- Volume 93, Issue 4, 2012

Human polyomavirus 9 (HPyV9) was discovered recently in immunocompromised patients and shown to be genetically closely related to B-lymphotropic polyomavirus (LPyV). No serological data are available for HPyV9, but human antibodies against LPyV have been reported previously. To investigate the seroepidemiology of HPyV9 and the sero-cross-reactivity between HPyV9 and LPyV, a capsomer-based IgG ELISA was established using the major capsid protein VP1 of HPyV9 and LPyV. VP1 of an avian polyomavirus was used as control. For HPyV9, a seroprevalence of 47 % was determined in healthy adults and adolescents (n = 328) and 20 % in a group of children (n = 101). In both groups, the seroreactivities for LPyV were less frequent and the ELISA titres of LPyV were lower. Of the HPyV9-reactive sera, 47 % reacted also with LPyV, and the titres for both PyVs correlated. Sera from African green monkeys, the natural hosts of LPyV, reacted also with both HPyV9 and LPyV, but here the HPyV9 titres were lower. This potential sero-cross-reactivity between HPyV9 and LPyV was confirmed by competition assays, and it was hypothesized that the reactivity of human sera against LPyV may generally be due to cross-reactivity between HPyV9 and LPyV. The HPyV9 seroprevalence of liver transplant recipients and patients with neurological dysfunctions did not differ from that of age-matched controls, but a significantly higher seroprevalence was determined in renal and haematopoietic stem-cell transplant recipients, indicating that certain immunocompromised patient groups may be at a higher risk for primary infection with or for reactivation of HPyV9.

The pleiotropic HBx protein of hepatitis B virus is linked functionally to the development of hepatocellular carcinoma (HCC) via effectors and signalling pathways of the host. To identify such effectors in a macrocarcinogenic environment, a PCR-based cDNA subtraction analysis was carried out in the X15-myc oncomouse model of HCC. Altogether, 19 categories of genes, mainly involved in protein biosynthesis and the electron-transport chain, were found to be upregulated in the liver of these mice. Ribosomal protein S27a (RPS27a), which is a natural fusion protein of N-terminal ubiquitin and C-terminal extension protein (CEP), topped the list of expressed genes, with >20-fold higher expression compared with its normal level. Sustained and elevated expression of RPS27a in the mouse liver and its moderate expression in cell culture in the presence of HBx suggested an indirect role of RPS27a in hepatocarcinogenesis. Nevertheless, a remarkable change in the intracellular distribution of ubiquitin from cytoplasm to late-endosomal lysosomes, and of CEP from nucleoli to the perinucleolar region/nuclear foci, was observed in the presence of HBx. RPS27a accelerated the progression of the cell cycle and cooperated with HBx in this process. Further, the knockdown of RPS27a expression by RNA interference in an HBx microenvironment led to retarded cell-cycle progression and reduced cell size. Thus, these results suggest strongly that RPS27a could be an effector of HBx-mediated hepatocarcinogenesis.

Human cytomegalovirus (HCMV) immediate-early (IE) 1 protein associates with chromosomes in mitotic cells using its carboxyl-terminal 16 aa region. However, the role of this IE1 activity in viral growth has not been evaluated in the context of mutant virus infection. We produced a recombinant HCMV encoding mutant IE1 with the carboxyl-terminal chromosome-tethering domain (CTD) deleted. This IE1(ΔCTD) virus grew like the wild-type virus in fibroblasts, indicating that the CTD is not essential for viral replication in permissive cells. Unlike wild-type virus infections, PML and STAT2, which interact with IE1, did not accumulate at mitotic chromosomes in IE1(ΔCTD) virus-infected fibroblasts, demonstrating that their associations with chromosomes are IE1 CTD-dependent. IE1 SUMOylation did not affect IE1 association with chromosomes. Our results provide genetic evidence that the CTD is required for the associations of IE1, PML and STAT2 with mitotic chromosomes, but that these IE1-related activities are not essential for viral replication in fibroblasts.

