Adaptation of novel H7N9 influenza A virus to human receptors.

The emergence of the novel H7N9 influenza A virus (IAV) has caused global concerns about the ability of this virus to spread between humans. Analysis of the receptor-binding properties of this virus using a recombinant protein approach in combination with fetuin-binding, glycan array and human tissue-binding assays demonstrates increased binding of H7 to both α2-6 and α2-8 sialosides as well as reduced binding to α2-3-linked SIAs compared to a closely related avian H7N9 virus from 2008. These differences could be attributed to substitutions Q226L and G186V. Analysis of the enzymatic activity of the neuraminidase N9 protein indicated a reduced sialidase activity, consistent with the reduced binding of H7 to α2-3 sialosides. However, the novel H7N9 virus still preferred binding to α2-3- over α2-6-linked SIAs and was not able to efficiently bind to epithelial cells of human trachea in contrast to seasonal IAV, consistent with its limited human-to-human transmission.

Acid-activated structural reorganization of the Rift Valley fever virus Gc fusion protein.

The entry of the enveloped Rift Valley fever virus (RVFV) into its host cell is mediated by the viral glycoproteins Gn and Gc. We investigated the RVFV entry process and, in particular, its pH-dependent activation mechanism using our recently developed nonspreading-RVFV-particle system. Entry of the virus into the host cell was efficiently inhibited by lysosomotropic agents that prevent endosomal acidification and by compounds that interfere with dynamin- and clathrin-dependent endocytosis. Exposure of plasma membrane-bound virions to an acidic pH (

Heparan sulfate facilitates Rift Valley fever virus entry into the cell.

Rift Valley fever virus (RVFV), an emerging arthropod-borne pathogen, has a broad host and cell tropism. Here we report that the glycosaminoglycan heparan sulfate, abundantly present on the surface of most animal cells, is required for efficient entry of RVFV. Entry was significantly reduced by preincubating the virus inoculum with highly sulfated heparin, by enzymatic removal of heparan sulfate from cells and in cells genetically deficient in heparan sulfate synthesis.

Retargeting of viruses to generate oncolytic agents.

Oncolytic virus therapy is based on the ability of viruses to effectively infect and kill tumor cells without destroying the normal tissues. While some viruses seem to have a natural preference for tumor cells, most viruses require the modification of their tropism to specifically enter and replicate in such cells. This review aims to describe the transductional targeting strategies currently employed to specifically redirect viruses towards surface receptors on tumor cells. Three major strategies can be distinguished; they involve (i) the incorporation of new targeting specificity into a viral surface protein, (ii) the incorporation of a scaffold into a viral surface protein to allow the attachment of targeting moieties, and (iii) the use of bispecific adapters to mediate targeting of a virus to a specified moiety on a tumor cell. Of each strategy key features, advantages and limitations are discussed and examples are given. Because of their potential to cause sustained, multiround infection-a desirable characteristic for eradicating tumors-particular attention is given to viruses engineered to become self-targeted by the genomic expression of a bispecific adapter protein.

A comparative infection study of pigeon and avian paramyxovirus type 1 viruses in pigeons: evaluation of clinical signs, virus shedding and seroconversion.

The pathogenesis of pigeon paramyxovirus type 1 (PPMV-1) isolate AV324/96 and of its recombinant derivative, rgAV324, was studied in pigeons. For comparison, the virulent chicken virus FL-Herts, which is a recombinant derivative of strain Herts/33, was also included. After inoculation by the combined intraocular, intranasal and intratracheal route, clinical signs, virus shedding and serological responses were examined. Clinical signs were observed only in the FL-Herts-infected group. All virus-inoculated pigeons had positive tracheal swabs until 5 days post infection. However, only the AV324/96-infected and rgAV324-infected birds, and not the FL-Herts-infected birds, shed virus in the cloaca. The AV324/96-infected pigeons showed higher mean antibody titres than the rgAV324-infected birds, whereas the antibody titres of the FL-Herts-infected group were rather low. The results show that the pigeon strain AV324 is not virulent for pigeons, but underlines the potential risk of poultry becoming infected by PPMV-1 shed by non-symptomatic pigeons.

Vaccination with a soluble recombinant hemagglutinin trimer protects pigs against a challenge with pandemic (H1N1) 2009 influenza virus.

