Fibropapillomatosis (FP) is a tumor disease associated with a herpesvirus (chelonid herpesvirus 5 [ChHV5]) that affects mainly green turtles globally. Understanding the epidemiology of FP has been hampered by a lack of robust serological assays to monitor exposure to ChHV5. This is due in part to an inability to efficiently culture the virus in vitro for neutralization assays. Here, we expressed two glycoproteins (FUS4 and FUS8) from ChHV5 using baculovirus. These proteins were immobilized on enzyme-linked immunosorbent assay plates in their native form and assayed for reactivity to two types of antibodies, full-length 7S IgY and 5.7S IgY, which has a truncated Fc region. Turtles from Florida were uniformly seropositive to ChHV5 regardless of tumor status. In contrast, in turtles from Hawaii, we detected strong antibody reactivity mainly in tumored animals, with a lower antibody response being seen in nontumored animals, including those from areas where FP is enzootic. Turtles from Hawaii actively shedding ChHV5 were more seropositive than nonshedders. In trying to account for differences in the serological responses to ChHV5 between green turtles from Hawaii and green turtles from Florida, we rejected the cross-reactivity of antibodies to other herpesviruses, differences in viral epitopes, or differences in procedure as likely explanations. Rather, behavioral or other differences between green turtles from Hawaii and green turtles from Florida might have led to the emergence of biologically different viral strains. While the strains from turtles in Florida apparently spread independently of tumors, the transmission of the Hawaiian subtype relies heavily on tumor formation. IMPORTANCE Fibropapillomatosis (FP) is a tumor disease associated with chelonid herpesvirus 5 (ChHV5) that is an important cause of mortality in threatened green turtles globally. FP is expanding in Florida and the Caribbean but declining in Hawaii. We show that Hawaiian turtles mount antibodies to ChHV5 mainly in response to tumors, which are the only sites of viral replication, whereas tumored and nontumored Floridian turtles are uniformly seropositive. Tumor viruses that depend on tumors for replication and spread are rare, with the only example being the retrovirus causing walleye dermal sarcoma in fish. The Hawaiian strain of ChHV5 may be the first DNA virus with such an unusual life history. Our findings, along with the fundamental differences in the life histories between Floridian turtles and Hawaiian turtles, may partly explain the differential dynamics of FP between the two regions.

One of the control mechanisms of cathepsin B biosynthesis and trafficking operates through alternative splicing of pre-mRNA. An mRNA lacking exon 2 is more efficiently translated than that containing all exons, and may be responsible for elevated biosynthesis and enzyme routing to the extracellular space, with critical consequences for connective tissue integrity in pathologies such as cancer and arthritis. mRNA missing exons 2 and 3 encodes a truncated procathepsin B form that is targeted to mitochondria. This enzyme variant is catalytically inactive because it cannot properly fold. However, it provokes a cascade of events, which result first in morphological changes in intracellular organelles and the nucleus, finally leading to cell death.

Besides acting as an inhibitor, the propeptide of human cathepsin B exerts an important auxiliary function as a chaperone in promoting correct protein folding. To explore the ability of N-terminally truncated forms of procathepsin B to fold into enzymatically active proteins, we produced procathepsin B variants progressively lacking N-terminal structural elements in baculovirus-infected insect cells. N-terminal truncation of the propeptide by up to 22 amino acids did not impair the production of activable procathepsin B. Secreted forms lacking the first 20, 21, or 22 amino acids spontaneously generated mature cathepsin B through autocatalytic processing, demonstrating that the first ␣-helix (Asp 11 -Arg 20 ) is necessary for efficient inhibition of the enzyme by its propeptide. In contrast, proenzymes lacking the N-terminal part including the first ␤-sheet (Trp 24 -Ala 26 ) of the propeptide or containing an amino acid mutation directly preceding this ␤-sheet were no longer properly folded. This shows that interactions between Trp 24 of the propeptide and Tyr 183 , Tyr 188 , and Phe 180 of the mature enzyme are important for stabilization and essential for procathepsin B folding. Thus, proenzyme forms missing more than the N-terminal 22 amino acids of the propeptide (notably truncated cathepsin B produced by the mRNA splice variant lacking exons 2 and 3, resulting in a propeptide shortened by 34 amino acids) are devoid of proteolytic activity because they cannot fold correctly. Thus, any pathophysiological involvement of truncated cathepsin B must be ascribed to properties other than proteolysis.The cysteine peptidase family C1 (the papain family, merops.sanger.ac.uk) contains two groups of enzymes within subfamily C1A characterized by the length of their propeptides: cathepsin B-like and cathepsin L-like enzymes. They show very little sequence homology in their proregions, and in contrast to cathepsin L-like proenzymes, which have a propeptide of about 100 amino acids in length, the propeptide region of the cathepsin B-like proenzymes is about 60 amino acids long (1). Studies on cathepsins B, L, and S demonstrated that the propeptides or parts of them are effective inhibitors of their cognate enzymes (2-5). The three-dimensional structure of human procathepsin B shows that the propeptide (Arg 1p-Lys 62p, where p stands for propeptide) is jacketed around the catalytic domain of the enzyme in a C-shaped fold, positioned in the direction opposite to bound substrates, thereby shielding the active site (6). Two regions of rat procathepsin B were found to be particularly important for inhibition, the NTTWQ sequence spanning residues 21p-25p, and the CGTVL sequence (amino acids 42p-46p) (2).Besides their function as intramolecular inhibitors, propeptides of several peptidases have been shown to assist in folding. Early in vitro refolding experiments showed that deletions in the propeptide of human cathepsin L resulted in the loss of enzymatic activity due to the lack of refolding capacity of the mature enzyme (7). In ...

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