Anti-envelope antibody responses in individuals at high risk of hepatitis C virus who resist infection.

Injection drug users uninfected by hepatitis C virus (HCV) despite likely repeated exposure through high-risk behaviour are well documented. Factors preventing infection in these individuals are incompletely understood. Here, we looked for anti-HCV-envelope antibody responses in a cohort of repeatedly exposed but uninfected subjects. Forty-two hepatitis C diagnostic antibody- and RNA-negative injection drug users at high risk of exposure were studied and findings compared to healthy controls and cases with chronic HCV infection. Purified IgGs from sera were tested by ELISA for binding to genotype 1a and 3a envelope glycoproteins E1E2 with further testing for IgG and IgM reactivity against soluble E2. Virus-neutralizing activity was assessed using an HCV pseudoparticle system. Uninfected subjects demonstrated significantly greater IgG and IgM reactivities to envelope glycoproteins than healthy controls with IgG from 6 individuals additionally showing significant neutralization. This study is the first to describe humoral immunological responses targeting the HCV envelope, important for viral neutralization, in exposed uninfected individuals. A subset of these cases also had evidence of viral neutralization via anti-envelope antibodies. In addition to confirming viral exposure, the presence of specific anti-envelope antibodies may be a factor that helps these individuals resist HCV infection.

Elevated interferon-stimulated gene transcription in peripheral blood mononuclear cells occurs in patients infected with genotype 1 but not genotype 3 hepatitis C virus.

Hepatitis C virus (HCV) can be classified into seven distinct genotypes that are associated with differing pathologies and respond differently to antiviral therapy. In the UK, genotype 1 and 3 are present in approximately equal proportions. Chronic infection with HCV genotype 3 is associated with increased liver steatosis and reduced peripheral total cholesterol levels, which potentially influences peripheral immune responses. To understand these differences, we investigated host gene transcription in peripheral blood mononuclear cells by microarray and quantitative PCR in patients with genotype 1 (n = 22) or genotype 3 infection (n = 22) and matched healthy controls (n = 15). Enrichment of genes involved in immune response and inflammatory pathways were present in patients infected with HCV genotype 1; however, no differences in genes involved in lipid or cholesterol metabolism were detected. This genotype-specific induction of genes is unrelated to IL28B genotype or previous treatment failure. Our data support the hypothesis that genotype 1 infection drives a skewed Type I interferon response and provides a foundation for future investigations into the host-pathogen interactions that underlie the genotype-specific clinical outcomes of chronic HCV infection.

Recurrent osteoid osteoma of the lunate: a case report and review of the literature.

Osteoid osteoma is a benign tumour of bone that rarely localises in the carpal bones. Its treatment by curettage and bone grafting is considered to be curative and its recurrence is thought to be rare. We report a case of an osteoid osteoma of the lunate, which recurred seven years after the initial operation. Recurrent osteoid osteoma of the lunate bone has not been reported in the literature. We present this case report for its atypical presentation and diagnostic difficulty and also to alert the readers of the possibility of an osteoid osteoma as a cause of the chronic unexplained wrist pain in young adults.

The transmembrane domain of the hepatitis C virus E2 glycoprotein is required for correct folding of the E1 glycoprotein and native complex formation.

Hepatitis C virus (HCV) encodes two glycoproteins, E1 and E2, that interact to form both native and aggregated complexes in tissue culture cells. In native complexes, E1 and E2 are associated by noncovalent interactions and such complexes are considered to constitute the authentic interactions between the proteins. By contrast, the proteins are linked by covalent, disulfide bonds in aggregated complexes. From studies with a mutant in which cysteine residues in E1 have been substituted with other amino acids, we show that E1 continues to associate with E2, although the migratory patterns of the proteins on gels are consistent with the formation of aggregated complexes. Therefore, such complexes can be stabilized by noncovalent as well as covalent interactions. To further examine the requirements for native complex formation, segments of foreign glycoproteins were linked to regions of E2. Our data provide direct evidence for the requirement of C-terminal sequences in E2 that contain the transmembrane domain to permit oxidation of E1 and assembly of a native complex. By contrast, native complexes and oxidized E1 are not found in the presence of chimeric proteins containing the E2 ectodomain. These data suggest that interaction of E1 with the E2 transmembrane domain is critical for native complex formation.

