Cara T. Pager

Cathepsin L Is Involved in Proteolytic Processing of the Hendra Virus Fusion Protein

ABSTRACT Proteolytic processing of paramyxovirus fusion (F) proteins is essential for the generation of a mature and fusogenic form of the F protein. Although many paramyxovirus F proteins are proteolytically processed by the cellular protease furin at a multibasic cleavage motif, cleavage of the newly emerged Hendra virus F protein occurs by a previously unidentified cellular protease following a single lysine at residue 109. We demonstrate here that the cellular protease cathepsin L is involved in converting the Hendra virus precursor F protein (F0) to the active F1 + F2 disulfide-linked heterodimer. To initially identify the class of protease involved in Hendra virus F protein cleavage, Vero cells transfected with pCAGGS-Hendra F or pCAGGS-SV5 F (known to be proteolytically processed by furin) were metabolically labeled and chased in the absence or presence of serine, cysteine, aspartyl, and metalloprotease inhibitors. Nonspecific and specific protease inhibitors known to decrease cathepsin activity inhibited proteolytic processing of Hendra virus F but had no effect on simian virus 5 F processing. We next designed shRNA oligonucleotides to cathepsin L which dramatically reduced cathepsin L protein expression and enzyme activity. Cathepsin L shRNA-expressing Vero cells transfected with pCAGGS-Hendra F demonstrated a nondetectable amount of cleavage of the Hendra virus F protein and significantly decreased membrane fusion activity. Additionally, we found that purified human cathepsin L processed immunopurified Hendra virus F0 into F1 and F2 fragments. These studies introduce a novel mechanism for primary proteolytic processing of viral glycoproteins and also suggest a previously unreported biological role for cathepsin L.

Host‐guest scale of left‐handed polyproline II helix formation

Despite the clear importance of the left‐handed polyproline II (PPII) helical conformation in many physiologically important processes as well as its potential significance in protein unfolded states, little is known about the physical determinants of this conformation. We present here a scale of relative PPII helix‐forming propensities measured for all residues, except tyrosine and tryptophan, in a proline‐based host peptide system. Proline has the highest measured propensity in this system, a result of strong steric interactions that occur between adjacent prolyl rings. The other measured propensities are consistent with backbone solvation being an important component in PPII helix formation. Side chain to backbone hydrogen bonding may also play a role in stabilizing this conformation. The PPII helix‐forming propensity scale will prove useful in future studies of the conformational properties of proline‐rich sequences as well as provide insights into the prevalence of PPII helices in protein unfolded states. Proteins 2003.

Anticipating rotavirus vaccines: epidemiology and surveillance of rotavirus in South Africa

Rotavirus infection is associated with acute infantile gastroenteritis in infants and young children globally. In South Africa, rotavirus infection has been shown to be associated with approximately one-quarter of all diarrhoeal admissions to hospital. Rotavirus infection predominantly occurs in infants less than 12 months of age (75%) and has a peak of shedding during the cooler, drier months of the year. A secondary peak during the spring has been observed. Multiple infections with rotavirus and at least one other microbial agent are common. The circulating VP7 serotypes and VP4 genotypes have been determined in various regions of South Africa and show a geographic specific distribution. A decade previously, P[8]G1 or G4 strains predominated, and P[4]G2 strains occurred in an epidemic pattern in one region. More recently, rotavirus strains with P[6] genotype have become common and novel VP7/VP4 genotype combinations are occurring across the country. G9 strains have been reported from Cape Town to Vendaland. The circulating rotavirus types observed in this study add to the knowledge of the natural history of rotavirus infection and provide the groundwork to consider future vaccine strategies.

