Medical Virology Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA1
Author for correspondence: Jeffrey Cohen.Fax +1 301 496 7383. e-mail jcohen{at}niaid.nih.gov
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Abstract |
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The bcl-2 family of proteins includes both anti-apoptotic (e.g. bcl- 2, bcl-XL) and pro-apoptotic (e.g. bak, bax) proteins. Members of this family contain up to four bcl-2 homology domains (BH1 to BH4). The BH1 and BH2 domains are important for bcl-2 and bcl-XL to form heterodimers with bax (reviewed in Kroemer, 1997 ). The BH3 domain of bak and bax is critical for these proteins to form heterodimers with bcl-2 and bcl-XL. The BH4 domain of bcl-2 is important for interactions with other cellular proteins.
Most herpesvirus bcl-2 homologues show conservation of sequences in both the BH1 and BH2 domains with respect to bcl-2, but have little homology with other regions of bcl-2. The viral bcl-2 proteins have poorly conserved BH3 domains and lack BH4 domains. Each of the gammaherpesviruses for which sequence is available, including EpsteinBarr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), herpesvirus saimiri (HVS), murine herpesvirus-68 (MHV-68), herpesvirus aeteles, alcelaphine herpesvirus-1, bovine herpesvirus-4 and equine herpesvirus-2, encodes a bcl-2 homologue (reviewed in Neipel et al., 1997 ; Tschopp et al., 1998
). However, only the EBV BHRF1 (Henderson et al., 1993
; Kawanishi, 1997
), KSHV ORF16 (Cheng et al., 1997b
; Sarid et al., 1997
) and HVS ORF16 (Nava et al., 1997
) proteins, which have both BH1 and BH2 domains, have been shown to inhibit apoptosis.
The MHV-68 genome contains an open reading frame, M11 (Virgin et al., 1997 ), that is predicted to encode a 171 amino acid protein that has limited homology to bcl-2 (Fig. 1
). While the M11 protein has a well conserved BH1 domain, the BH2 domain is apparently absent, unlike most of the other gammaherpesvirus bcl-2 homologues. Although MHV-68 M11 is located in a similar position in its genome as the EBV BHRF1 bcl-2 homologue is in the EBV genome, the degree of similarity was insufficient to consider it a true homologue of other gammaherpesvirus bcl-2-like proteins, and Virgin et al. (1997
) postulated that the M11 protein may have functions distinct from the other viral bcl-2 homologues. Here we show that the MHV-68 M11 protein, like its gammaherpesvirus counterparts, can inhibit apoptosis.
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Transfection of HeLa cells with plasmid expressing the HA-tagged M11 fusion protein followed by incubation with anti-HA antibody (HA.11; Berkeley Antibody Co.) and a fluorescein isothiocyanate-conjugated goat anti-mouse antibody showed that M11 was expressed diffusely throughout the cytoplasm, possibly with a small amount of nuclear staining, in transfected cells (Fig. 2 ). In virus-infected cells, the pattern of localization may be different in the presence of other viral proteins; however, the EBV bcl-2 homologue also localizes to the cytoplasm of virus-infected cells (Hickish et al., 1994
) . Transfection of HeLa cells with empty vector (pCI) showed only background immunofluorescent staining (not shown).
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Treatment of cells transfected with the control vector followed by anti-Fas antibody or TNF resulted in <5% protection from apoptosis. In contrast, treatment of cells transfected with either M11 or bcl-X L resulted in 70% protection from apoptosis (Fig. 3
a). Since TNF has been shown to be a potent antiviral agent (Wong & Goeddel, 1986
) and Fas can be used by cytotoxic T cells (CTLs) to initiate killing of virus-infected cells (Nagata, 1997
), the ability of M11 to block Fas- and TNF-induced apoptosis may protect MHV-68 virus-infected cells from destruction by CTLs or TNF. A recent report showed that the EBV BHRF1 bcl-2 homologue can also block apoptosis induced by anti-Fas antibody and TNF (Kawanishi, 1997
).
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MHV-68 infects mice and induces a latent infection in B cells (Sunil- Chandra et al., 1992 ) and splenocytes (Weck et al., 1996
). The virus establishes a persistent infection in lung epithelial cells (Stewart et al., 1998)
. The M11 protein, if similar to its other gammaherpesvirus homologues, is likely to be expressed during lytic but not latent infection with MHV-68. Thus, the M11 protein may protect virus-infected cells from TNF or Fas-induced apoptosis, allowing the virus to complete its replication cycle. MHV-68 DNA has been detected in some lymphomas in mice (Sunil-Chandra et al., 1994)
. Other gammaherpesviruses with bcl-2 homologues (e.g. EBV, HVS) are tumorigenic in certain animal models. Since bcl-2 enhances oncogenesis in certain models (Vaux et al., 1988)
, the M11 protein may enhance tumorigenesis by MHV-68.
MHV-68 M11 has a number of features that may make it a better inhibitor of apoptosis than bcl-2. The lack of BH2, BH3 and BH4 domains in M11 may prevent interactions of the viral protein with bax and bak which could otherwise inhibit the pro-apoptotic function of the viral protein. M11 lacks the loop structure located between the BH4 and BH3 domains of bcl-2. Cleavage of this loop in bcl-2 by caspases results in conversion of bcl-2 to a pro-apoptotic protein (Cheng et al., 1997a ). Thus, MHV-68 may have taken the bcl-2 gene from the murine genome and modified it to produce a more potent anti-apoptotic protein.
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References |
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Received 7 December 1998;
accepted 9 June 1999.