Intraocular T cells of patients with herpes simplex virus (HSV)-induced acute retinal necrosis recognize HSV tegument proteins VP11/12 and VP13/14.

It has previously been shown that T cells specific for the triggering virus infiltrate the eye of patients with herpes simplex virus type 1 (HSV-1)-induced acute retinal necrosis (ARN). The T cells were mainly directed against 0.67-0.73 HSV-1 map region encoded antigens. The fine specificities of genetically different T cell clones (TCC), obtained from affected eyes of 3 patients with HSV-induced ARN and reactive toward this genomic region of HSV-1, were analyzed with recombinant HSV viruses and synthetic peptides. For 1 patient, the HSV-1 UL46 gene encoded tegument protein VP11/12 was identified as the target antigen. Two separate CD4(+) T cell epitopes were defined in VP11/12. TCC from the other 2 patients recognized the HSV-1 UL47 gene encoded tegument protein VP13/14. Two separate CD4(+) VP13/14 T cell epitopes were identified in these patients. Analysis of the data indicates that HSV-1 VP11/12 and VP13/14 are major target antigens for T cells obtained from vitreous fluid samples of the HSV-induced ARN patients studied.

Properties of the hepatitis C virus core protein: a structural protein that modulates cellular processes.

The core protein of hepatitis C virus (HCV) is believed to form the capsid shell of virus particles. Maturation of the protein is achieved through cleavage by host cell proteases to give a product of 21 000 MW, which is found in tissue culture systems and sera from infected individuals. However, efficient propagation of the virus is not possible at present in tissue culture. Hence, studies have focused on the properties of the core protein and its possible role in pathologies associated with HCV infection. This review describes key features of the polypeptide and the status of current knowledge on its ability to influence several cellular processes.

New insights into the mechanisms of lipid-body biogenesis in plants and other organisms.

A comparative approach has been used to study the role of several lipid-body-binding proteins in plants and animals. Caleosins are a newly discovered class of calcium-binding lipid-body proteins found in plants and fungi, which we now report to have separate endoplasmic reticulum- and lipid-body-associated isoforms. We also compare the lipid-body targeting of oleosin from plants and the core protein of the hepatitis C virus when they were expressed separately in lipid-accumulating animal cell lines. This is a novel and powerful approach to investigating the factors that determine the lipid-body targeting of a wide range of proteins.

Covalent interactions are not required to permit or stabilize the non-covalent association of hepatitis C virus glycoproteins E1 and E2.

Hepatitis C virus (HCV) encodes two glycoproteins, E1 and E2, which are thought to locate to the envelope of virus particles. These proteins form two complexes in tissue culture systems, a high molecular mass aggregate that contains intermolecular covalent bonds and a native complex in which E1 and E2 associate by non-covalent interactions. The contribution of either complex to the structures of the proteins on virus particles is not known. Using dithiothreitol to reduce inter- and intramolecular disulphide bonds in situ, we have studied the nature of the interactions within the aggregate and the role of covalent bonds in the early stages of E1-E2 association. Results with two HCV type 1a strains, Glasgow and H77, showed that the aggregate contains not only covalent interactions but also non-covalent associations between E1 and E2. These non-covalent associations are complex since deletion mutant analysis failed to identify any single region which was required for non-covalent interaction. Complex formation by de novo synthesized proteins was not arrested under reducing conditions which prevented the production of inter- and intramolecular disulphide bonds. Moreover, a conformation-specific antibody continued to recognize the E2 protein in reduced complexes, indicating that covalent bonds do not stabilize certain structures of E2 that can interact with E1. These data suggest that disulphide bonds are not required either to allow association between the proteins or to stabilize E1-E2 complexes.

The abundance of the herpes simplex virus type 1 UL37 tegument protein in virus particles is closely controlled.

