- Volume 88, Issue 1, 2007

p7 protein is a small protein encoded by Hepatitis C virus (HCV) that functions as an ion channel in planar lipid bilayers. The function of p7 is vital for the virus life cycle. In this study, the p7 protein of genotype 2a (strain JFH1; the only strain that replicates and produces virus progeny in vitro) was tagged with either an enhanced green fluorescent protein (eGFP) or a haemagglutinin (HA) epitope to facilitate tracking of the protein in the intracellular environment. The tagged viral polyprotein was expressed transiently in the cells after transfection with the recombinant RNA transcripts. Confocal microscopy revealed that the tagged p7 protein was localized in the endoplasmic reticulum (ER) but not associated with mitochondria. Immunoelectron microscopy confirmed the p7 localization data and, moreover, showed that intracellular virus-like particles formed in the cells transfected with the wild-type, but not the recombinant, transcripts. Following a few passages of the transfected cells, the recombinant genome with the HA tag reverted to wild-type and the entire tag was deleted. Therefore, in this study, it has been demonstrated that the p7 protein in the context of the full-length polyprotein encoded by a replication competent genome is only localized to the ER and has a possible role in HCV particle formation.

Genetic recombination between different strains of Hepatitis C virus (HCV) was investigated in three chimpanzees inoculated experimentally with factor VIII concentrate containing HCV subgenotypes 1a, 1b, 2b and 3a. A 750 bp long fragment from the HCV envelope region was amplified by RT-PCR and quasispecies were isolated by plasmid cloning. Nucleotide sequences derived from isolated quasispecies were screened for the presence of inter-subgenotypic recombination by using sequence analysis. Recombination between HCV subgenotype 1a and 1b was found in two animals; each recombinant variant differed by location of predicted crossover region or order of subgenotype 1a and 1b sequences.

The yellow fever virus attenuated 17D vaccine strain is a safe and effective vaccine and a valuable model system for evaluating immune responses against attenuated viral variants. This study compared the in vitro interactions of the commercially available yellow fever vaccine (YF-VAX), Dengue virus and the live-attenuated dengue vaccine PDK50 with dendritic cells (DCs), the main antigen-presenting cells at the initiation of immune responses. Similar to PDK50, infection with YF-VAX generated activated DCs; however, for YF-VAX, activation occurred with limited intracellular virus replication. The majority of internalized virus co-localized with endolysosomal markers within 90 min, suggesting that YF-VAX is processed rapidly in DCs. These results indicate that restricted virus replication and lysosomal compartmentalization may be important contributing factors to the success of the YF-VAX vaccine.

This study analysed the transport behaviour of the glycoprotein E2 of Bovine viral diarrhea virus (BVDV) expressed from recombinant vesicular stomatitis virus (rVSV). E2 protein was found to be retained at an intracellular compartment. A chimeric protein containing the membrane anchor and cytoplasmic tail of the VSV G protein, E2–G(MT), was transported to the cell surface. Only the latter protein was incorporated into rVSV particles in significant amounts. A soluble form of E2 lacking the membrane anchor, E2(MTdel), appeared to be affected in conformational stability. In contrast to both membrane-anchored forms of E2, expression of the soluble form was detectable only by immunofluorescence microscopy but not by Western blotting. These results are in agreement with reports of intracellular retention of the E2 protein due to a retention signal in the membrane anchor. However, in another analysis of E2 expressed from rVSV, E2 protein was reported to be transported to the cell surface and incorporated into VSV particles [ Grigera, P. R., Marzocca, M. P., Capozzo, A. V. E., Buonocore, L., Donis, R. O. & Rose, J. K. (2000). Virus Res 69, 3–15 ]. Reasons for these contradictory results are discussed.