In 2009 a new influenza A/H1N1 virus strain ("pandemic (H1N1) 2009", H1N1v) emerged that rapidly spread around the world. The virus is suspected to have originated in swine through reassortment and to have subsequently crossed the species-barrier towards humans. Several cases of reintroduction into pigs have since been reported, which could possibly create a reservoir for human exposure or ultimately become endemic in the pig population with similar clinical disease problems as current swine influenza strains. A soluble trimer of hemagglutinin (HA), derived from the H1N1v, was used as a vaccine in pigs to investigate the extent to which this vaccine would be able to protect pigs against infection with the H1N1v influenza strain, especially with respect to reducing virus replication and excretion. In a group of unvaccinated control pigs, no clinical symptoms were observed, but (histo)pathological changes consistent with an influenza infection were found on days 1 and 3 after inoculation. Live virus was isolated from the upper and lower respiratory tract, with titres up to 10(6) TCID(50) per gram of tissue. Furthermore, live virus was detected in brain samples. Control pigs were shedding live virus for up to 6 days after infection, with titres of up to 10(5) TCID(50) per nasal or oropharyngeal swab. The soluble H1N1v HA trimer diminished virus replication and excretion after a double vaccination and subsequent challenge. Live virus could not be detected in any of the samples taken from the vaccinated pigs. Vaccines based on soluble HA trimers provide an attractive alternative to the current inactivated vaccines.

Passaging of a Newcastle disease virus pigeon variant in chickens results in selection of viruses with mutations in the polymerase complex enhancing virus replication and virulence.

Some Newcastle disease virus (NDV) variants isolated from pigeons (pigeon paramyxovirus type 1; PPMV-1) do not show their full virulence potential for domestic chickens but may become virulent upon spread in these animals. In this study we examined the molecular changes responsible for this gain of virulence by passaging a low-pathogenic PPMV-1 isolate in chickens. Complete genome sequencing of virus obtained after 1, 3 and 5 passages showed the increase in virulence was not accompanied by changes in the fusion protein--a well known virulence determinant of NDV--but by mutations in the L and P replication proteins. The effect of these mutations on virulence was confirmed by means of reverse genetics using an infectious cDNA clone. Acquisition of three amino acid mutations, two in the L protein and one in the P protein, significantly increased virulence as determined by intracerebral pathogenicity index tests in day-old chickens. The mutations enhanced virus replication in vitro and in vivo and increased the plaque size in infected cell culture monolayers. Furthermore, they increased the activity of the viral replication complex as determined by an in vitro minigenome replication assay. Our data demonstrate that PPMV-1 replication in chickens results in mutations in the polymerase complex rather than the viral fusion protein, and that the virulence level of pigeon paramyxoviruses is directly related to the activity of the viral replication complex.

The viral replication complex is associated with the virulence of Newcastle disease virus.

Virulent strains of Newcastle disease virus ([NDV] also known as avian paramyxovirus type 1) can be discriminated from low-virulence strains by the presence of multiple basic amino acid residues at the proteolytic cleavage site of the fusion (F) protein. However, some NDV variants isolated from pigeons (pigeon paramyxovirus type 1 [PPMV-1]) have low levels of virulence, despite the fact that their F protein cleavage sites contain a multibasic amino acid sequence and have the same functionality as that of virulent strains. To determine the molecular basis of this discrepancy, we examined the role of the internal proteins in NDV virulence. Using reverse genetics, the genes encoding the nucleoprotein (NP), phosphoprotein (P), matrix protein (M), and large polymerase protein (L) were exchanged between the nonvirulent PPMV-1 strain AV324 and the highly virulent NDV strain Herts. Recombinant viruses were evaluated for their pathogenicities and replication levels in day-old chickens, and viral genome replication and plaque sizes were examined in cell culture monolayers. We also tested the contributions of the individual NP, P, and L proteins to the activity of the viral replication complex in an in vitro replication assay. The results showed that the replication proteins of Herts are more active than those of AV324 and that the activity of the viral replication complex is directly related to virulence. Although the M protein affected viral replication in vitro, it had only a minor effect on virulence.

Rift Valley fever virus subunit vaccines confer complete protection against a lethal virus challenge.

