- Volume 91, Issue 5, 2010

The mechanisms by which cyclophilin A (CypA) governs hepatitis C virus (HCV) replication remain unknown. Since CypA binds two essential components of the HCV replication complex (RC) – the polymerase NS5B and the phosphoprotein NS5A – we asked in this study whether CypA regulates their RC association. We found that CypA, via its isomerase pocket, locates in a protease-resistant compartment similar to that where HCV replicates. CypA association with this compartment is not mediated by HCV. Moreover, CypA depletion of RC does not influence NS5A and NS5B RC association, arguing against a model where CypA governs HCV replication by recruiting NS5A or NS5B into RC.

Immunological control of hepatitis C virus (HCV) is possible and is probably mediated by host T-cell responses, but the genetic diversity of the virus poses a major challenge to vaccine development. We considered monovalent and polyvalent candidates for an HCV vaccine, including natural, consensus and synthetic ‘mosaic’ sequence cocktails. Mosaic vaccine reagents were designed using a computational approach first applied to and demonstrated experimentally for human immunodeficiency virus type 1 (HIV-Δ). Mosaic proteins resemble natural proteins, but are assembled from fragments of natural sequences via a genetic algorithm and optimized to maximize the coverage of potential T-cell epitopes (all 9-mers) found in natural sequences and to minimize the inclusion of rare 9-mers to avoid vaccine-specific responses. Genotype 1-specific and global vaccine cocktails were evaluated. Among vaccine candidates considered, polyvalent mosaic sequences provided the best coverage of both known and potential epitopes and had the fewest rare epitopes. A global vaccine based on conserved proteins across genotypes may be feasible, as a five-antigen mosaic cocktail provided 90, 77 and 70 % coverage of the Core, NS3 and NS4 proteins, respectively; protein coverage diminished with increased protein variability, dropping to 38 % for NS2. For the genotype 1-specific vaccine, the H77 prototype vaccine sequence matched only 50 % of the potential epitopes in the population, whilst a polyprotein three-antigen mosaic cocktail increased potential epitope coverage to 83 %. More than 75 % coverage of all HCV proteins was achieved with a three-antigen mosaic cocktail, suggesting that genotype-specific vaccines could also include the more variable proteins.

The hepatitis C virus NS5B RNA-dependent RNA polymerase (RdRp) is a key enzyme involved in viral replication. Interaction between NS5B RdRp and the viral RNA sequence is likely to be an important step in viral RNA replication. The C-terminal half of the NS5B-coding sequence, which contains the important cis-acting replication element, has been identified as an NS5B-binding sequence. In the present study, we confirm the specific binding of NS5B to one of the RNA stem–loop structures in the region, 5BSL3.2. In addition, we show that NS5B binds to the complementary strand of 5BSL3.2 (5BSL3.2N). The bulge structure of 5BSL3.2N was shown to be indispensable for tight binding to NS5B. In vitro RdRp activity was inhibited by 5BSL3.2N, indicating the importance of the RNA element in the polymerization by RdRp. These results suggest the involvement of the RNA stem–loop structure of the negative strand in the replication process.

Here we report on a segment in the genomic 3′ non-translated region (3′NTR) of bovine viral diarrhea virus (BVDV) that is accessible for the insertion of foreign sequence elements such as the 5′NTR of hepatitis C virus. Recombinant viruses exhibited replication kinetics similar to those of the parental strain, and characterization of RNA species after several passages revealed that foreign inserts had the same genetic stability as the BVDV 3′NTR. The generation of such BVDV recombinants is relevant for several applications.