In an earlier report, different variants of echovirus 30 (E-30), an enterovirus serotype, were identified during two outbreaks in 1997 and 2000. Here, the diversity of E-30 was investigated over a longer period (1991–2005) and the variations in four genomic segments were determined in 52 isolates involved in meningitis cases, to characterize the evolutionary processes underlying the emergence of lineages. Phylogenetic analysis of the VP1 sequences showed that five phylogenetic variants succeeded one another. When a partial 3CD segment was examined, the five variants split further into 10 lineages. Phylogenetic groupings observed with both the VP1 and 3CD sequences were clearly related to the calendar time of virus isolation. The rapid turnover of lineages during the study period was not associated with variations in amino acid residues in either the VP1 or the 3CD sequences, indicating major evolutionary contraints in E-30. The variation patterns were examined further along a subgenomic segment of 4878 nt in 13 virus isolates, representative of the 10 lineages. Breakpoints detected in the similarity profiles were investigated by bootscanning and maximum-likelihood phylogenetic analysis of virus genes. Evidence of several past recombination events was observed in the middle of the genome and predicted recombination crossover sites were mapped with precision. The contribution of recombination to the evolution of E-30 is substantial. It is associated tightly with the emergence of new genetic lineages and certain recombinants have undergone epidemic spread.

Feline calicivirus (FCV) is a major causative agent of respiratory disease in cats. It is also one of the few cultivatable members of the family Caliciviridae. It has recently been reported that FCV binding is in part due to interaction with junction adhesion molecule-A. This report describes the characterization of additional receptor components for FCV. Chemical treatment of cells with sodium periodate showed that FCV recognized carbohydrate moieties on the surface of permissive cells. Enzymic treatment with Vibrio cholerae neuraminidase demonstrated that sialic acid was a major determinant of virus binding. Further characterization using linkage-specific lectins from Maackia amurensis and Sambucus nigra revealed that FCV recognized sialic acid with an α2,6 linkage. Using various proteases and metabolic inhibitors, it was shown that α2,6-linked sialic acid recognized by FCV is present on an N-linked glycoprotein.

Small immunoproteins (SIPs) are single-chain molecules comprising the variable regions of an antibody assembled in a single polypeptide (scFv) and joined to the immunoglobulin heavy-chain dimerizing domain. To investigate the potential of these molecules to provide protection against enteric infections when supplied orally, SIPs were generated against Transmissible gastroenteritis virus (TGEV), a highly pathogenic porcine virus. Different variants of TGEV-specific SIPs were created, of ε and α isotypes, by exploiting the dimerizing domains εCH4 and αCH3 of human and swine origin. Transfected cells secreted these recombinant mini-antibodies efficiently, mainly as dimers stabilized covalently by inter-chain disulphide bridges. The specificity and functionality of the recombinant TGEV-specific SIPs were determined by in vitro binding, neutralization and infection-interference assays. The neutralization indices of the TGEV-specific SIPs were all very similar to that of the original TGEV-specific mAb, thus confirming that the immunological properties have been preserved in the recombinant SIPs. In vivo protection experiments on newborn piglets have, in addition, demonstrated a strong reduction of virus titre in infected tissues of animals treated orally with TGEV-specific SIPs. It has therefore been demonstrated that it is possible to confer passive immunization to newborn pigs by feeding them with recombinant SIPs.

The respiratory syncytial virus (RSV) phosphoprotein (P) is a major polymerase co-factor that interacts with both the large polymerase fragment (L) and the nucleoprotein (N). The N-binding domain of RSV P has been investigated by co-expression of RSV P and N proteins in Escherichia coli. Pull-down assays performed with a series of truncated forms of P fused to glutathione S-transferase (GST) revealed that the region comprising the last nine C-terminal amino acid residues of P (233-DNDLSLEDF-241) is sufficient for efficient binding to N. Site-directed mutagenesis shows that the last four residues of this peptide are crucial for binding and must be present at the end of a flexible C-terminal tail. The presence of the P oligomerization domain (residues 100–160) was an important stabilizing factor for the interaction. The tetrameric full-length P fused to GST was able to pull down both helical and ring structures, whereas a monomeric C-terminal fragment of P (residues 161–241) fused to GST pulled down exclusively RNA–N rings. Electron-microscopy analysis of the purified rings showed the presence of two types of complex: undecamers (11N) and decamers (10N). Mass-spectrometry analysis of the RNA extracted from rings after RNase A treatment showed two peaks of 22 900 and 24 820 Da, corresponding to a mean RNA length of 67 and 73 bases, respectively. These results suggest strongly that each N subunit contacts 6 nt, with an extra three or four bases further protected from nuclease digestion by the ring structure at both the 5′ and 3′ ends.