Rift Valley fever virus (RVFV) is an emerging mosquito-borne virus causing significant morbidity and mortality in livestock and humans. Rift Valley fever is endemic in Africa, but also outside this continent outbreaks have been reported. Here we report the evaluation of two vaccine candidates based on the viral Gn and Gc envelope glycoproteins, both produced in a Drosophila insect cell expression system. Virus-like particles (VLPs) were generated by merely expressing the Gn and Gc glycoproteins. In addition, a soluble form of the Gn ectodomain was expressed and affinity-purified from the insect cell culture supernatant. Both vaccine candidates fully protected mice from a lethal challenge with RVFV. Importantly, absence of the nucleocapsid protein in either vaccine candidate facilitates the differentiation between infected and vaccinated animals using a commercial recombinant nucleocapsid protein-based indirect ELISA.

Virulence of pigeon paramyxovirus type 1 does not always correlate with the cleavability of its fusion protein.

Some pigeon paramyxovirus type 1 (PPMV-1) strains exhibit low virulence in chickens, despite their fusion (F) protein's multi-basic cleavage site. To elucidate the molecular basis of the low pathogenicity of these strains, we constructed an infectious full-length cDNA clone of PPMV-1 strain AV324. This strain is non-virulent for chickens, although its F protein contains the typical virulence motif (112)RRKKRF(117). By using reverse genetics, we exchanged the F genes of AV324 and a virulent Newcastle disease virus (NDV) strain (Herts) and evaluated the recovered chimeric viruses for their pathogenicity in 1-day-old chickens and in embryonated eggs. Our results show that the F protein of AV324, and probably those of similar PPMV-1 strains, are functionally not different from those of virulent NDV strains and that the difference in pathogenicity must be determined by other factors.

Cleavage of group 1 coronavirus spike proteins: how furin cleavage is traded off against heparan sulfate binding upon cell culture adaptation.

A longstanding enigmatic feature of the group 1 coronaviruses is the uncleaved phenotype of their spike protein, an exceptional property among class I fusion proteins. Here, however, we show that some group 1 coronavirus spike proteins carry a furin enzyme recognition motif and can actually be cleaved, as demonstrated for a feline coronavirus. Interestingly, this feature can be lost during cell culture adaptation by a single mutation in the cleavage motif; this, however, preserves a heparan sulfate binding motif and renders infection by the virus heparan sulfate dependent. We identified a similar cell culture adaptation for the human coronavirus OC43.

Localization and membrane topology of coronavirus nonstructural protein 4: involvement of the early secretory pathway in replication.

The coronavirus nonstructural proteins (nsp's) derived from the replicase polyproteins collectively constitute the viral replication complexes, which are anchored to double-membrane vesicles. Little is known about the biogenesis of these complexes, the membrane anchoring of which is probably mediated by nsp3, nsp4, and nsp6, as they contain several putative transmembrane domains. As a first step to getting more insight into the formation of the coronavirus replication complex, the membrane topology, processing, and subcellular localization of nsp4 of the mouse hepatitis virus (MHV) and severe acute respiratory syndrome-associated coronavirus (SARS-CoV) were elucidated in this study. Both nsp4 proteins became N glycosylated, while their amino and carboxy termini were localized to the cytoplasm. These observations imply nsp4 to assemble in the membrane as a tetraspanning transmembrane protein with a Nendo/Cendo topology. The amino terminus of SARS-CoV nsp4, but not that of MHV nsp4, was shown to be (partially) processed by signal peptidase. nsp4 localized to the endoplasmic reticulum (ER) when expressed alone but was recruited to the replication complexes in infected cells. nsp4 present in these complexes did not colocalize with markers of the ER or Golgi apparatus, while the susceptibility of its sugars to endoglycosidase H indicated that the protein had also not traveled trough the latter compartment. The important role of the early secretory pathway in formation of the replication complexes was also demonstrated by the inhibition of coronaviral replication when the ER export machinery was blocked by use of the kinase inhibitor H89 or by expression of a mutant, Sar1[H79G].

Assembly of HCV E1 and E2 glycoproteins into coronavirus VLPs.

Hepatitis C virus (HCV) is believed to assemble by budding into membranes of the early secretory pathway, consistent with the membrane location where the viral envelope glycoproteins E1 and E2 accumulate when expressed. Coronavirus assembly also takes place at pre-Golgi membranes. Here, we generated coronavirus-like particles carrying in their envelope chimeric HCV glycoproteins composed of the ectodomains of E1 and E2, each fused to the transmembrane plus endodomain of the mouse hepatitis coronavirus spike glycoprotein. The chimeric particle system will enable structural and functional studies of the HCV glycoproteins.