RNA elements within the flavivirus genome may play essential regulatory roles during virus replication. Here, recombinant West Nile virus (WNV) NS5 protein was used in combination with WNV subgenomic RNA templates to establish in vitro RNA-dependent RNA polymerase and RNA-binding assays. These assays identified mutations in the stem–loop A (SLA) region of the 5′ untranslated region (5′UTR) altering NS5 RNA synthesis and RNA-binding capability. These mutations were then introduced into the full-length WNV genome by reverse genetics. Further analysis of the mutant viruses showed that deletion of nt 46–60, which disrupted the stem and side loop of SLA, greatly compromised virus replication, whereas mutations that destroyed the top loop of SLA required for RNA synthesis in vitro did not significantly alter virus replication. These results suggest that SLA present in the 5′UTR of WNV is essential for RNA synthesis in vitro and for virus replication.

Eukaryotic initiation factor (eIF) 4E is a subunit of the cap-binding protein complex, eIF4F, which recognizes the cap structure of cellular mRNAs to facilitate translation initiation. eIF4E is assembled into the eIF4F complex via its interaction with eIF4G, an event that is under Akt/mTOR regulation. The eIF4E–eIF4G interaction is regulated by the eIF4E binding partners, eIF4E-binding proteins and eIF4E-transporter. Cleavage of eIF4G occurs upon poliovirus infection and is responsible for the shut-off of host-cell protein synthesis observed early in infection. Here, we document that relocalization of eIF4E to the nucleus occurs concomitantly with cleavage of eIF4G upon poliovirus infection. This event is not dependent upon virus replication, but is dependent on eIF4G cleavage. We postulate that eIF4E nuclear relocalization may contribute to the shut-off of host protein synthesis that is a hallmark of poliovirus infection by perturbing the circular status of actively translating mRNAs.

Human parechoviruses (HPeVs) are highly prevalent RNA viruses classified in the family Picornaviridae. Several antigenically distinct types circulate in human populations worldwide, whilst recombination additionally contributes to the genetic heterogeneity of the virus. To investigate factors influencing the likelihood of recombination and to compare its dynamics among types, 154 variants collected from four widely geographically separated referral centres (UK, The Netherlands, Thailand and Brazil) were typed by VP3/VP1 amplification/sequencing with recombination groups assigned by analysis of 3Dpol sequences. HPeV1B and HPeV3 were the most frequently detected types in each referral region, but with marked geographical differences in the frequencies of different recombinant forms (RFs) of types 1B, 5 and 6. HPeV1B showed more frequent recombination than HPeV3, in terms both of evolutionary divergence and of temporal/geographical indicators of population separation. HPeV1 variants showing between 10 and 20 % divergence in VP3/VP1 almost invariably fell into different recombination groups, compared with only one-third of similarly divergent HPeV3 variants. Substitution rates calculated by beast in the VP3/VP1 region of HPeV1 and HPeV3 allowed half-lives of the RFs of 4 and 20 years, respectively, to be calculated, estimates fitting closely with their observed lifespans based on population sampling. The variability in recombination dynamics between HPeV1B and HPeV3 offers an intriguing link with their markedly different seasonal patterns of transmission, age distributions of infection and clinical outcomes. Future investigation of the epidemiological and biological opportunities and constraints on intertypic recombination will provide more information about its influence on the longer term evolution and pathogenicity of parechoviruses.

Both entero- and cardioviruses have been shown to suppress host mRNA synthesis. Enteroviruses are also known to inhibit the activity of rRNA genes, whereas this ability of cardioviruses is under debate. This study reported that mengovirus (a cardiovirus) suppressed rRNA synthesis but less efficiently than poliovirus (an enterovirus). In contrast to poliovirus infection, the incorporation of BrUTP, fluorouridine and [14C]uridine in rRNA precursors was observed even during the late stages of mengovirus infection, although at a significantly reduced level. The cleavage of TATA-binding protein, considered to be one of the central events in poliovirus-induced transcription shutoff, was not detected in mengovirus-infected cells, indicating a difference in the mechanisms of host RNA synthesis inhibition caused by these viruses. The results also showed that functional leader protein is redundant for the suppression of host RNA synthesis by cardiovirus.