Structural data support a model where – following proteolytic cleavage – the amino-terminal domain of human immunodeficiency virus type 1 (HIV-1) capsid protein refolds into a β-hairpin/helix tertiary structure that is stabilized by a buried salt bridge forming between the positively charged primary imino group of a proline residue and the negatively charged carboxyl group of a conserved aspartate. In order to evaluate the contribution of either side-chain length or charge to the formation of infectious virus capsids, aspartate 183 was substituted for glutamate or asparagine in the viral context. It was found that both modifications abolished infectivity of the corresponding viruses in permissive T lymphocytes, although none of particle assembly and release, RNA encapsidation, incorporation of Env glycoproteins and packaging of cyclophilin A were impaired. However, whereas biophysical analyses of mutant virions yielded wild-type-like particle sizes and densities, electron microscopy revealed aberrant core morphologies that could be attributed to either increased (D183N) or reduced (D183E) capsid stability. Although the two amino acid substitutions had opposing effects upon core stability, both mutants were shown to exhibit a severe block in early reverse transcription, underscoring the importance of correct salt-bridge formation for early steps of virus replication.

Live-vector human immunodeficiency virus (HIV) vaccines are an integral part of a number of HIV vaccine regimens currently under evaluation that have yielded promising results in pre-clinical testing. In this report, a non-pathogenic protozoan parasitic vector, Leishmania tarentolae, which shares common target cells with HIV-1, was used to express full-length HIV-1 Gag protein. Immunization of BALB/c mice with recombinant L. tarentolae led to the expansion of HIV-1 Gag-specific T cells and stimulated CD8+ T cells to produce gamma interferon in response to specific viral Gag epitopes. A booster immunization with recombinant L. tarentolae elicited effector memory HIV-1 Gag-specific CD4+ T lymphocytes and increased antibody titres against HIV-1 Gag. Most importantly, immunization of human tonsillar tissue cultured ex vivo with Gag-expressing L. tarentolae vaccine vector elicited a 75 % decrease in virus replication following exposure of the immunized tonsils to HIV-1 infection. These results demonstrated that recombinant L. tarentolae is capable of eliciting effective immune responses in mice and human systems, respectively, and suggest that this novel non-pathogenic recombinant vaccine vector shows excellent promise as a vaccination strategy against HIV-1.

Expression of the pol-encoded proteins of human immunodeficiency virus type 1 (HIV-1) requires a programmed –1 ribosomal frameshift at the junction of the gag and pol coding sequences. Frameshifting takes place at a heptanucleotide slippery sequence, UUUUUUA, and is enhanced by a stimulatory RNA structure located immediately downstream. In patients undergoing viral protease (PR) inhibitor therapy, a p1/p6gag L449F cleavage site (CS) mutation is often observed in resistant isolates and frequently generates, at the nucleotide sequence level, a homopolymeric and potentially slippery sequence (UUUUCUU to UUUUUUU). The mutation is located within the stimulatory RNA downstream of the authentic slippery sequence and could act to augment levels of pol-encoded enzymes to counteract the PR deficit. Here, RNA secondary structure probing was employed to investigate the structure of a CS-containing frameshift signal, and the effect of this mutation on ribosomal frameshift efficiency in vitro and in tissue culture cells was determined. A second mutation, a GGG insertion in the loop of the stimulatory RNA that could conceivably lead to resistance by enhancing the activity of the structure, was also tested. It was found, however, that the CS and GGG mutations had only a very modest effect on the structure and activity of the HIV-1 frameshift signal. Thus the increased resistance to viral protease inhibitors seen with HIV-1 isolates containing mutations in the frameshifting signal is unlikely to be accounted for solely by enhancement of frameshift efficiency.

