- Volume 84, Issue 1, 2003

The immune system has a variety of tools at its disposal to combat virus infections. These can be subdivided roughly into two categories: ‘first line defence’, consisting of the non-specific, innate immune system, and ‘adaptive immune response’, acquired over time following virus infection or vaccination. During evolution, viruses have developed numerous, and often very ingenious, strategies to counteract efficient recognition of virions or virus-infected cells by both innate and adaptive immunity. This review will focus on the different strategies that viruses use to avoid recognition by one of the components of the immune system: the complement system. Complement evasion is of particular importance for viruses, since complement activation is a crucial component of innate immunity (alternative and mannan-binding lectin activation pathway) as well as of adaptive immunity (classical, antibody-dependent complement activation).

Alphaviruses are small enveloped viruses that have been used extensively as model enveloped viruses. During infection, virus particles are taken up into endosomes, where a low pH activates the viral fusion protein, E1. Fusion of the viral and the endosomal membranes releases the viral core into the cytoplasm where cores are disassembled by interaction with 60S ribosomal subunits. Recently, we have shown that in vitro this disassembly is strongly stimulated by low pH. We have proposed that after entry of the core into the cytoplasm, the viral membrane proteins that have been transferred to the endosomal membrane form an ion-permeable pore in the endosome. The resulting flow of protons from the endosome into the cytoplasm through this pore could generate a low-pH environment for core disassembly in vivo. Here we report two types of analysis aimed at the identification of such pores. First, the release of [3H]choline from the interior of liposomes was analysed in the presence of virus particles and viral proteins. Secondly, cells were infected with Sindbis or Semliki Forest alphaviruses at the plasma membrane and the possible generation of ion-permeable pores during this process was analysed by whole-cell voltage clamp analysis of the membrane current. The results obtained indicated that the proposed pores are in fact generated and allowed us to identify the formation of individual pores. Available evidence indicates that the alphavirus E1 protein probably forms these pores. Proteins homologous to the alphavirus E1 protein are present in flaviviruses and hepatitis C virus.

Flavivirus particles are synthesized in an immature form containing heterodimers of the proteins prM and E. Shortly before release from the cell, prM is cleaved by the host protease furin to yield mature virions. In this study, the furin-mediated cleavage of the tick-borne encephalitis (TBE) virus protein prM was prevented by specific mutagenesis of the cleavage site. This resulted in the production of immature TBE virions, which were shown to be completely non-infectious in BHK-21 cells. This finding contrasted with previous studies in which immature flavivirus particles produced by other techniques were shown to have considerable residual infectivity. The structural integrity of the mutant virus particles was confirmed by the characterization of physical and antigenic properties. Most importantly, infectivity could be restored by the addition of trypsin, which presumably cleaved protein prM at one of the monobasic sites retained in the mutated sequence. In the presence of trypsin, the mutant could be passaged repeatedly in BHK-21 cells, but if the protease was removed, the activated particles could initiate only a single round of infection, which again generated non-infectious virus progeny. These observations provide evidence that the infectivity of flaviviruses depends on the endoproteolytic cleavage of protein prM, which probably has a regulatory function rather than a direct role in virus entry. Moreover, the results illustrate that mutation of the furin cleavage site is a convenient way to produce single-round infectious flavivirus particles, which may be useful in vaccine and vector development.

Inactivated influenza A virus and fixed, virus-infected cells induce type 1 interferon (IFN-α/β) production in murine splenocytes. In this study, we have explored the nature of the virus–spleen cell interaction that leads to IFN-α/β induction and the reason for the poor response to some virus strains. IFN-α/β induction by horse serum-sensitive, but not -resistant, strains of influenza virus was inhibited in the presence of horse serum, indicating that binding of the virus to sialylated cell receptors is a necessary step in the induction process. Furthermore, influenza viruses A/PR/8/34 (H1N1) and A/WS/33 (H1N1), which were poor inducers of IFN-α/β in spleen cells, were shown to have a more active neuraminidase than strains that induced higher IFN levels, and IFN-α/β induction by A/PR/8/34 (H1N1) and A/WS/33 (H1N1) was restored in the presence of a neuraminidase inhibitor. Growth of virus in different cell types altered the level of IFN-α/β induced in spleen cells by particular virus strains, suggesting that the nature of the carbohydrate moieties on the viral glycoproteins may also influence IFN-α/β induction in this system. Consistent with this notion, treatment of egg-grown virus with periodate to oxidize viral carbohydrate greatly reduced its capacity for IFN-α/β induction. Furthermore, induction of IFN-α/β was inhibited in the presence of the saccharides yeast mannan and laminarin. Together these findings indicate: (i) a requirement for interaction of the virus with sialylated receptors on the IFN-producing cell; (ii) an influence of viral carbohydrate on the response; and (iii) possible involvement of a lectin-like receptor on the IFN-producing cell in the induction of IFN-α/β or in regulation of this response.

