An InternalRibosome Entry Site Directs Translation of the Murine Gammaherpesvirus68 MK3 Open ReadingFrame

Abstract

The gammaherpesviruses characteristically drive the proliferation of latently infected lymphocytes. The murine gammaherpesvirus 68 (MHV-68) MK3 protein contributes to this process in vivo by evading CD8؉ -T-cell recognition during latency, as well as during lytic infection. We analyzed some of the molecular mechanisms that control MK3 expression. No dedicated MK3 mRNA was detected. Instead, the MK3 open reading frame (ORF) was transcribed as part of a bicistronic mRNA, downstream of a previously unidentified ORF, 13M. The 13M/MK3 promoter appeared to extend approximately 1 kb 5 of the transcription start site and included elements both dependent on and independent of the ORF50 lytic transactivator. MK3 was translated from the bicistronic transcript by virtue of an internal ribosome entry site (IRES) element. RNA structure mapping identified two stem-loops between 13M and MK3 that were sufficient for IRES activity in a bicistronic reporter plasmid and a third stem-loop just within the MK3 coding sequence, with a subtler, perhaps regulatory role. Overall, translation of the MHV-68 MK3 bore a striking resemblance to that of the Kaposi's sarcoma-associated herpesvirus vFLIP, suggesting that IRES elements are a common theme of latent gammaherpesvirus immune evasion in proliferating cells.The murine gammaherpesvirus 68 (MHV-68) is a natural parasite of mice (4, 5) that is related to the Kaposi's sarcomaassociated herpesvirus (KSHV). Thus, we can learn from MHV-68 something of how KSHV persists in immunocompetent hosts and causes disease. Some 90% of MHV-68 genes have clear position or sequence homologs in KSHV (42). However, the homology is greatest for the genes encoding structural virion components and essential lytic replication enzymes; there is much less conservation of host interaction genes such as those concerned with immune evasion. Of course, it is precisely these functions that are difficult to define in vitro and about which MHV-68 can be most informative. Thus, much of the utility of MHV-68 as a model for human disease mechanisms depends on identifying how the host interaction functions of each virus-assumed to have a greater commonality than is apparent from DNA sequence alignments-are distributed among their more variable genes.Immune evasion is a case in point. The list of immune evasion genes for either MHV-68 or KSHV genes is far from complete, but already the general impression is that those of each virus have evolved as a coordinated set, with the acquisition of a new gene leading to modified functions for the others. KSHV has two lytic cycle genes that downregulate major histocompatibility complex (MHC) class I expression, K3 and K5, while MHV-68 has just one, MK3 (10,17,37). An MHV-68 chemokine binding protein, M3, also mediates CD8 ϩ -T-cell evasion (7, 32) and may compensate for the lack of a K5, although exactly where it fits into in vivo pathogenesis remains controversial (41). M3 may also overlap in function with the KSHV vMIPs (26). In addition to its lytic cycle repertoire, KSHV has a late...

Stress-Inducible Alternative Translation Initiation of Human Cytomegalovirus Latency Protein pUL138

“…Interestingly, polycistronic transcripts generated from latencyassociated regions of herpesviruses are not uncommon and may represent a conserved strategy for the regulation of protein expression during latency (4,8,23,31,41,69,70). For example, Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a cluster of gene products, LANA (ORF73), v-cyclin (ORF72), and v-flip (ORF71), that are important for latency (1,48).…”

Dendritic Cells Present Lytic Antigens and Maintain Function throughout Persistent γ-Herpesvirus Infection

“…This approach incorporates an ectopic copy of the 500 bp upstream of the MHV-68 M3 open reading frame (ORF) as a strong, ORF50-dependent lytic cycle promoter (31). The full-length OVA-encoding sequence was PCR-cloned between the M3 promoter and a bovine growth hormone poly-A site derived from pcDNA3 (Invitrogen Life Technologies).…”

CD4+ T cells specific for a model latency‐associated antigen fail to control a gammaherpesvirus in vivo

“…The ORF73-IRES-eGFP control virus has been described [5]. OVA production in the MHV-68 lytic cycle (MHV-OVA) was from an intergenic expression cassette [62] that incorporates an ectopic copy of the 500 bp upstream of the MHV-68 M3 ORF as a strong, ORF50-dependent lytic cycle promoter [63]. The full-length OVA-encoding sequence was PCR-cloned as an Eco RI/Xho I fragment into Eco RI/Sal I sites between the M3 promoter and a bovine growth hormone poly-A site derived from pcDNA3 (Invitrogen).…”