Inefficient Codon Usage Impairs mRNA Accumulation: the Case of the v-FLIP Gene of Kaposi's Sarcoma-Associated Herpesvirus

Abstract

Like cellular FLIPs, the v-FLIP contains death effector domains (DEDs) (9), but unlike its host homologs, it does not appear to directly modulate caspase activation from the plasma membrane. Rather, it interacts with cytosolic IKK-␥ (10), leading to phosphorylation and proteasomal degradation of IB, the inhibitor of the transcription factor NF-B. As a result, NF-B undergoes constitutive activation, leading to the expression of a proinflammatory (11) and antiapoptotic (12) program. The antiapoptotic aspect of this program is essential for B-cell survival in primary effusion lymphoma (PEL) (13), and the proinflammatory signaling is speculated to play a role in the inflammatory phenotype of Kaposi's sarcoma (KS). NF-B activation in human primary endothelial cells is also responsible for the spindle-like elongation characteristic of the infected endothelial cells of KS (14). Finally, v-FLIP-mediated NF-B activation helps to stabilize the latent state by impairing lytic reactivation (15,16), and inhibition of NF-B triggers enhanced lytic induction (14). Therefore, v-FLIP expression is considered to be an important feature of KSHV latency in most lineages examined to date.Despite this, how v-FLIP is expressed in infection has been difficult to rigorously define. Expression of the protein appears to be extremely inefficient; it is difficult to detect by immunoblotting in latently infected cells, and attempts to express it from potent recombinant vectors generally result in unexpectedly low levels of protein accumulation (see reference 17 and Fig. 3 below). Northern blots of latently infected cells do not show the presence of a monocistronic mRNA for v-FLIP; rather, v-FLIP sequences detected by this method are found as the 3=-most open reading frame (ORF) in bi-or tricistronic transcripts directed from the LANA promoter (a map of the known transcripts from the MLL is shown in Fig. 1) (18-22). This seeming paradox led to the identification of sequences in the v-cyclin gene that can function as an internal ribosome entry site (IRES) element in recombinant vectors (17,23), and since that time, it has been presumed that v-FLIP is expressed from polycistronic mRNAs via IRES-mediated internal translational initiation. Direct evidence for this mechanism in infected cells has been lacking, however. In fact, reverse transcription (RT)-PCR-based studies have detected low-level expression of a spliced monocistronic v-FLIP RNA (Fig. 1) in some latently infected cells (17). Moreover, the reasons for the difficulty in

The Itch ubiquitin ligase is required for KSHV RTA induced vFLIP degradation

“…However, vFLIP expression is reduced in infected cells due to inefficient codon usage. This low level expression is advantageous to the virus, as codon optimization resulted in inefficient lytic induction (Bellare et al, 2015). These observations suggest that vFLIP must maintain a delicate balance.…”

Transcriptional and Post-Transcriptional Regulation of Viral Gene Expression in the Gamma-Herpesvirus Kaposi’s Sarcoma-Associated Herpesvirus

“…Interestingly, Vogt et al found a similar effect, reporting that ORF57 is required to sustain the levels of some viral mRNAs containing higher AT nucleotide content, which they propose is suboptimal compared to cellular nucleotide content [86]. In a separate study vFLIP was demonstrated to have suboptimal codon usage, which impaired vFLIP RNA accumulation [87]. In this case, the resulting low levels of vFLIP were optimal for lytic virus production [87].…”

“…In a separate study vFLIP was demonstrated to have suboptimal codon usage, which impaired vFLIP RNA accumulation [87]. In this case, the resulting low levels of vFLIP were optimal for lytic virus production [87]. This suggests that codon and sequence composition may be used by KSHV to further modulate its gene expression.…”

Azidothymidine Sensitizes Primary Effusion Lymphoma Cells to Kaposi Sarcoma-Associated Herpesvirus-Specific CD4+ T Cell Control and Inhibits vIRF3 Function

“…Some of these genes show intrinsic features which likely minimize exposure to CD8+ T cells by restricting synthesis of their encoded protein, reducing the supply of defective ribosomal products (DRiPs) that are thought to be the source of CD8+ T cell peptide-epitopes[6]. Firstly vFLIP utilizes inefficient codons resulting in the production of unstable mRNA and low levels of protein expression[7]. Secondly LANA encodes extensive repeat sequences which restrict translation and proteasomal mediated destruction[8], minimizing epitope presentation from this protein to CD8+ T cells[9].…”

“…The finding that knock down of vFLIP induced sensitivity to Fas ligation indicates that this viral protein can interfere with this apoptotic pathway[18], however vFLIP is thought to be expressed at low levels within infected cells[7]; whether other viral proteins contribute to this inhibition is unclear. An understanding of whether other latent proteins inhibit pathways induced by extrinsic apoptotic stimuli would allow the rational design of interventions to inhibit these functions and restore sensitivity to T cell effector mechanisms.…”