Envelopes of primary R5-tropic human immunodeficiency virus type 1 (HIV-1) isolates may be particularly relevant for vaccine purposes and should be evaluated for immunogenicity in animals including macaques before carrying out human vaccine trials. In the present study, the immunogenicities of synthetic HIV-1 env DNA vaccines, which had been derived from the early primary isolate Bx08 and contain humanized codons, were evaluated in mice, guinea pigs and rhesus macaques. Neutralization sensitivity of the HIV-1Bx08 isolate was found to resemble that of other primary isolate prototypes. Immunogenicity of gp120 delivered as codon-optimized DNA vaccine was comparable to that of recombinant gp120 protein plus adjuvant in mice. Similarly, DNA vaccination of guinea pigs with synthetic gp140Bx08 and gp150Bx08 DNA induced a strong antibody response independent of the gene construct and DNA immunization route. Mamu-A*01 rhesus macaques were DNA vaccinated with synthetic gp150Bx08 or gp140Bx08 DNA and boosted with a replication-deficient recombinant human adenovirus type 5 expressing a synthetic gp120Bx08 gene. DNA-vaccinated rhesus macaques developed specific CD8+ T lymphocyte responses and anti-rgp120IIIb antibody responses. Both the humoral and cellular responses were significantly improved following intramuscular boosting with the recombinant adenovirus. The demonstrated humoral and cellular immunogenicities of these HIV Bx08 Env vaccines in non-human primates encourages their further development as one component in candidate HIV vaccines for humans.

Recent studies have revealed that ‘human retrovirus-5’ sequences found in human samples belong to a rabbit endogenous retrovirus family named RERV-H. A part of the gag–pro region of the RERV-H genome was amplified by PCR from DNA in human samples and several forms of RERV-H protease were expressed in bacteria. The RERV-H protease was able to cleave itself from a precursor protein and was also able to cleave the RERV-H Gag polyprotein precursor in vitro whereas a form of the protease with a mutation engineered into the active site was inactive. Potential N- and C-terminal autocleavage sites were characterized. The RERV-H protease was sensitive to pepstatin A, showing it to be an aspartic protease. Moreover, it was strongly inhibited by PYVPheStaAMT, a pseudopeptide inhibitor specific for Mason–Pfizer monkey virus and avian myeloblastosis-associated virus. A structural model of the RERV-H protease was constructed that, together with the activity data, confirms that this is a retroviral aspartic protease.

Bluetongue virus (BTV) and several other Orbivirus species are transmitted between mammalian hosts via bites from adults of certain species of Culicoides midges. However, BTV can survive for 9–12 months (typically during the winter), in the absence of adult vectors, with no detectable cases of viraemia, disease or seroconversion in the host. The survival of the virus from one ‘vector season’ to the next is called ‘overwintering’ but the mechanism involved is not fully understood. It is demonstrated that BTV can persistently infect ovine γδ T-cells in vitro, a process that may also occur during infection and viraemia in mammalian hosts, thus providing a mechanism for virus persistence. Interaction of persistently BTV-infected γδ T-cells with antibody to the γδ T-cell-specific surface molecule WC-1 resulted in conversion to a lytic infection and increased virus release. Skin fibroblasts induce a similar conversion, indicating that they express a counter ligand for WC-1. Feeding of Culicoides midges induces skin inflammation, which is accompanied by recruitment of large numbers of activated γδ T-cells. The interaction of persistently infected γδ T-cells with skin fibroblasts would result in increased virus production at ‘biting sites’, favouring transmission to the insect vector. This suggested mechanism might also involve up-regulation of the WC-1 ligand at inflamed sites. It has been shown previously that cleavage of virus surface proteins by protease enzymes (which may also be associated with inflammation) generates infectious subvirus particles that have enhanced infectivity (100 times) for the insect vector.

The gene complement of wild-type human cytomegalovirus (HCMV) is incompletely understood, on account of the size and complexity of the viral genome and because laboratory strains have undergone deletions and rearrangements during adaptation to growth in culture. We have determined the sequence (241 087 bp) of chimpanzee cytomegalovirus (CCMV) and have compared it with published HCMV sequences from the laboratory strains AD169 and Toledo, with the aim of clarifying the gene content of wild-type HCMV. The HCMV and CCMV genomes are moderately diverged and essentially collinear. On the basis of conservation of potential protein-coding regions and other sequence features, we have discounted 51 previously proposed HCMV ORFs, modified the interpretations for 24 (including assignments of multiple exons) and proposed ten novel genes. Several errors were detected in the published HCMV sequences. We presently recognize 165 genes in CCMV and 145 in AD169; this compares with an estimate of 189 unique genes for AD169 made in 1990. Our best estimate for the complement of wild-type HCMV is 164 to 167 genes.

