1996, 12: 79-85.PubMedGoogle Scholar
- Krijnse-Locker J, Ericsson M, Rottier PJ, Griffiths G: Characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the RER to the Golgi complex requires only one vesicular transport step. J Cell Biol 1994,124(1-2):55-70. 10.1083/jcb.124.1.55View ArticlePubMedGoogle Scholar
- Moss B: Poxviridae: the viruses and their replication. In Fields virology. Edited by: Knipe DMPMH. Philadelphia , Lippincott Williams and Wilkins; 2001:2849-2871.Google Scholar
- Hobman TC, Zhao B, Chan H, Farquhar MG: Immunoisolation and characterization of a subdomain of the endoplasmic reticulum that concentrates proteins involved in COPII vesicle biogenesis. Mol Biol Cell 1998,9(6):1265-1278.PubMed CentralView ArticlePubMedGoogle Scholar
- Jackson MR, Nilsson T, Peterson PA: Identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum. Embo J 1990,9(10):3153-3162.PubMed CentralPubMedGoogle Scholar
- Hobman TC, Woodward L, Farquhar MG: The rubella virus E1 glycoprotein is arrested in a novel post-ER, pre-Golgi compartment. J Cell Biol 1992,118(4):795-811. 10.1083/jcb.118.4.795View ArticlePubMedGoogle Scholar
- Hobman TC, Woodward L, Farquhar MG: The rubella virus E2 and E1 spike glycoproteins are targeted to the Golgi complex. J Cell Biol 1993,121(2):269-281. 10.1083/jcb.121.2.269View ArticlePubMedGoogle Scholar
- Niwa H, Yamamura K, Miyazaki J: Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 1991,108(2):193-199. 10.1016/0378-1119(91)90434-DView ArticlePubMedGoogle Scholar
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