It has previously been shown by our laboratory that OTK18, a human immunodeficiency virus (HIV)-inducible zinc-finger protein, reduces progeny-virion production in infected human macrophages. OTK18 antiviral activity is mediated through suppression of Tat-induced HIV-1 long terminal repeat (LTR) promoter activity. Through the use of LTR-scanning mutant vectors, the specific regions responsible for OTK18-mediated LTR suppression have been defined. Two different LTR regions were identified as potential OTK18-binding sites by an enhanced DNA–transcription factor ELISA system; the negative-regulatory element (NRE) at −255/−238 and the Ets-binding site (EBS) at −150/−139 in the LTR. In addition, deletion of the EBS in the LTR blocked OTK18-mediated LTR suppression. These data indicate that OTK18 suppresses LTR activity through two distinct regulatory elements. Spontaneous mutations in these regions might enable HIV-1 to escape from OTK18 antiretroviral activity in human macrophages.

Natural killer (NK) cells are a major component of the host innate immune defence against various pathogens. Several viruses, including Human immunodeficiency virus 1 (HIV-1), have developed strategies to evade the NK-cell response. This study was designed to evaluate whether HIV-1 could interfere with the expression of NK cell-activating ligands, specifically the human leukocyte antigen (HLA)-I-like MICA and ULBP molecules that bind NKG2D, an activating receptor expressed by all NK cells. Results show that the HIV-1 Nef protein downmodulates cell-surface expression of MICA, ULBP1 and ULBP2, with a stronger effect on the latter molecule. The activity on MICA and ULBP2 is well conserved in Nef protein variants derived from HIV-1-infected patients. In HIV-1-infected cells, cell-surface expression of NKG2D ligands increased to a higher extent with a Nef-deficient virus compared with wild-type virus. Mutational analysis of Nef showed that NKG2D ligand downmodulation has structural requirements that differ from those of other reported Nef activities, including HLA-I downmodulation. Finally, data demonstrate that Nef expression has functional consequences on NK-cell recognition, causing a decreased susceptibility to NK cell-mediated lysis. These findings provide a novel insight into the mechanisms evolved by HIV-1 to escape from the NK-cell response.

In the pathogenesis of feline immunodeficiency virus (FIV) infection, feline dendritic cells (feDCs) are thought to play an important role. As with DCs in other species, feDCs are believed to transport virus particles to lymph nodes and transfer them to lymphocytes. Our investigation has focused on the ability of feDCs to influence the infection of syngeneic peripheral blood mononuclear cells (PBMCs) and allogeneic thymocytes. feDCs were derived from bone marrow mononuclear cells that were cultured under the influence of feline interleukin-4 and feline granulocyte–macrophage colony-stimulating factor. By using these feDCs in co-culture with resting PBMCs, an upregulation of FIV replication was shown. An enhancement of FIV infection was also detected when co-cultures of feDCs/feline thymocytes were infected. To obtain this enhancement, direct contact of the cells in the co-culture was necessary; transwell cultures showed that the involvement of only soluble factors produced by feDCs in this process is not likely. These feDCs were also able to induce the proliferation of resting thymocytes, which might explain the enhanced FIV replication observed. Together, these data suggest that feDCs have abilities similar to those shown for simian and human DCs in the interaction with leukocytes. This system is suitable for further investigations of the interplay of DC and T cells during FIV infection in vitro.

It was shown recently that retrovirus transactivators interact with transcriptional coactivators, such as histone acetyltransferases (HATs). Foamy viruses (FVs) direct gene expression from the long terminal repeat and from an internal promoter. The activity of both promoters is strictly dependent on the DNA-binding transactivator Tas. Recently, it was shown that Tas interacts with the HATs p300 and PCAF. Based on these findings, it is demonstrated here that PCAF has the ability to acetylate Tas in vitro and in vivo. Tas acetylation resulted in enhanced DNA binding to the virus promoters. In vitro transcription reactions on non-chromatinized template showed that only acetylated Tas enhanced transcription significantly. These results demonstrate that acetylation of the FV transactivator Tas may be an effective means to regulate virus transcription.