The human cytomegalovirus (HCMV) UL36–38 immediate early (IE) locus encodes proteins required for virus growth. The UL37 IE promoter drives production of differentially spliced and unspliced RNAs. To study their post-transcriptional processing, we generated target minigenes encoding each UL37 RNA splicing substrate. Target 1 RNA, spanning UL37 exon 1 (x1) donor and 2 (x2) acceptor as well as adjacent intronic sequences, but not the UL38 gene, accurately reproduced UL37 x1/x2 RNA splicing in transfected permissive cells. Surprisingly, deletion of distal intronic sequences nt −82 to −143 from the UL37x2 acceptor resulted in aberrant splicing to an upstream non-consensus exonic donor. Target 1 RNAs carry the UL37x1 polyadenylation (PA) signal and site as well as a downstream SV40 early PA signal. Both the UL37x1 and SV40 PA signals are used in wild-type target 1 RNAs but inhibited in UL37x1 PA signal mutants. Alternative RNA splicing of UL37 exons 2 to 3 or 3A as well as exons 3 to 4, observed in HCMV mature UL37 and UL36 spliced RNAs, is accurately reproduced with target minigene RNAs carrying the corresponding UL37 exonic and intronic sequences. Moreover, alternative splicing using two novel UL37 exon 3 consensus splice donors (di and dii) was found in target and in HCMV-infected cell RNA. These results demonstrate that: (i) target minigene RNAs accurately recapitulate the processing of UL37 IE RNAs in the HCMV-infected cell; (ii) precise UL37x1 donor selection is modulated by 3′-distal UL37 intronic sequences; and (iii) UL37 exon 3 contains multiple alternative consensus splice donors.

The repression of human cytomegalovirus immediate-early (IE) lytic gene expression is crucial for the maintenance of the latent viral state. By using conditionally permissive cell lines, which provide a good model for the differentiation state-dependent repression of IE gene expression, we have identified several cellular factors that bind to the major immediate-early promoter (MIEP) and whose expression is down-regulated after differentiation to a permissive phenotype. Here we show that the cellular protein Ets-2 Repressor Factor (ERF) physically interacts with the MIEP and represses MIEP activity in undifferentiated non-permissive T2 embryonal carcinoma cells. This factor binds to the dyad element and the 21 bp repeats within the MIEP – regions known to be important for the negative regulation of MIEP activity. Finally, we show that following differentiation to a permissive phenotype ERF's repressive effects are severely abrogated.

In human cytomegalovirus (HCMV) infection, the isolated expression of the viral immediate-early protein 2 (IE2) 86 kDa regulatory protein coincides with an up-regulation of cyclin E gene expression, both in fibroblasts and U373 cells. Since IE2 also interferes with cell-cycle progression, it is unclear whether IE2 is a genuine activator of cyclin E or whether IE2-arrested cells contain elevated levels of cyclin E primarily as a consequence of them being arrested at the beginning of S phase. It is important to distinguish between these possibilities in order to define and analyse at a mechanistic level the proliferative and anti-proliferative capacities of IE2. Here we have shown that IE2 can activate cyclin E independent of the cell-cycle state and can therefore function as a genuine activator of cyclin E gene expression. A mutant of IE2 that failed to activate cyclin E also failed to promote G1/S transition. Instead, cells became arrested in G1. S-phase entry could be rescued in these cells by co-expression of cyclin E, but these cells still arrested in early S phase, as is the case with wild-type IE2. Our data demonstrate that IE2 can promote two independent cell-cycle functions at the same time: (i) the induction of G1/S transition via up-regulation of cyclin E, and (ii) a block in cell-cycle progression in early S phase. In G1, the proliferative activity of IE2 appears to be dominant over the anti-proliferative force, whereas after G1/S transition, this situation is reversed.

