Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Mailstop G18, Herpesvirus Section, 1600 Clifton Road, Atlanta, GA 30333, USA
Correspondence
Naoki Inoue
nai0{at}cdc.gov
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ABSTRACT |
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Present address: Department of Pediatrics, Asahikawa Medical College, Japan.
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MAIN TEXT |
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Expression of HHV-8 gM (gM8) (ORF39) was analysed by immunoblotting of cell lysates prepared from BCBL-1 cells, a primary effusion lymphoma cell line containing latent HHV-8 (Renne et al., 1996). The lytic infection was induced by treatment of the cells with 20 ng 12-O-tetradecanoylphorbol 13-acetate (TPA) ml-1. Anti-gM8 serum was prepared by immunization of rabbits with a gM8 oligopeptide (KDISTPAPRTQYQSD) conjugated with keyhole limpet haemocyanin, as described previously (Inoue et al., 2000
). Two proteins with molecular masses of 46 and 80 kDa (46K and 80K proteins, respectively) were detected in TPA-treated but not in untreated BCBL-1 cells (Fig. 1
A). To confirm that these were gM8 products, we constructed pcDNA-gM8 by PCR amplification with primers 5'-ccgaattcATCAGATTGGCTCTCCCGCCGCAG-3' (GenBank U75698, nt 6023360210) and 5'-ccgctcgagCTAAATGAATATCATTTGCGTTTCG-3' (nt 5897358997), followed by cloning into pcDNA3 (Invitrogen). Two identical 46K and 80K proteins were detected in 293T cells transfected with pcDNA-gM8 (Fig. 1B
), indicating that these are gM8 products. We also constructed pCMV-gM8F, which expresses gM8 with a FLAG tag, by PCR with the same forward primer and a reverse primer, 5'-ccgctcgagAATGAATATCATTTGCGTTTCGTCG-3', followed by cloning into pCMV-tag4 (Stratagene). Anti-FLAG and anti-gM8 antibodies reacted with the same 46K and 80K proteins in 293T cells transfected with pCMV-gM8F (data not shown), which confirmed the specificity of the anti-gM8 antibody and the integrity of the plasmids. Culturing the cells in the presence of tunicamycin (TM) for 48 h decreased the molecular mass of the gM8 products to 39 and 71 kDa (Fig. 1A, B
), suggesting N-glycosylation of gM8.
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Next, we examined whether gM8 is a virion component. Virion samples were prepared from culture supernatant of TPA-treated BCBL-1 cells by passage through 0·45 µm membranes and centrifugal precipitation, followed by two sucrose gradient centrifugations. The virion-associated gM8 products were 48 kDa and 86 kDa proteins, slightly larger than those expressed in TPA-induced BCBL-1 cells (Fig. 1E).
HHV-8 virions and the cell extracts prepared from 293T cells transfected with pcDNA-gM8 were enzymatically deglycosylated with peptide: N-glycosidase F (PNGaseF) and with endoglycosidase H (EndoH). Only the 80K protein and its digested products were analysable because the incubation at 37 °C decreased the amount of 46K protein (Fig. 1C). Although gM8 in the virions was EndoH-resistant, gM8 in the transfected 293T cells was EndoH-sensitive (Fig. 2
A), suggesting that transiently expressed gM8 is post-translationally processed differently from the form present in virions.
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The extracts prepared from 293T cells transfected with the gM8 and/or gN8Myc constructs were reacted with anti-Myc tag antibody conjugated with beads (Santa Cruz Biotechnology) for 2 h at 4 °C. gM8 was immunoprecipitated only from the extract of the cells co-transfected with gN8Myc, indicating complex formation between gM8 and gN8Myc (Fig. 2C). To confirm the specificity of the immunoprecipitation, 293T cells were transfected with pcDNA-gM8 and pA3M-K1, which expresses HHV-8 K1 glycoprotein with the same tags (K. Fries, F. R. Stamey & P. E. Pellett, unpublished). Immunoprecipitation of this extract did not co-precipitate gM8 (data not shown). In addition, gN8Myc was immunoprecipitated with anti-FLAG antibody conjugated with agarose beads (Sigma) only from the extract of the cells co-transfecte with gM8-FLAG (Fig. 2D
). Therefore, our results indicated that gM8 forms a complex with gN8 as demonstrated in other herpesviruses. However, further study will be required to determine whether this complex is formed by direct gM8/gN8 interaction or by indirect interaction mediated through a cellular factor. gM8 in 293T cells co-transfected with the gM8 and gN8Myc plasmids was detected as a broader band with a slightly slower migration than when expressed alone (Fig. 2C
). Also gM8 co-precipitated with gN8Myc was larger than gM8 expressed alone and it was the same as that in virion samples (Fig. 2A, C
). It is possible that gN8 co-expression alters the post-translational modifications of gM8 and that only gM8 forming a complex with gN8 obtained the additional modifications, resulting in the migration difference. To examine this hypothesis, extracts of 293T cells expressing gM8 alone or gM8/gN8 together were digested with glycosidases. The PNGaseF digestion generated products with an identical size in both cases (Fig. 2A, E
). However, gM8 co-expressed with gN8, but not gM8 expressed alone, was EndoH-resistant (Fig. 2E
), indicating that gN8 is required for the additional modification of gM8. Because EndoH resistance indicates that a glycoprotein has moved from the endoplasmic reticulum to the trans-Golgi network (TGN) (Dunphy & Rothman, 1985
), our results suggest that: (i) authentic trafficking and post-translational processing of gM8 depend on gN8 expression; and (ii) HHV-8 virions acquire gM8 from the TGN.