Prevalence of unusual human rotavirus strains in Ghanaian children

Sixty‐seven rotavirus‐positive fecal samples, collected between January and April 1999, from children with diarrhea in the Upper East Region of Ghana were examined for rotavirus VP7 and VP4 types. Sufficient viral RNA could be obtained from 46 (68.7%) of the samples and all the isolates had short electrophoretic pattern and typed as subgroup I rotaviruses by subgroup ELISA. Three rotavirus strains with G8 specificity were identified for the first time in Ghana. G and P typing by PCR identified two distinct strains, P[6]G2 (50%) and P[6]G8 (4.3%). Eighty‐two percent of the isolates (n = 38) were of the “putative” neonatal P[6] genotype. Two of these G8 isolates carried the VP4 P[6] genotype whereas the third could not be assigned a P type. Mixed infections of G1, G2, G3 and G8 were detected amongst the stool samples. The presence of these unusual strains, especially the high incidence of G2 rotavirus strains in Ghana, reinforces the need to put in place a surveillance system for the detection of new and exotic rotavirus strains, that will provide information on the spread of these strains in West Africa as well as useful data for the formulation of the next generation of rotavirus vaccines. J. Med. Virol. 63:67–71, 2001.

Comparative studies of human rotavirus serotype G8 strains recovered in South Africa and the United Kingdom.

Epidemiological studies on the VP7 serotype prevalence of human rotaviruses in South Africa and the United Kingdom identified several strains which could not be serotyped as G1-G4 by monoclonal antibodies. Further analysis of these strains with a G8-specific monoclonal antibody and with probes for human rotaviruses confirmed them as G8 rotaviruses. These G8 strains exhibited a high degree of sequence identity when compared with each other and with other rotavirus G8 strains. Five South African strains were further characterized as VP6 subgroup I, but with a long RNA electropherotype, which is similar to the G8 strains previously isolated in Finland. In the UK strains, one was VP6 subgroup II with a long RNA electropherotype (similar to the Italian G8 strain). The other two were subgroup I with a short RNA electropherotype. None of these strains exhibited the super-short RNA electropherotype described in the prototype G8 strains recovered from Indonesia (69M).

Modulation of Hepatitis C Virus RNA Abundance and Virus Release by Dispersion of Processing Bodies and Enrichment of Stress Granules

Components of cytoplasmic processing bodies (P-bodies) and stress granules can be subverted during viral infections to modulate viral gene expression. Because hepatitis C virus (HCV) RNA abundance is regulated by P-body components such as microRNA miR-122, Argonaute 2 and RNA helicase RCK/p54, we examined whether HCV infection modulates P-bodies and stress granules during viral infection. It was discovered that HCV infection decreased the number of P-bodies, but induced the formation of stress granules. Immunofluorescence studies revealed that a number of P-body and stress granule proteins co-localized with viral core protein at lipid droplets, the sites for viral RNA packaging. Depletion of selected P-body proteins decreased overall HCV RNA and virion abundance. Depletion of stress granule proteins also decreased overall HCV RNA abundance, but surprisingly enhanced the accumulation of infectious, extracellular virus. These data argue that HCV subverts P-body and stress granule components to aid in viral gene expression at particular sites in the cytoplasm.

Subcellular Localization and Calcium and pH Requirements for Proteolytic Processing of the Hendra Virus Fusion Protein

ABSTRACT Proteolytic cleavage of the Hendra virus fusion (F) protein results in the formation of disulfide-linked F1 and F2 subunits, with cleavage occurring after residue K109 in the sequence GDVK↓L. This unusual cleavage site and efficient propagation of Hendra virus in a furin-deficient cell line indicate that the Hendra F protein is not cleaved by furin, the protease responsible for proteolytic activation of many viral fusion proteins. To identify the subcellular site of Hendra F processing, Vero cells transfected with pCAGGS-Hendra F or pCAGGS-SV5 F were metabolically labeled and chased in the absence and presence of inhibitors of exocytosis. The addition of carbonyl-cyanide-3-chlorophenylhydrazone, monensin, brefeldin A, or NaF-AlCl3 or incubation of cells at 20°C all inhibited processing of the Hendra F protein, suggesting that cleavage of Hendra F occurs either in secretory vesicles budding from the trans-Golgi network or at the cell surface. In contrast to proteolytic cleavage of the simian virus 5 (SV5) F protein by the Ca2+-dependent protease furin, proteolytic cleavage of the Hendra F protein was not significantly inhibited by decreases in Ca2+ levels following incubation with EGTA or A23187. However, in the presence of weak amines and H+ V-ATPase inhibitors, known to raise intracellular pH, cleavage of Hendra F protein was inhibited while processing of the SV5 F protein was not significantly affected. The subcellular location, sensitivity to pH changes, and decreased Ca2+ requirement suggest that the protease responsible for cleavage of Hendra F protein differs from proteases previously shown to be involved in the processing of other viral glycoproteins.