Human cytomegalovirus (HCMV) infection of transplant recipients is frequently associated with allograft vasculopathy and rejection. One potential mechanism is vascular injury from HCMV-triggered, immunologically mediated processes. HCMV infection has been shown to increase the expression of intercellular adhesion molecule-1 (ICAM-1). The objective of this study was to determine the molecular basis of HCMV-enhanced ICAM-1 gene expression. Transient transfection experiments identified the IE2p86 protein as a potent activator of the ICAM-1 promoter. The tegument protein pp71 showed a strong synergistic effect on IE2p86-mediated ICAM-1 promoter activation. Mutagenesis experiments defined a DNA element from −110 to −42 relative to the transcription start site as responsive for IE2p86. Further point mutations within this DNA element identified an Sp1-binding site that was essential for strong synergistic activation by IE2p86 and pp71. To confirm the activation of ICAM-1 gene expression, human fibroblasts (HFF) as well as endothelial cells (HUVEC) were infected with recombinant IE2p86- and pp71-expressing baculoviruses, respectively. In FACS analysis, a synergistic induction of ICAM-1 was detectable when cells were co-infected with the two recombinant baculoviruses. These findings clearly demonstrate that IE2p86 and pp71 are crucial regulatory factors for HCMV-induced ICAM-1 upregulation.

Live attenuated strains of human cytomegalovirus are under development as vaccines to prevent birth defects resulting from congenital infections. These strains encode four proteins that inhibit surface expression of MHC class I, presumably to evade cytotoxic T-cell recognition and, perhaps, attenuate induction of immunity. To initiate studies of the role of class I down-regulation on congenital infection and vaccine efficacy, the ability of guinea pig cytomegalovirus to down-regulate class I was examined. Surface class I was specifically down-regulated on infected cells up to 8-fold. Sensitivity to UV irradiation and insensitivity to a viral DNA synthesis inhibitor revealed that immediate early or early viral gene(s) are responsible. Identification of these genes will permit future experiments to evaluate the role of class I down-regulation in congenital cytomegalovirus disease and its impact on vaccine efficacy. These findings should be pertinent to understanding human cytomegalovirus disease and may help guide the design of candidate vaccines.

Non-human primate herpesviruses establish and maintain a lifelong persistent infection in immunocompetent hosts in the absence of clinical signs of disease. A fundamental issue for understanding the natural history of non-human primate herpesviruses is whether the viruses are maintained in a truly latent state or one characterized by a low level of chronic expression. To address this issue, a real-time PCR assay was developed to quantify Cercopithecine herpesvirus type 1 (B virus) DNA in mucosal fluids of rhesus macaques. This assay was rapid, sensitive (10 genome copies) and specific for B virus obtained from multiple species of macaques. The shedding profile of B virus was compared to another endemic herpesvirus, rhesus cytomegalovirus (RhCMV), in colony-reared monkeys. Mucosal swabs or saliva samples were taken daily from two groups of seropositive monkeys undergoing either a stressful relocation (group 1) or daily chair restraint (group 2). B virus DNA was detected in mucosal fluids from four animals relocated during the breeding season (group 1) but not from 10 animals moved at other times of the year. No B virus DNA was detected in any group 2 monkey. In contrast, RhCMV DNA was detected in the majority of animals of both groups 1 and 2. Detection of B virus DNA shedding is a relatively rare event associated with the breeding season, while RhCMV DNA is persistently detected in mucosal fluids of most monkeys.

Equine herpesvirus-1 (EHV-1) may cause abortion in vaccination- and infection-immune horses. EHV-1-infected peripheral blood mononuclear cells (PBMCs) play an important role in virus immune evasion. The mechanisms by which infected PBMCs can avoid destruction by EHV-1-specific antibody and equine complement were examined. The majority of EHV-1-infected PBMCs (68·6 %) lacked surface expression of viral antigens and these cells were not susceptible to complement-mediated lysis. In infected PBMCs with surface expression of viral antigens, 63 % showed focal surface expression, whereas 37 % showed general surface expression. General surface expression rendered infected PBMCs susceptible to lysis by antibody and complement (from 5·4 to 31·2 % lysed cells depending on the concentration of antibody and complement). Infected PBMCs with focal surface expression showed significant lysis only in the presence of high concentrations of antibody and complement. Thus, the absence of surface expression protects infected PBMCs against complement-mediated lysis.

The murine gammaherpesvirus-68 genome encodes 73 protein-coding open reading frames with extensive similarities to human γ2 herpesviruses, as well as unique genes and cellular homologues. We performed transcriptome analysis of stage-specific viral RNA during permissive infection using an oligonucleotide-based microarray. Using this approach, M4, K3, ORF38, ORF50, ORF57 and ORF73 were designated as immediate-early genes based on cycloheximide treatment. The microarray analysis also identified 10 transcripts with early expression kinetics, 32 transcripts with early-late expression kinetics and 29 transcripts with late expression kinetics. The latter group consisted mainly of structural proteins, and showed high expression levels relative to other viral transcripts. Moreover, we detected all eight tRNA-like transcripts in the presence of cycloheximide and phosphonoacetic acid. Lytic infection with MHV-68 also resulted in a significant reduction in the expression of cellular transcripts included in the DNA chip. This global approach to viral transcript analysis offers a powerful system for examining molecular transitions between lytic and latent virus infections associated with disease pathogenesis.