We have investigated the possible role of trans-acting factors interacting with the untranslated regions (UTRs) of coxsackievirus B3 (CVB3) RNA. We show here that polypyrimidine tract-binding protein (PTB) binds specifically to both 5′ and 3′ UTRs, but with different affinity. We have demonstrated that PTB is a bona fide internal ribosome entry site (IRES) trans-acting factor (ITAF) for CVB3 RNA by characterizing the effect of partial silencing of PTB ex vivo in HeLa cells. Furthermore, IRES activity in BSC-1 cells, which are reported to have a very low level of endogenous PTB, was found to be significantly lower than that in HeLa cells. Additionally, we have mapped the putative contact points of PTB on the 5′ and 3′ UTRs by an RNA toe-printing assay. We have shown that the 3′ UTR is able to stimulate CVB3 IRES-mediated translation. Interestingly, a deletion of 15 nt at the 5′ end or 14 nt at the 3′ end of the CVB3 3′ UTR reduced the 3′ UTR-mediated enhancement of IRES activity ex vivo significantly, and a reduced interaction was shown with PTB. It appears that the PTB protein might help in circularization of the CVB3 RNA by bridging the ends necessary for efficient translation of the viral RNA.

Increased vascular permeability is a key feature of the pathological symptoms caused by hantaviruses. Here, we analysed the interaction between hantaviruses and mast cells, which regulate vascular homeostasis. In highly purified human skin mast cells increasing amounts of Hantaan (HTNV) and, to a lower extent, Prospect Hill (PHV) virions were produced. Replication was confirmed by the production of viral plus-strand RNA as determined by a virus strand-specific RT-PCR. PHV but not HTNV elicited early expression of beta interferon, MxA, ISG15 and CCL5 consistent to studies with other cell types. The data demonstrate that mature mast cells are permissive to infection with hantaviruses. This interaction might contribute to the development of vascular leakage syndrome.

The genus Hantavirus (family Bunyaviridae) includes negative-strand RNA viruses that are carried by persistently infected rodent and insectivore species. Puumala virus (PUUV), carried by bank voles (Myodes glareolus), is a pathogenic hantavirus that causes outbreaks of mild haemorrhagic fever with renal syndrome across Europe. In northern Europe, PUUV is represented by several genetic lineages that are maintained by distinct phylogroups of bank voles. The present study describes sequences of new PUUV strains recovered from northern and southern regions of Scandinavia and compares phylogenetic relationships between north-European PUUV strains and M. glareolus. This analysis revealed contradictions in phylogenetic clustering and remarkable differences in estimated divergence times between the lineages of PUUV and its host, suggesting that the established PUUV lineages did not co-diverge with the distinct phylogroups of M. glareolus that carry them at present.

Sandflies are widely distributed around the Mediterranean Basin. Therefore, human populations in this area are potentially exposed to sandfly-transmitted diseases, including those caused by phleboviruses. Whilst there are substantial data in countries located in the northern part of the Mediterranean basin, few data are available for North Africa. In this study, a total of 1489 sandflies were collected in 2008 in Tunisia from two sites, bioclimatically distinct, located 235 km apart, and identified morphologically. Sandfly species comprised Phlebotomus perniciosus (52.2 %), Phlebotomus longicuspis (30.1 %), Phlebotomus papatasi (12 .0%), Phlebotomus perfiliewi (4.6 %), Phlebotomus langeroni (0.4 %) and Sergentomyia minuta (0.5 %). PCR screening, using generic primers for the genus Phlebovirus, resulted in the detection of ten positive pools. Sequence analysis revealed that two pools contained viral RNA corresponding to a novel virus closely related to sandfly fever Naples virus. Virus isolation in Vero cells was achieved from one pool. Genetic and phylogenetic characterization based on sequences in the three genomic segments showed that it was a novel virus distinct from other recognized members of the species. This novel virus was provisionally named Punique virus. Viral sequences in the polymerase gene corresponding to another phlebovirus closely related to but distinct from sandfly fever Sicilian virus were obtained from the eight remaining positive pools.