The tegument region of herpes simplex virus type 1 virus particles contains approximately 15 virus-encoded polypeptide species. One of the less abundant species is a 120 kDa protein specified by gene UL37. The abundance of the UL37 protein in infected cells was increased about 20-fold by replacing the native promoter for gene UL37 with the strong immediate early promoter of human cytomegalovirus. This rise in abundance did not induce any detectable increase in the amount of UL37 protein incorporated into virus particles. These data contrast with those previously obtained for a second tegument protein VP22 whose level of incorporation was elevated by increasing its abundance in infected cells. Thus, for the UL37 protein, a mechanism exists to control its abundance in the tegument. Moreover, data obtained with light particles which lack the viral capsid suggest that this controlled incorporation is not directed by interaction with the capsid structure.

Overexpression of the herpes simplex virus type 1 tegument protein VP22 increases its incorporation into virus particles.

The tegument of herpes simplex virus type 1 (HSV-1) virus particles is a complex assemblage of virus proteins whose relative proportions within virions are essentially constant for a particular strain of virus. To examine the processes controlling incorporation into the tegument, we constructed a HSV-1 recombinant that expresses two copies of gene UL49, which encodes the major tegument protein VP22. One copy specifies the unmodified form of VP22 under the control of the native promoter while the second expresses an epitope-tagged version of the protein via the human cytomegalovirus immediate early promoter. In cells infected with the recombinant virus, the overall levels of VP22 synthesized were about fivefold higher than those for wild-type virus, due to the high levels of expression of tagged protein. Analysis of virus particles revealed that the amount of VP22 in the tegument was approximately two- to threefold higher in recombinant virions and L-particles than in particles produced by wild-type virus. These results provide the first evidence that, for certain proteins, the level of polypeptide synthesis can act as a controlling factor for the amount of protein incorporated into tegument.

The herpes simplex virus type 1 UL37 gene product is a component of virus particles.

The herpes simplex virus type 1 UL37 gene encodes a protein with an M(r) of 120K that is produced at late times after infection. To study the properties of this protein we have linked a 10 amino acid epitope derived from a human cytomegalovirus protein to the UL37 polypeptide coding sequences by inserting an oligonucleotide at a SpeI site that is unique in the virus genome and lies close to the 3' end of the open reading frame. From studies on the resultant virus recombinant using a monoclonal antibody that recognizes the inserted epitope we find that, contrary to a previous report, the UL37 protein is a structural component of both virions and L particles and is present in the tegument of virus particles. Indirect immunofluorescence analysis revealed that the protein is distributed throughout infected cells but is more abundant in the cytoplasm than the nucleus.

Characterization of a herpes simplex virus type 1 deletion variant (1703) which under-produces Vmw63 during immediate early conditions of infection.

The herpes simplex virus type 1 deletion variant 1703 apparently fails to synthesize the essential IE2 gene product Vmw63 despite the deletion leaving the gene intact. Sequence analysis revealed that the deletion removes a region to the right of IE2 comprising the 3' end of IE1, UL56 and the 3' part of UL55, stopping 555 bp downstream of the IE2 polyadenylation signal. Further DNA sequencing has shown that there is no secondary mutation in the IE2 gene. Western blot analysis demonstrated that Vmw63 is made at reduced levels compared to that produced by the wild-type virus during immediate early conditions of infection. S1 nuclease protection mapping has revealed that this reduction is also apparent at the level of mRNA synthesis. A direct link between the deletion and the change in mRNA synthesis was provided by the insertion of a deletion-spanning fragment from 1703 into a 17+ genome, which resulted in the recombinant having a 1703-like phenotype. Evidence that down-regulation of IE2 mRNA during immediate early conditions of infection could be due to antisense RNA initiating from the IE1 promoter was obtained by the insertion of a novel transcriptional termination signal between IE1 and IE2 in the variant and the subsequent detection of wild-type levels of IE2 mRNA and protein.

Comparing the mortality and morbidity of cemented and uncemented hemiarthroplasties.

