Alternative Proteolytic Processing of Mouse Mammary Tumor Virus Superantigens

Abstract

Mouse mammary tumor viruses express a superantigen essential for their life cycle. It has been proposed that viral superantigens (vSags) require processing by prohormone convertases (PCs) for activity. We now observe, using a panel of mutant forms of potential PC cleavage sites and in vitro cleavage assays, that only the CS1 (position 68 to 71) and CS2 (position 169 to 172) sites are utilized by furin and PC5. Other members of the convertase family that are expressed in lymphocytes are not endowed with this activity. Furthermore, mutant forms of two different viral superantigens, vSag7 and vSag9, which completely abrogated in vitro processing by convertases, were efficient in functional presentation to responsive T-cell hybridomas. This effect was observed in both endogenous presentation and paracrine transfer of the vSag. Processing by convertases thus appears not to be essential for vSag function. Finally, we have identified the purified endosomal protease cathepsin L as another protease that is able to cleave convertase mutant vSag in vitro, yielding fragments similar to those detected in vivo, thus suggesting that proteases other than convertases are involved in the activation of vSags.

A Defective Viral Superantigen-Presenting Phenotype in HLA-DR Transfectants Is Corrected by CIITA

“…This last result suggests that if vSAgs gain access to the endocytic pathway, their passage must be swift and is probably not compatible with their intercalation in a putative class II-Ii-MTV nonamer. In furin-negative cells, it was proposed that alternative cleavage by cathepsins could generate an active form of vSAg (62,63). HEK 293T cells are furin negative, and the efficient transfer of vSAg is in accordance with the existence of alternative cleavage sites.…”

“…The nature of the active fragment remains ill defined; redundancy has been reported regarding the potency of the furin cleavage sites in activating vSAgs (61). Also, cathepsin L was shown to liberate a 27-kDa fragment, which could explain the activity of vSAgs in furin-deficient cells or after mutagenesis of the furin recognition motifs (62,63). In addition to activating the vSAg, processing would explain the paracrine transfer of a soluble active C-terminal fragment to class II-positive APCs (64 -66).…”

“…Accordingly, it was later proposed that successful presentation of vSAg7 is conditional to the existence of a diverse, class II-bound peptide repertoire (71). Indeed, endogenous vSAg-induced T cell deletion was not taking place in modified mice expressing class II molecules almost exclusively loaded with CLIP (DM knockout (KO)) or loaded with a covalently linked peptide (E␣ [52][53][54][55][56][57][58][59][60][61][62][63][64][65][66][67][68] ). These results contrast with those obtained in vitro by Hsu et al (72), which suggest that class II-vSAg interactions occur in the ER through a CLIP-like region of vSAg occupying the peptide binding groove.…”

The Type B Leukemogenic Virus Truncated Superantigen Is Dispensable for T-Cell Lymphomagenesis

“…This ORF encodes a 37-kDa, type II transmembrane glycoprotein known as superantigen (Sag) that functions in the milk-borne transmission of MMTV (1,7,9,10,16) by the stimulation of T cells through the T-cell receptor (TCR) (33,35). The truncated Sag protein encoded in the TBLV LTR lacks the entire C-terminal region that interacts with the TCR, yet it retains the transmembrane region, a part of the extracellular region that includes the five glycosylation sites, a single proteolytic processing site, and the major histocompatibility complex class II protein binding motif (12,20). This study shows that the truncated TBLV Sag protein is dispensable for virally induced T-cell lymphomas.…”

Nipah virus fusion protein: Influence of cleavage site mutations on the cleavability by cathepsin L, trypsin and furin

“…Many viral and cellular proteins are known to be processed by the cysteine proteases cathepsin L or B as well as by trypsin or furin-related prohormone convertases which belong to the family of serine proteases. For example, the viral superantigen 7 (vSag7) of the mouse mammary tumor virus can be processed by furin or by cathepsin L. However, activation by both enzymes has different sequence requirements and does likely not occur at exactly the same position (Denis et al, 2000). The requirement for pHdependent cathepsin L cleavage to restore the fusion activity of the SARS coronavirus spike protein can be circumvented by trypsin treatment demonstrating that the spike protein provides recognition sites for both, cathepsins and trypsin (Simmons et al, 2005).…”