Potent T cell response to a class I-binding 13-mer viral epitope and the influence of HLA micropolymorphism in controlling epitope length

The BZLF1 antigen of Epstein-Barr virus includes three overlapping sequences of different lengths that conform to the binding motif of human leukocyte antigen (HLA) B*3501. These 9-mer ((56)LPOGQLTAy(64)), 11-mer ((54)EPLPQGQLTAy(64)), and 13-mer ((52)LPEPLPQGQLTAY(64)) peptides all bound well to B*3501; however, the CTL response in individuals expressing this HILA allele was directed strongly and exclusively towards the 11-mer peptide. In contrast, EBV-exposed donors expressing HLA B*3503 showed no significant CTL response to these peptides because the single amino acid difference between B*3501 and B*3503 within the F pocket inhibited HLA binding by these peptides. The extraordinarily long 13-mer peptide was the target for the CTL response in individuals expressing B*3508, which differs from B*3501 at a single position within the D pocket (B*3501, 156 Leucine; B*3508, 156 Arginine). This minor difference was shown to enhance binding of the 13-mer peptide, presumably through a stabilizing interaction between the negatively charged glutamate at position 3 of the peptide and the positively charged arginine at HLA position 156. The 13-mer epitope defined in this study represents the longest class I-binding viral epitope identified to date as a minimal determinant. Furthermore, the potency of the response indicates that peptides of this length do not present a major structural barrier to CTL recognition.

Sequence variation in primer targets affects the accuracy of viral quantitative PCR

This study was designed to assess the effect of sequence mismatches between primers and their targets on viral quantitative PCR. Numerous primers were constructed incorporating various mismatches with a target sequence on the BKV T antigen gene. When using these primers in standard Taqman two-step cycling conditions, as few as two mismatches in a single primer increased cycle threshold values and significantly influenced the calculation of viral load. (C) 2005 Elsevier B.V. All rights reserved.

Gene codon composition determines differentiation-dependent expression of a viral capsid gene in keratinocytes in vitro and in vivo

By establishing mouse primary keratinocytes (KCs) in culture, we were able, for the first time, to express papillomavirus major capsid (L1) proteins by transient transfection of authentic or codon-modified L1 gene expression plasmids. We demonstrate in vitro and in vivo that gene codon composition is in part responsible for differentiation-dependent expression of L1 protein in KCs. L1 mRNA was present in similar amounts in differentiated and undifferentiated KCs transfected with authentic or codon-modified L1 genes and had a similar half-life, demonstrating that L1 protein production is posttranscriptionally regulated. We demonstrate further that KCs substantially change their tRNA profiles upon differentiation. Aminoacyl-tRNAs from differentiated KCs but not undifferentiated KCs enhanced the translation of authentic L1 mRNA, suggesting that differentiation-associated change to tRNA profiles enhances L1 expression in differentiated KCs. Thus, our data reveal a novel mechanism for regulation of gene expression utilized by a virus to direct viral capsid protein expression to the site of virion assembly in mature KCs. Analysis of two structural proteins of KCs, involucrin and keratin 14, suggests that translation of their mRNAs is also regulated, in association with KC differentiation in vitro, by a similar mechanism

CTL recognition of a bulged viral peptide involves biased TCR selection

MHC class I molecules generally present peptides of 8-10 aa long, forming an extended coil in the HLA cleft. Although longer peptides can also bind to class I molecules, they tend to bulge from the cleft and it is not known whether the TCR repertoire has sufficient plasticity to recognize these determinants during the antiviral CTL response. In this study, we show that unrelated individuals infected with EBV generate a significant CTL response directed toward an HLA-B*3501-restricted, 11-mer epitope from the BZLF1 Ag. The 11-mer determinant adopts a highly bulged conformation with seven of the peptide side chains being solvent-exposed and available for TCR interaction. Such a complex potentially creates a structural challenge for TCR corecognition of both HLA-B*3501 and the peptide Ag. Surprisingly, unrelated B*3501 donors recognizing the 11-mer use identical or closely related alpha beta TCR sequences that share particular CDR3 motifs. Within the small number of dominant CTL clonotypes observed, each has discrete fine specificity for the exposed-side chain residues of the peptide. The data show that bulged viral peptides are indeed immunogenic but suggest that the highly constrained TCR repertoire reflects a limit to TCR diversity when responding to some unusual MHC peptide ligands.

Real-time reverse transcriptase PCR for the endogenous koala retrovirus reveals an association between plasma viral load and neoplastic disease in koalas

Koala retrovirus (KoRV) is a newly described endogenous retrovirus and is unusual in that inserts comprise a full-length replication competent genome. As koalas are known to suffer from an extremely high incidence of leukaemia/lymphoma, the association between this retrovirus and disease in koalas was examined. Using quantitative real-time reverse transcriptase PCR it was demonstrated that KoRV RNA levels in plasma are significantly increased in animals suffering from leukaemia or lymphoma when compared with healthy animals. Increased levels of KoRV were also seen for animals with clinical chlamydiosis. A significant positive association between viral RNA levels and age was also demonstrated. Real-time PCR demonstrated as much as 5 log variation in KoRV proviral DNA levels in genomic DNA extracted from whole blood from different animals. Taken together these data indicate that KoRV is an active endogenous retrovirus and suggests that it may be causally linked to neoplastic disease in koalas.