Multiple sclerosis (MS)-associated retrovirus (MSRV)/HERV-W (human endogenous retrovirus W) and Human herpesvirus 6 (HHV-6) are the two most studied (and discussed) viruses as environmental co-factors that trigger MS immunopathological phenomena. Autopsied brain tissues from MS patients and controls and peripheral blood mononuclear cells (PBMCs) were analysed. Quantitative RT-PCR and PCR with primers specific for MSRV/HERV-W env and pol and HHV-6 U94/rep and DNA-pol were used to determine virus copy numbers. Brain sections were immunostained with HERV-W env-specific monoclonal antibody to detect the viral protein. All brains expressed MSRV/HERV-W env and pol genes. Phylogenetic analysis indicated that cerebral MSRV/HERV-W-related env sequences, plasmatic MSRV, HERV-W and ERVWE1 (syncytin) are related closely. Accumulation of MSRV/HERV-W-specific RNAs was significantly greater in MS brains than in controls (P=0.014 vs healthy controls; P=0.006 vs pathological controls). By immunohistochemistry, no HERV-W env protein was detected in control brains, whereas it was upregulated within MS plaques and correlated with the extent of active demyelination and inflammation. No HHV-6-specific RNAs were detected in brains of MS patients; one healthy control had latent HHV-6 and one pathological control had replicating HHV-6. At the PBMC level, all MS patients expressed MSRV/HERV-W env at higher copy numbers than did controls (P=0.00003). Similar HHV-6 presence was found in MS patients and healthy individuals; only one MS patient had replicating HHV-6. This report, the first to study both MSRV/HERV-W and HHV-6, indicates that MSRV/HERV-W is expressed actively in human brain and activated strongly in MS patients, whilst there are no significant differences between these MS patients and controls for HHV-6 presence/replication at the brain or PBMC level.

Natural, aqueous extracts of Quillaja saponaria, the Chilean soapbark tree, contain several physiologically active triterpenoid saponins that display strong adjuvant activity when used in either human or animal vaccines. In this paper, we describe studies that demonstrate a novel antiviral activity of Quillaja extracts against six viruses: vaccinia virus, herpes simplex virus type 1, varicella zoster virus, human immunodeficiency viruses 1 and 2 (HIV-1, HIV-2) and reovirus. We demonstrate that microgram amounts of extract, while exhibiting no cell cytotoxicity or direct virucidal activity, prevent each of the six viruses tested from infecting their host cells. In addition, the presence of residual amounts of extract continue to block virus infection and render cells resistant to infection for at least 16 h after the removal of the extract from the cell culture medium. We demonstrate that a Quillaja extract possesses strong antiviral activity at concentrations more than 100-fold lower than concentrations that exhibit cell cytotoxicity. Extract concentrations as high as 100 μg ml−1 are not cytotoxic, but concentrations as low as 0.1 μg ml−1 are able to block HIV-1 and HIV-2 virus attachment and infection.

Biochemical studies have suggested that annexin 2 (A2) may participate in cytomegalovirus (CMV) infection. In the current work, effects of A2 monomer (p36) and heterotetramer (A2t; p362p112) were investigated. Demonstrating a role for endogenous A2, the four stages of infection that were followed were each inhibited by anti-p36 or anti-p11 at 37 °C. Immuno-inhibition was attenuated when the virus and cells were pre-incubated at 4 °C to coordinate virus entry initiated afterwards at 37 °C, reconciling controversy in the literature. As an explanation, CMV-induced phosphorylation of p36 was prevented by the 4 °C treatment. Supporting these immuno-inhibition data, purified A2t or p11 increased CMV infectious-progeny generation and CMV gene expression. A specific role for A2t was indicated by purified p36 having no effect. Unlike other steps, primary plaque formation was not enhanced by purified A2t or p11, possibly because of undetectable phosphorylation. As annexins 1 (A1) and 5 (A5) interact with A2, their effect on CMV was also tested. Both purified proteins inhibited CMV infection. In each experiment, the concentration of A1 required for half-maximal inhibition was five- to 10-fold lower than that of A5. Addition of A2 opposed A1- or A5-mediated inhibition of CMV, as did certain A2-specific antibodies that had no effect in the absence of added A1 or A5. Transfection of the p36-deficient cell line HepG2 increased CMV infection and was required for inhibition by the other annexins. These data suggest that CMV exploits A2t at physiological temperature to oppose the protection of cells conferred by A1 or A5.