Infection of laboratory mice by the Murid herpesvirus 4 (MHV-4) is a much studied model system for gammaherpesvirus pathogenesis. Little, however, is known about its natural host range, epidemiology and pathogenesis outside the laboratory. We have studied MHV-4 infection in free-living murids in the UK. Using a combination of serology and PCR analysis, we found that MHV-4 was endemic in wood mice (Apodemus sylvaticus) but not in two species of voles (Clethrionomys glareolus, Microtus agrestis). The sites of detection of viral DNA were the lungs and, less commonly, the spleen, emphasizing the importance of the former in virus persistence during natural infection and confirming similar data in laboratory mice.

Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) and its closely related variant H. zea SNPV (HzSNPV) contain 20 open reading frames (ORFs) unique among baculoviruses. In this report, the function of HaSNPV ORF 122 (Ha122) is investigated. Ha122 was transcribed as a polyadenylated transcript from 8 h post-infection in infected H. armigera insect cells. 5′RACE analysis indicated that Ha122 transcription starts predominantly in the consensus major late transcription initiation motif DTAAG, around 47 nt upstream of the putative translation start codon, with a minor start at position −89. Using 3′RACE, the transcription stop site mapped 27 nt downstream of the putative translation stop codon. By Western blot analysis using a chicken-derived polyclonal antibody, the product of Ha122 was found in infected cells to be a 21 kDa protein, close to the theoretical size of 21.6 kDa. The Ha122 protein, when fused to green fluorescent protein, was observed in the nuclei of H. armigera cells but only in conjunction with wild-type HaSNPV infection. The 21 kDa protein was located specifically in the nucleocapsid of occlusion-derived virions (ODVs) and not in that of budded virus. The available data suggest that Ha122 is a functional ORF of HaSNPV and HzSNPV and that the 21 kDa protein is a novel specific component of baculovirus ODVs.

Enhancin genes have been identified in a number of baculoviruses and enhancin proteins are characterized by their ability to enhance the oral infectivity of heterologous baculoviruses in various lepidopteran insects. Here, we describe the putative enhancin gene from Mamestra configurata nucleopolyhedrovirus (MacoNPV), only the second NPV in which an enhancin-like ORF has been identified. The putative enhancin gene from MacoNPV has a typical baculovirus late promoter (ATAAG) 15 bp upstream from the ATG codon. The enhancin ORF encodes an 847 amino acid protein with a predicted molecular mass of 98 kDa and contains a conserved zinc-binding domain (HEIAH) common to metalloproteases. The MacoNPV enhancin shows approximately 20 % amino acid identity with other baculovirus enhancins. An Autographa californica M nucleopolyhedrovirus (AcMNPV) recombinant, AcMNPV-enMP2, expressing the MacoNPV enhancin gene under control of its native promoter was developed and characterized. Northern blot analysis showed expression of enhancin from 24 through 72 h post-infection. In 2nd-instar Trichoplusia ni larvae, the LD50 of the AcMNPV-enMP2 recombinant was 2·8 polyhedral inclusion bodies (PIB) per larva, 4·4 times lower than that of AcMNPV E2 wild-type virus (12·4 PIB per larva). At biologically equivalent doses, i.e. LD90, the survival time 50 % (ST50) of AcMNPV-enMP2 recombinant and AcMNPV E2 wild-type viruses were not significantly different.

Adeno-associated virus type 2 (AAV-2) establishes latency by site-specific integration into a unique locus, AAVS1, on human chromosome 19 (chr19). To study the kinetics and frequency of chr19-specific integration, a rapid, sensitive and quantitative real-time PCR assay specific for AAV inverted terminal repeat (ITR)–chr19 junction sequences was developed. Since the assay only detected right-hand AAV ITR-specific integration events, the development of a complementary left-hand ITR-specific real-time PCR assay is described. The time-course of left-hand ITR-dependent AAV integration at AAVS1 of chr19 was determined in AAV-2-infected HeLa cells. Both the kinetics and frequencies of left-hand ITR-dependent integration were found to be similar to those of the right-hand ITR. In addition, left-hand ITR-specific fusion sequences and chromosomal breakpoints within AAVS1 were variable, yet were the same as those found in right-hand ITR–chr19 junction sequences. Thus, the AAV-2 genome integrates site-specifically into chr19 with similar efficiency in either orientation.