Human cytomegalovirus (HCMV) is a large DNA virus belonging to the subfamily Betaherpesvirinae. Haematopoietic cells of the myeloid lineage have been shown to harbour latent HCMV. However, following terminal differentiation of these cells, virus is reactivated, and in an immunocompromised host acute infection can occur. It is currently unknown which viral and cellular factors are involved in regulating the switch between lytic and latent infections. Cyclophilin A (CyPA) is a cellular protein that acts as a major factor in virus replication and/or virion maturation for a number of different viruses, including human immunodeficiency virus, hepatitis C virus, murine cytomegalovirus, influenza A virus and vaccinia virus. This study investigated the role of CyPA during HCMV infection. CyPA expression was silenced in human foreskin fibroblast (HF) and THP-1 cells using small interfering RNA (siRNA) technology, or the cells were treated with cyclosporin A (CsA) to inhibit CyPA activity. Silencing CyPA in HF cells with siRNA resulted in an overall reduction in virus production characterized by delayed expression of immediate-early (IE) proteins, decreased viral DNA loads and reduced titres. Furthermore, silencing of CyPA in THP-1 cells pre- and post-differentiation prevented IE protein expression and virus reactivation from a non-productive state. Interestingly, it was observed that treatment of THP-1 cells with CsA prevented the cells from establishing a fully latent infection. In summary, these results demonstrate that CyPA expression is an important factor in HCMV IE protein expression and virus production in lytically infected HF cells, and is a major component in virus reactivation from infected THP-1 cells.

The outer envelope of vaccinia virus extracellular virions is derived from intracellular membranes that, at late times in infection, are enriched in several virus-encoded proteins. Although palmitoylation is common in vaccinia virus envelope proteins, little is known about the role of palmitoylation in the biogenesis of the enveloped virus. We have studied the palmitoylation of B5, a 42 kDa type I transmembrane glycoprotein comprising a large ectodomain and a short (17 aa) cytoplasmic tail. Mutation of two cysteine residues located in the cytoplasmic tail in close proximity to the transmembrane domain abrogated palmitoylation of the protein. Virus mutants expressing non-palmitoylated versions of B5 and/or lacking most of the cytoplasmic tail were isolated and characterized. Cell-to-cell virus transmission and extracellular virus formation were only slightly affected by those mutations. Notably, B5 versions lacking palmitate showed decreased interactions with proteins A33 and F13, but were still incorporated into the virus envelope. Expression of mutated B5 by transfection into uninfected cells showed that both the cytoplasmic tail and palmitate have a role in the intracellular transport of B5. These results indicate that the C-terminal portion of protein B5, while involved in protein transport and in protein–protein interactions, is broadly dispensable for the formation and egress of infectious extracellular virus and for virus transmission.

To infect per os, baculovirus virions cross the peritrophic matrix (PM) to reach the midgut epithelium. Insect intestinal mucins (IIMs) are PM proteins that protect the PM and aid passage of the food bolus through the gut. Some baculoviruses, including Mamestra configurata nucleopolyhedrovirus (MacoNPV-A), encode metalloproteases, known as enhancins, that facilitate infection by degrading IIMs. We examined the interaction between MacoNPV-A enhancin and M. configurata IIMs both in vivo and in vitro. Per os inoculation of M. configurata larvae with MacoNPV-A occlusion bodies (OBs) resulted in the degradation of McIIM4 within 4 h of OB ingestion, while McIIM2 was unaffected. The PM recovered by 8 h post-inoculation. To investigate whether enhancin was responsible for the degradation of IIM, a recombinant Autographa californica multiple nucleopolyhedrovirus expressing MacoNPV enhancin (AcMNPV-enMP2) was constructed. Enhancin was found to be a component of occlusion-derived virions in AcMNPV-enMP2 and MacoNPV-A. In in vitro assays, McIIM4 was degraded after MacoNPV-A and AcMNPV-enMP2 treatments. Degradation of McIIM4 was inhibited by EDTA, a metalloprotease inhibitor, indicating that the degradation was due to enhancin activity. Thus, MacoNPV-A enhancin is able to degrade major structural PM proteins, but exhibits target substrate specificity.