Role of N-Linked Glycosylation of the Hendra Virus Fusion Protein

ABSTRACT The Hendra virus fusion (F) protein contains five potential sites for N-linked glycosylation in the ectodomain. Examination of F protein mutants with single asparagine-to-alanine mutations indicated that two sites in the F2 subunit (N67 and N99) and two sites in the F1 subunit (N414 and N464) normally undergo N-linked glycosylation. While N-linked modification at N414 is critical for protein folding and transport, F proteins lacking carbohydrates at N67, N99, or N464 remained fusogenically active. As N464 lies within heptad repeat B, these results contrast with those seen for several paramyxovirus F proteins.

South African G4P[6] asymptomatic and symptomatic neonatal rotavirus strains differ in their NSP4, VP8*, and VP7 genes.

Over the past decade, a G4P[6] strain has been found to be circulating in different neonatal wards in the Pretoria area. This endemic strain was associated with both asymptomatic and symptomatic infection, providing the opportunity to undertake a molecular study of some of the putative “virulence” genes. The genes encoding NSP4, VP8*, and VP7 of two asymptomatic and two Symptomatic strains were sequenced and compared with ST3. Within each of these genes, amino acid substitutions unique to South African strains were recorded. Four conserved amino acid differences between asymptomatic and symptomatic strains at aa 82 (serine to leucine), aa 114 (aspartic acid to glutamic acid), aa 138 (proline to threonine), and aa 169 (leucine to serine) were identified within the NSP4 gene. The hypervariable region of VP8* exhibited 10 specific amino acid differences (at aa 73, 78, 98, 111, 116, 142, 145, 167, 169, and 188) between asymptomatic and symptomatic strains, while three amino acid substitutions within VP7 were noted. These changes to VP7 occurred within the glycosylation site at aa 70 (leucine to serine), at antigenic region A (aa 96, asparagine to threonine), and at aa 318 (aspartic acid to glycine). It may be speculated that these changes are specific to G4P[6] strains. Furthermore, the observed substitutions may also be particular to South African strains. NSP4, VP8*, and VP7 have been associated with virulence and the amino acid substitutions within these genes correlate with both asymptomatic and symptomatic infection observed in neonates. J. Med. Virol. 62:208–216, 2000.

Reduced DEAF1 function during type 1 diabetes inhibits translation in lymph node stromal cells by suppressing Eif4g3

The transcriptional regulator deformed epidermal autoregulatory factor 1 (DEAF1) has been suggested to play a role in maintaining peripheral tolerance by controlling the transcription of peripheral tissue antigen genes in lymph node stromal cells (LNSCs). Here, we demonstrate that DEAF1 also regulates the translation of genes in LNSCs by controlling the transcription of the poorly characterized eukaryotic translation initiation factor 4 gamma 3 (Eif4g3) that encodes eIF4GII. Eif4g3 gene expression was reduced in the pancreatic lymph nodes of Deaf1-KO mice, non-obese diabetic mice, and type 1 diabetes patients, where functional Deaf1 is absent or diminished. Silencing of Deaf1 reduced Eif4g3 expression, but increased the expression of Caspase 3, a serine protease that degrades eIF4GII. Polysome profiling showed that reduced Eif4g3 expression in LNSCs resulted in the diminished translation of various genes, including Anpep, the gene for aminopeptidase N, an enzyme involved in fine-tuning antigen presentation on major histocompatibility complex (MHC) class II. Together these findings suggest that reduced DEAF1 function, and subsequent loss of Eif4g3 transcription may affect peripheral tissue antigen (PTA) expression in LNSCs and contribute to the pathology of T1D.