Recent large-scale sequence analyses revealed ‘signature’ amino acids at specific positions in viral proteins that distinguish human influenza viruses from avian viruses. To determine the role of these host lineage-specific amino acids in the pathogenicity of H5N1 avian influenza viruses, we generated mutant viruses possessing signature amino acids in the PB2, PA and NP proteins of human influenza isolates (‘human-like amino acids’) in the genetic background of an avian H5N1 virus, and tested their pathogenicity in mice. We found that some of these mutants exhibited enhanced pathogenicity in mice, suggesting the involvement of these host lineage-specific amino acids in the pathogenicity of H5N1 avian influenza viruses in mammals.

The influenza A virus RNA-dependent RNA polymerase produces capped and polyadenylated mRNAs in the nucleus of infected cells that resemble mature cellular mRNAs, but are made by very different mechanisms. Furthermore, only two of the 10 viral protein-coding mRNAs are spliced: most are intronless, while two contain unremoved introns. The mechanism(s) by which any of these mRNAs are exported from the nucleus is uncertain. To probe the involvement of the primary cellular mRNA export pathway, we treated cells with siRNAs against NXF1, Aly or UAP56, or with the drug 5,6-dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB), an inhibitor of RNA polymerase II phosphorylation previously shown to inhibit nuclear export of cellular mRNA as well as influenza virus segment 7 mRNAs. Depletion of NXF1 or DRB treatment had similar effects, inhibiting the nuclear export of several of the viral mRNAs. However, differing degrees of sensitivity were seen, depending on the particular segment examined. Intronless HA mRNA and spliced M2 or unspliced M1 transcripts (all encoding late proteins) showed a strong requirement for NXF1, while intronless early gene mRNAs, especially NP mRNA, showed the least dependency. Depletion of Aly had little effect on viral mRNA export, but reduction of UAP56 levels strongly inhibited trafficking and/or translation of the M1, M2 and NS1 mRNAs. Synthesis of NS2 from the spliced segment 8 transcript was, however, resistant. We conclude that influenza A virus co-opts the main cellular mRNA export pathway for a subset of its mRNAs, including most but not all late gene transcripts.

Human metapneumovirus (hMPV) is a major cause of upper and lower respiratory-tract infection in infants, the elderly and immunocompromised individuals. Virus-directed cellular immunity elicited by hMPV infection is poorly understood, in contrast to the phylogenetically and clinically related pathogen human respiratory syncytial virus (hRSV). In a murine model of acute lower respiratory-tract infection with hMPV, we demonstrate the accumulation of gamma interferon (IFN-γ)-producing CD8+ T cells in the airways and lungs at day 7 post-infection (p.i.), associated with cytotoxic T lymphocytes (CTLs) directed to an epitope of the M2-1 protein. This CTL immunity was accompanied by increased pulmonary expression of Th1 cytokines IFN-γ and interleukin (IL)-12 and antiviral cytokines (IFN-β), as well as chemokines Mip-1α, Mip-1β, Mig, IP-10 and CX3CL1. There was also a moderate increase in Th2-type cytokines IL-4 and IL-10 compared with uninfected mice. At 21 days p.i., a strong CTL response could be recalled from the spleen. A similar pattern of CTL induction to the homologous M2-1 CTL epitope of hRSV, and of cytokine/chemokine induction, was observed following infection with hRSV, highlighting similarities in the cellular immune response to the two related pathogens.