Hepatitis A virus (HAV) infections result in different courses of the disease, varying between normal, prolonged and relapsing. However, the reason for these heterogeneous clinical appearances is not understood. As HAV–anti-HAV IgA immunocomplexes (HAV–IgA) infect hepatocytes, IgA was postulated as a carrier supporting hepatotropic transport of HAV, and it was speculated that this carrier mechanism contributes to the various clinical outcomes. In this study, the IgA-carrier mechanism was investigated in a mouse model. We show that HAV–IgA immunocomplexes efficiently reached the liver not only in HAV-seronegative mice, but also, and this is in contrast to free-HAV particles, in immunized HAV-seropositive animals. This IgA-mediated transport of HAV to the liver in the presence of immunity depended on the stage of development of the immune response. We conclude that over a period of several weeks after infection, anti-HAV IgA is able to promote an enterohepatic cycling of HAV, resulting in continuous endogenous reinfections of the liver. Our experiments indicate that highly avid IgG antibodies, which are present at later times of the infection, can terminate the reinfections. However, the endogenous reinfections in the presence of a developing neutralizing immunity might contribute to prolonged as well as to relapsing courses of HAV infections. Furthermore, the results show that serum IgA may act as an infection protracting factor.

Dengue virus (DENV) causes fever and severe haemorrhagic symptoms in humans. The DEN2 16681 strain, derived from a dengue haemorrhagic fever patient, has been widely used in studies related to DENV pathogenesis, such as mouse and non-human primate haemorrhagic models and human vascular endothelial-cell permeability. To clarify the entry mechanism of the 16681 strain, we characterized a novel cell receptor for this strain. Our two major findings were as follows: firstly, the SDC2 membrane protein was an effective DEN2 16681 receptor in a cloned K562 cell line. Secondly, a heparan sulfate (HS) glycochain (of four glycochains in SDC2) is the specific binding site of DENV and seems to be involved in tissue-culture adaptation. Our findings present an entry mechanism that could be implicated for DENV adaptation and HS-mediated DENV infection.

The flavivirus non-structural protein 1 (NS1) is a glycoprotein that is secreted as a soluble hexameric complex during the course of natural infection. Growing evidence indicates that this secreted form of NS1 (sNS1) plays a significant role in immune evasion and modulation during infection. Attempts to determine the crystal structure of NS1 have been unsuccessful to date and relatively little is known about the macromolecular organization of the sNS1 hexamer. Here, we have applied single-particle analysis to images of baculovirus-derived recombinant dengue 2 virus NS1 obtained by electron microscopy to determine its 3D structure to a resolution of 23 Å. This structure reveals a barrel-like organization of the three dimeric units that comprise the hexamer and provides further insights into the overall organization of oligomeric sNS1.

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is part of the viral replicative complex and plays a crucial role in HCV replication. It has been described that NS5B interacts with cellular proteins, and that interactions between NS5B and host proteins are crucial for viral replication. Some of the host factors involved in the HCV replication cycle include the oestrogen receptor alpha (ESR1), protein kinases (c-Src) and chaperones (Hsp70). In this report, we determine the requirements for the interplay between NS5B and the domain C of ESR1 (ESR1C) by using Förster Resonance Energy Transfer. NS5B–ESR1C and ESR1C–ESR1C interactions are dependent on ionic strength, indicating that contacts are mainly electrostatic. Additionally, NS5B residues involved in NS5B oligomerization were also essential for NS5B–ESR1C interaction. The study of the interactions among viral and host factors will provide data to establish innovative therapeutic strategies and the development of new antiviral drugs.