Oral viral infections and the therapeutic use of antiviral agents in dentistry

This paper reviews the current concepts of viral classification, infection and replication. The clinical presentation of common oral viral infections encountered in the dental practice are discussed, including: herpes simplex virus types 1 and 2; Epstein-Barr virus; varicella-zoster virus; Coxsackie virus; human papilloma virus; and human immunodeficiency virus. The diagnosis, principles of management and pharmacological agents available for the treatment of oral viral infections are also discussed.

Endosome-to-cytosol transport of viral nucleocapsids

During viral infection, fusion of the viral envelope with endosomal membranes and nucleocapsid release were thought to be concomitant events. We show here that for the vesicular stomatitis virus they occur sequentially, at two successive steps of the endocytic pathway. Fusion already occurs in transport intermediates between early and late endosomes, presumably releasing the nucleocapsid within the lumen of intra- endosomal vesicles, where it remains hidden. Transport to late endosomes is then required for the nucleocapsid to be delivered to the cytoplasm. This last step, which initiates infection, depends on the late endosomal lipid lysobisphosphatidic acid ( LBPA) and its putative effector Alix/ AIP1, and is regulated by phosphatidylinositol- 3-phosphate ( PtdIns( 3) P) signalling via the PtdIns( 3) P- binding protein Snx16. We conclude that the nucleocapsid is exported into the cytoplasm after the back- fusion of internal vesicles with the limiting membrane of late endosomes, and that this process is controlled by the phospholipids LBPA and PtdIns( 3) P and their effectors.

Systemic activation of dendritic cells by Toll-like receptor ligands or malaria infection impairs cross-presentation and anti-viral immunity

The mechanisms responsible for the immunosuppression associated with sepsis or some chronic blood infections remain poorly understood. Here we show that infection with a malaria parasite (Plasmodium berghei) or simple systemic exposure to bacterial or viral Toll-like receptor ligands inhibited cross-priming. Reduced cross-priming was a consequence of downregulation of cross-presentation by activated dendritic cells due to systemic activation that did not otherwise globally inhibit T cell proliferation. Although activated dendritic cells retained their capacity to present viral antigens via the endogenous major histocompatibility complex class I processing pathway, antiviral responses were greatly impaired in mice exposed to Toll-like receptor ligands. This is consistent with a key function for cross-presentation in antiviral immunity and helps explain the immunosuppressive effects of systemic infection. Moreover, inhibition of cross-presentation was overcome by injection of dendritic cells bearing antigen, which provides a new strategy for generating immunity during immunosuppressive blood infections.

Delipidation of a hepadnavirus: Viral inactivation and vaccine development

Many viruses including HIV, hepatitis C and hepatitis B, have an outer lipid envelope which maintains inserted viral peptides in the “correct” functional conformation and orientation. Disruption of the lipid envelope by most solvents destroys infectivity and often results in a loss of antigenicity. This communication outlines a novel approach to viral inactivation by specific solvent delipidation which modifies the whole virion rendering it non-infective, but antigenic. Duck hepatitis B virus (DHBV) was delipidated using a diisopropylether (DIPE) and butanol mixture and residual infectivity tested by inoculation into day-old ducks. Delipidation completely inactivated the DHBV (p < 0.001). Delipidated DHBV was then used to vaccinate ducks. Three doses of delipidated DHBV induced anti-DHBs antibody production and prevented high dose challenge infection in five out of six ducks. In comparison, five of six ducks vaccinated with undelipidated DHBV and four of four ducks vaccinated with glutaraldehyde inactivated DHBV were unprotected (p < 0.05). Although this solvent system completely inactivated DHBV, viral antigens were retained in an appropriate form to induce immunity. Delipidation of enveloped viruses with specific organic solvents has potential as the basis for development of vaccines.

The role of histidine residues in low-pH-mediated viral membrane fusion

A central event in the invasion of a host cell by an enveloped virus is the fusion of viral and cell membranes. For many viruses, membrane fusion is driven by specific viral surface proteins that undergo large-scale conformational rearrangements, triggered by exposure to low pH in the endosome upon internalization. Here, we present evidence suggesting that in both class I (helical hairpin proteins) and class 11 (beta-structure-rich proteins) pH-dependent fusion proteins the protonation of specific histidine residues triggers fusion via an analogous molecular mechanism. These histidines are located in the vicinity of positively charged residues in the prefusion conformation, and they subsequently form salt bridges with negatively charged residues in the postfusion conformation. The molecular surfaces involved in the corresponding structural rearrangements leading to fusion are highly conserved and thus might provide a suitable common target for the design of antivirals, which could be active against a diverse range of pathogenic viruses.

RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway

RNA interference (RNAi) is widely used to silence genes in plants and animals. it operates through the degradation of target mRNA by endonuclease complexes guided by approximately 21 nucleotide (nt) short interfering RNAs (siRNAs). A similar process regulates the expression of some developmental genes through approximately 21 nt microRNAs. Plants have four types of Dicer-like (DCL) enzyme, each producing small RNAs with different functions. Here, we show that DCL2, DCL3 and DCL4 in Arabidopsis process both replicating viral RNAs and RNAi-inducing hairpin RNAs (hpRNAs) into 22-, 24- and 21 nt siRNAs, respectively, and that loss of both DCL2 and DCL4 activities is required to negate RNAi and to release the plant's repression of viral replication. We also show that hpRNAs, similar to viral infection, can engender long-distance silencing signals and that hpRNA-induced silencing is suppressed by the expression of a virus-derived suppressor protein. These findings indicate that hpRNA-mediated RNAi in plants operates through the viral defence pathway.