Inhibition of cell fusion was reported previously as one of the gM functions (Klupp et al., 2000). Because the inhibition was demonstrated only for alphaherpesviruses, namely pseudorabies virus (PRV) and equine herpesvirus 1, we examined whether this is the common function of gM among the herpesvirus subfamilies. To compare the inhibition between alpha- and gammaherpesviruses, we amplified herpes simplex virus type 1 (HSV-1) gM (gM1) and gN (gN1) genes by PCR with primer sets 5'-cggaattcgccaccATGGGACGCCCGGCCCCCAG-3' and 5'-gctctagaCTACCAACGGCGGACGGTGCTGTACA-3', and 5'-cggaattcgccaccATGG GCCCCCCCAGAAGGGT-3' and 5'-gctctagaTCAGGCGTGCCCGGCAGCCAGTAGCC-3', respectively and cloned into pcDNA3. pSMH-gB, -gD, -gH and -gL, which encode HSV-1 gB, gD, gH and gL, respectively, were gifts of H. Browne (University of Cambridge, UK) (Turner et al., 1998
) and each HSV-1 glycoprotein gene was recloned into pcDNA3. 293T-T7RP was established by transfection of 293T cells with a plasmid encoding the T7 RNA polymerase (T7RP) gene (a gift of B. Moss, NIH, USA) (Elroy-Stein & Moss, 1990
) and a plasmid encoding hygromycin B phosphotransferase. A clone expressing T7RP at the highest level was selected from hygromycin B-resistant clones. Forty-eight hours after transfection of 293T-T7RP cells with plasmid(s) encoding viral glycoproteins, the cells were mixed with an equal number of 293T cells that had been transfected with pOS8, a plasmid containing the lacZ gene under control of the T7 promoter (a gift of B. Moss) (Wyatt et al., 1995
). Fusion events were measured 24 h after mixing the cells as T7RP-dependent
-galactosidase expression, using a chemiluminescent assay (Luminescent
-galactosidase detection kit II; Clontech). As shown in Fig. 3
(A), co-expression of gM1 and gN1 inhibited cell fusion by 60 %. Co-expression of gM8 and gN8 also decreased cell fusion, although more weakly than HSV-1 gM/gN. gM1 or gM8 alone did not inhibit fusion significantly (Fig. 3A
and data not shown). Similar results were obtained by X-Gal staining of fused cells. The fusion inhibition was not due to toxicity of these proteins, because the
-galactosidase expression level in 293T cells transfected with pCMV
, a plasmid containing lacZ under the control of the cytomegalovirus (CMV) promoter, and the pcDNA-gM and -gN constructs was the same as in the cells transfected with pCMV
and pcDNA3 vector (data not shown). In our preliminary experiments, human CMV gM/gN strongly inhibited the fusion induced by HSV-1 glycoproteins. Therefore, the fusion inhibition is likely to be one of the conserved gM/gN characteristics across the herpesvirus family.
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PRV gM inhibited fusion induced by the F protein of bovine respiratory syncytial virus (BRSV) (Klupp et al., 2000) and in the presence of BRSV F protein expression, bovine herpesvirus 1 (BHV-1) lacking gM formed larger syncytia than did wild-type BHV-1 (König et al., 2002
). These and our results suggest that there is a common mechanism of fusion inhibition by the gM/gN complex among some enveloped viruses. Potential mechanisms include inhibition of authentic glycoprotein processing and modification of viral glycoprotein distribution on the plasma membrane. Integration of highly hydrophobic gM proteins into membranes has been hypothesized to reduce their fluidity and consequently their ability to fuse (Klupp et al., 2000
). Interestingly, a gM mutant of BHV-1 was more susceptible to antibody-mediated neutralization (König et al., 2002
), suggesting differences in the topological distribution of glycoproteins on the viral envelope. To explain the apparently diverse defects in the EpsteinBarr virus gN mutant, Lake & Hutt-Fletcher (2000)
hypothesized that gM plays a role in association and dissociation of capsids with membranes for budding through the inner nuclear membrane during egress and for penetration through the plasma membrane during infection. Brack et al. (1999)
hypothesized that PRV gM is required for directing intracytoplasmic capsids to the TGN. Taken together with the fusion inhibition, it is possible that the gM/gN complex plays a role in virus entry and egress through modulation of glycoprotein trafficking and/or membrane fusion. Further studies to identify cellular proteins that interact with gM/gN may clarify the exact mechanisms.
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ACKNOWLEDGEMENTS |
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Received 1 November 2002;
accepted 7 February 2003.