Tick-borne encephalitis virus (TBEV) is a member of the family Flaviviridae. It is transmitted by Ixodes spp. ticks in a cycle involving rodents and small mammals. TBEV has three subtypes: European, Siberian and Far Eastern. The virus causes thousands of cases of meningoencephalitis in Europe annually, with an increasing trend. The increase may be attributed to a complex network of elements, including climatic, environmental and socio-economic factors. In an attempt to understand the evolutionary history and dispersal of TBEV, to existing genetic data we add two novel complete ORF sequences of TBEV strains from northern Europe and the completion of the genome of four others. Moreover, we provide a unique measure for the natural rate of evolution of TBEV by studying two isolations from the same forest on an island in Åland archipelago 44 years apart. For all isolates, we analysed the phylogeny, rate of evolution and probable time of radiation of the different TBEV strains. The results show that the two lineages of TBEV in different Ixodes species have evolved independently for approximately 3300 years. Notably, rapid radiation of TBEV-Eur occurred approximately 300 years ago, without the large-scale geographical clustering observed previously for the Siberian subtype. The measurements from the natural rate of evolution correlated with the estimates done by phylogenetic programs, demonstrating their robustness.

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne RNA virus of the genus Alphavirus, family Togaviridae, that is responsible for sporadic outbreaks in human and equid populations in Central and South America. In order to ascertain the role that complement plays in resolving VEEV-induced disease, complement-deficient C3−/− mice were infected with a VEEV mutant (V3533) that caused mild, transient disease in immunocompetent mice. In the absence of a functional complement system, peripheral inoculation with V3533 induced much more severe encephalitis. This enhanced pathology was associated with a delay in clearance of infectious virus from the serum and more rapid invasion of the central nervous system in C3−/− mice. If V3533 was inoculated directly into the brain, however, disease outcome in C3−/− and wild-type mice was identical. These findings indicate that complement-dependent enhancement of peripheral virus clearance is critical for protecting against the development of severe VEEV-induced encephalitis.

A proline-rich region (PRR) within the rubella virus (RUBV) P150 replicase protein that contains three SH3 domain-binding motifs (PxxPxR) was investigated for its ability to bind cell proteins. Pull-down experiments using a glutathione S-transferase–PRR fusion revealed PxxPxR motif-specific binding with human p32 protein (gC1qR), which could be mediated by either of the first two motifs. This finding was of interest because p32 protein also binds to the RUBV capsid protein. Binding of p32 to P150 was confirmed and was abolished by mutation of the first two motifs. When mutations in the first two motifs were introduced into a RUBV cDNA infectious clone, virus replication was significantly impaired. However, virus RNA synthesis was found to be unaffected, and subsequent immunofluorescence analysis of RUBV-infected cells revealed co-localization of p32 and P150 but little overlap of p32 with RNA replication complexes, indicating that p32 does not participate directly in virus RNA synthesis. Thus, the role of p32 in RUBV replication remains unresolved.

GII.4 noroviruses are a major cause of acute gastroenteritis in humans. A new variant of GII.4, the 2008 variant, has recently increased its prevalence on a global scale. A previous study of this variant in Japan suggested that it might be of recombinant origin, with a breakpoint at the ORF1–ORF2 junction. Here, examination of the evolutionary origin of the 2008 variant based on a larger sample of worldwide GII.4 norovirus sequences revealed a more complex pattern of recombination between the 2006a- and 2006b-like variants of genotype GII.4, involving the P2 antigenic domain. Double (termed ‘2008i’) and triple (termed ‘2008ii’) recombinant forms of 2008 variants were identified. This study highlights the possible importance of intra-genotypic recombination over antigenic regions in driving norovirus evolution, and is suggestive of a process analogous to the antigenic shift of influenza A virus by reassortment.

The endodomain of several coronavirus (CoV) spike (S) proteins contains palmitylated cysteine residues and enables co-localization and interaction with the CoV membrane (M) protein. Depalmitylation of mouse hepatitis virus S proteins abolished this interaction, resulting in the failure of S incorporation into virions. In contrast, an immunofluorescence assay (IFA) showed that depalmitylated severe acute respiratory syndrome coronavirus (SCoV) S proteins still co-localized with the M protein in the budding site. Here, we determined the ability of depalmitylated SCoV S mutants to incorporate S into virus-like particles (VLPs). IFA confirmed that all SCoV S mutants co-localized with the M protein intracellularly. However, the mutants lacking two cysteine residues (C1234/1235) failed to incorporate S into VLPs. This indicated that these palmitylated cysteines are essential for S incorporation, but are not involved in S co-localization mediated by the M protein. Our findings suggest that M–S co-localization and S incorporation occur independently of one another in SCoV virion assembly.