A series of 207 consecutive patients admitted to Aberdeen Royal Infirmary over a 1-year period with a diagnosis of displaced subcapital fracture of the neck of the femur were treated by hemiarthroplasty with either a cemented Hasting or an uncemented Monk bipolar prosthesis. Patients were reviewed at an average of 19 months and differences in mortality and morbidity assessed. We found that there was a significant perioperative mortality in the cemented group and no perioperative mortality in the uncemented group. There was no significant difference in the morbidity or complication rate in the two groups, although surviving patients appeared significantly more satisfied with the cemented Hasting prosthesis.

Failures of screening and management of congenital dislocation of the hip.

We report the screening of 67,093 infants for congenital dislocation of the hip from 1980 to 1989 and compare the results with those during the preceding two decades. More dislocations have been missed at neonatal examination during the last decade (0.13% of live births). Operative treatment was needed in 54 children (0.08% of live births) some of whom had been diagnosed at birth. We discuss the reasons for the failure of neonatal screening.

Herpes simplex virus IE63 acts at the posttranscriptional level to stimulate viral mRNA 3' processing.

We have shown previously that a novel herpes simplex virus-induced activity, LPF, selectively increases RNA 3'-end processing at the poly(A) site of a late virus gene (J. McLauchlan, S. Simpson, and J. B. Clements, Cell 59:1093-1105, 1989). Here, our in vivo and in vitro analyses both demonstrate that LPF is induced during early stages of virus infection. Studies of virus mutants indicate that expression of the immediate-early IE63 gene is required for induction of this activity. The selective effects on 3' processing displayed in the presence of IE63 provide direct evidence that IE63 can influence this posttranscription process. This extends previous studies which reported increases in reporter gene activity with certain poly(A) sites by IE63 (R. M. Sandri-Goldin and G. E. Mendoza, Genes Dev. 6:848-863, 1992).

Noninfectious L-particles supply functions which can facilitate infection by HSV-1.

The tegument of herpes simplex virus contains proteins important for the efficient initiation of infection. L-particles are noninfectious virion-related particles that lack the nucleocapsid but do contain tegument and envelope. Their ability to deliver functional proteins into cells was compared to that of virions by testing the biological activities of two tegument proteins which are present in both types of particle. Results indicated that L-particles are as efficient as virions in supplying these in a fully functional state. Thus, L-particles are biologically competent and have the potential to participate in the early stages of virus infection.

Characterization of enveloped tegument structures (L particles) produced by alphaherpesviruses: integrity of the tegument does not depend on the presence of capsid or envelope.

Recent studies have shown that infection with herpes simplex virus type 1 (HSV-1) strain 17 generates in addition to virions a novel type of non-infectious particle. These particles, termed L particles, lack capsids and viral DNA, and consist predominantly of tegument and envelope proteins. We show that L particle production is not restricted to one strain of HSV-1, and that pseudorabies virus and equine herpesvirus type 1 also release particles which are similar in composition to and morphologically indistinguishable from HSV-1 L particles. Data obtained from monoclonal antibody analysis revealed that Vmw175, an immediate early HSV-1 polypeptide which had been previously identified as a virion component, is located predominantly in L particles and not in virions. Following removal of the envelope from L particles, the remaining tegument material largely retained its structural integrity, indicating that the structure of the tegument does not depend on the presence of the capsid or envelope.

Assembly of enveloped tegument structures (L particles) can occur independently of virion maturation in herpes simplex virus type 1-infected cells.

Cells infected with a number of alphaherpesviruses produce non-infectious virion-related particles, termed L particles, in addition to infectious virions. L particles consist of the tegument and envelope components, but lack the virus capsid and DNA. Using a herpes simplex virus type 1 (HSV-1) temperature-sensitive mutant, ts1201, which fails to produce mature virions, we show that L particle production is independent of virion formation. Moreover, the quantity and protein composition of L particles generated by this mutant at the non-permissive temperature are indistinguishable from those produced in wild-type HSV-1 infections. Electron microscopy studies suggest that the processes governing the assembly of tegument and envelope components into L particles are similar to those involved in virion maturation.