Chandipura virus (CHPV) is an emerging human pathogen associated with acute encephalitis and is related closely to vesicular stomatitis virus (VSV), a prototype rhabdovirus. Here, we demonstrate that the RNA polymerase L protein of CHPV exhibits a VSV-like RNA : GDP polyribonucleotidyltransferase (PRNTase) activity, which transfers the 5′-monophosphorylated (p-) viral mRNA start sequence to GDP to produce a capped RNA, and that the conserved HR motif in the CHPV L protein is essential for the PRNTase activity. Interestingly, the CHPV L protein was found to form two distinct SDS-resistant complexes with the CHPV mRNA and leader RNA start sequences; mutations in the HR motif significantly reduced the formation of the former complex (a putative covalent enzyme–pRNA intermediate in the PRNTase reaction), but not the latter complex. These results suggest that the rhabdoviral L proteins universally use the active-site HR motif for the PRNTase reaction at the step of the enzyme–pRNA intermediate formation.

Merino Walk virus (MWV), a proposed novel tentative species of the family Arenaviridae, was isolated from a rodent, Myotomys unisulcatus, collected at Merino Walk, Eastern Cape, South Africa, in 1985. Full-length genomic sequence confirmed MWV as an arenavirus related distantly to Mobala, Mopeia and Ippy viruses, all members of the Old World arenavirus complex. We propose MWV as a tentative novel species in the Lassa–lymphocytic choriomeningitis virus complex, based on its isolation from a novel rodent species and its genetic and serological characteristics.

The highly pathogenic Marburg virus (MARV) can only be investigated in high containment laboratories, which is time consuming and expensive. To investigate the MARV life cycle under normal laboratory conditions, an infectious virus-like particle (VLP) system was developed. The infectious VLP system is based on the T7-polymerase driven synthesis of a MARV-specific minigenome that encodes luciferase and is transcribed and replicated by the simultaneously expressed MARV nucleocapsid proteins NP, VP35, L and VP30. Transcription of the minigenome resulted in luciferase activity and replication resulted in encapsidated minigenomes. The encapsidated minigenomes, together with the viral matrix proteins VP40 and VP24 and the surface glycoprotein (GP), formed VLPs at the plasma membrane. Among the released pleomorphic VLPs, filamentous particles of 200–400 nm in length showed the highest capacity to induce reporter activity upon infection of target cells. To characterize the infectious VLP system, the intracellular concentration of one of the components was titrated, while all others were held constant. Intracellular concentrations of nucleocapsid proteins that resulted in highest replication and transcription activities also yielded VLPs with the highest ability to induce luciferase activity in target cells. High intracellular levels of VP40 maximized the release of VLPs, but reduced their ability to induce luciferase activity in target cells. The intracellular concentration of GP positively correlated with its incorporation into VLPs and their infectivity. Finally, we demonstrated that the infectious VLP system was suitable for rapid screening of neutralizing antibodies directed against MARV.

It has been reported that the addition of a potential N-linked glycosylation site (PNGS) to the gp120 human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein provides protection against neutralizing antibodies (NAbs) by acting as a ‘glycan shield’. In this study, we induced insertion of a PNGS into the V2 region of HIV-1BaL with the KD-247 anti-V3 neutralizing monoclonal antibody. In the presence of KD-247 (200 μg ml−1) at passage five, viruses with 3 aa mutations in the C2 (T240S and I283T) and V3 (T319A) regions expanded from pre-existing variants. After six passages with KD-247 (>300 μg ml−1), a PNGS emerged in the V2 region in addition to C2 (T240S) and V3 mutations (R315K and F317L). A variant with a PNGS insertion in V2, but no V3 mutations was sensitive to KD-247, whereas a clone with a V2 PNGS insertion and mutations in V3 demonstrated a high level of resistance to KD-247. Replication kinetic analysis revealed that the F317L mutation in V3 played a compensatory role for fitness-loss caused by the PNGS insertion in V2. The evading HIV-1 variant did not revert back to the wild-type virus after 14 passages without KD-247. These findings demonstrate that the virus with fitness-loss mutations can replicate equally as well as the wild-type virus to acquire some key mutations in the V3 stem and the C2 region, and the compensated variants containing PNGS do not revert back to the ancestral virus even in the absence of NAb.