The porcine reproductive and respiratory syndrome virus (PRRSV) replicase gene consists of two large ORFs, ORF1a and ORF1b, the latter of which is expressed by ribosomal frameshifting. The ORF1a-encoded part of the resulting replicase polyproteins (pp1a and pp1ab) is predicted to be processed proteolytically into ten non-structural proteins (nsps), known as nsp1–8, with both the nsp1 and nsp7 regions being cleaved internally (yielding nsp1α and nsp1β, and nsp7α and nsp7β, respectively). The experimental verification of these predictions depends strongly on the ability to identify individual cleavage products with specific antibodies. In this study, a panel of monoclonal and polyclonal antibodies was generated, which together were able to recognize eight ORF1a-encoded PRRSV nsps. Using these reagents, replicase cleavage products were detected in PRRSV-infected MARC-145 cells using a variety of immunoassays. By immunofluorescence microscopy, most nsps could be detected by 6 h post-infection. During the early stages of infection, nsp1β, nsp2, nsp4, nsp7α, nsp7β and nsp8 co-localized in distinct punctate foci in the perinuclear region of the cell, which were determined to be the site of viral RNA synthesis by in situ labelling. Western blot and immunoprecipitation analysis identified most individual nsps and several long-lived processing intermediates (nsp3–4, nsp5–7, nsp5–8 and nsp3–8). The identification and subcellular localization of PRRSV nsps in virus-infected cells documented here provides a basis for the further structure–function studies. Thus, this PRRSV antibody panel will be an important tool for future studies on the replication and pathogenesis of this major swine pathogen.

The influenza A virus genome comprises eight segments of negative-sense RNA that encode up to 12 proteins. RNA segment 2 encodes three proteins, PB1, PB1-F2 and N40, that are translated from the same mRNA by ribosomal leaky scanning and reinitiation. PB1 is a subunit of the trimeric viral RNA polymerase. PB1-F2 has been reported to be a potential virulence factor, and has been shown to be involved in a number of activities including induction of apoptosis, regulation of virus replication and modulation of the immune response. No function has yet been ascribed to N40, which represents an N-terminally deleted form of PB1. Previous studies on PB1-F2 function mainly used viruses genetically engineered to prevent PB1-F2 expression by mutation of the PB1-F2 start codon. However, ablation of the start codon was shown to increase the expression level of the downstream protein N40. In the present study, we generated recombinant A/WSN/33 viruses carrying different combinations of PB1-F2- and N40-knockout mutations. Overexpression of N40 in a PB1-F2-deficient background had a detrimental effect on virus growth in vitro and in vivo. However, ablation of PB1-F2 or N40 expression individually was not disadvantageous for the virus. Primer-extension analyses revealed an increase in vRNA production by viruses that overexpressed N40. Our data suggest that the observed attenuation of mutant viruses in vitro and in vivo results from these changes in transcription and replication.

Bats constitute a reservoir of zoonotic infections and some bat paramyxoviruses are capable of cross-species transmission, often with fatal consequences. Determining the level of viral diversity in reservoir populations is fundamental to understanding and predicting viral emergence. This is particularly relevant for RNA viruses where the adaptive mutations required for cross-species transmission can be present in the reservoir host. We report the use of non-invasively collected, pooled, neat urine samples as a robust sample type for investigating paramyxoviruses in bat populations. Using consensus PCR assays we have detected a high incidence and genetic diversity of novel paramyxoviruses in an urban fruit bat population over a short period of time. This may suggest a similarly unique relationship between bats and the members of the family Paramyxoviridae as proposed for some other viral families. Additionally, the high rate of bat–human contact at the study site calls for the zoonotic potential of the detected viruses to be investigated further.