Glycosylation of the severe acute respiratory syndrome coronavirus triple-spanning membrane proteins 3a and M.

The severe acute respiratory syndrome coronavirus (SARS-CoV) open reading frame 3a protein has recently been shown to be a structural protein. The protein is encoded by one of the so-called group-specific genes and has no sequence homology with any of the known structural or group-specific proteins of coronaviruses. It does, however, have several similarities to the coronavirus M proteins; (i) they are triple membrane spanning with the same topology, (ii) they have similar intracellular localizations (predominantly Golgi), (iii) both are viral structural proteins, and (iv) they appear to interact with the E and S proteins, as well as with each other. The M protein plays a crucial role in coronavirus assembly and is glycosylated in all coronaviruses, either by N-linked or by O-linked oligosaccharides. The conserved glycosylation of the coronavirus M proteins and the resemblance of the 3a protein to them led us to investigate the glycosylation of these two SARS-CoV membrane proteins. The proteins were expressed separately using the vaccinia virus T7 expression system, followed by metabolic labeling. Pulse-chase analysis showed that both proteins were modified, although in different ways. While the M protein acquired cotranslationally oligosaccharides that could be removed by PNGaseF, the 3a protein acquired its modifications posttranslationally, and they were not sensitive to the N-glycosidase enzyme. The SARS-CoV 3a protein, however, was demonstrated to contain sialic acids, indicating the presence of oligosaccharides. O-glycosylation of the 3a protein was indeed confirmed using an in situ O-glycosylation assay of endoplasmic reticulum-retained mutants. In addition, we showed that substitution of serine and threonine residues in the ectodomain of the 3a protein abolished the addition of the O-linked sugars. Thus, the SARS-CoV 3a protein is an O-glycosylated glycoprotein, like the group 2 coronavirus M proteins but unlike the SARS-CoV M protein, which is N glycosylated.

Redirecting coronavirus to a nonnative receptor through a virus-encoded targeting adapter.

Murine hepatitis coronavirus (MHV)-A59 infection depends on the interaction of its spike (S) protein with the cellular receptor mCEACAM1a present on murine cells. Human cells lack this receptor and are therefore not susceptible to MHV. Specific alleviation of the tropism barrier by redirecting MHV to a tumor-specific receptor could lead to a virus with appealing properties for tumor therapy. To demonstrate that MHV can be retargeted to a nonnative receptor on human cells, we produced bispecific adapter proteins composed of the N-terminal D1 domain of mCEACAM1a linked to a short targeting peptide, the six-amino-acid His tag. Preincubation of MHV with the adapter proteins and subsequent inoculation of human cells expressing an artificial His receptor resulted in infection of these otherwise nonsusceptible cells and led to subsequent production of progeny virus. To generate a self-targeted virus able to establish multiround infection of the target cells, we subsequently incorporated the gene encoding the bispecific adapter protein as an additional expression cassette into the MHV genome through targeted RNA recombination. When inoculated onto murine LR7 cells, the resulting recombinant virus indeed expressed the adapter protein. Furthermore, inoculation of human target cells with the virus resulted in a His receptor-specific infection that was multiround. Extensive cell-cell fusion and rapid cell killing of infected target cells was observed. Our results show that MHV can be genetically redirected via adapters composed of the S protein binding part of mCEACAM1a and a targeting peptide recognizing a nonnative receptor expressed on human cells, consequently leading to rapid cell death. The results provide interesting leads for further investigations of the use of coronaviruses as antitumor agents.

Luxury at a cost? Recombinant mouse hepatitis viruses expressing the accessory hemagglutinin esterase protein display reduced fitness in vitro.

Group 2 coronaviruses encode an accessory envelope glycoprotein species, the hemagglutinin esterase (HE), which possesses sialate-O-acetylesterase activity and which, presumably, promotes virus spread and entry in vivo by facilitating reversible virion attachment to O-acetylated sialic acids. While HE may provide a strong selective advantage during natural infection, many laboratory strains of mouse hepatitis virus (MHV) fail to produce the protein. Apparently, their HE genes were inactivated during cell culture adaptation. For this report, we have studied the molecular basis of this phenomenon. By using targeted RNA recombination, we generated isogenic recombinant MHVs which differ exclusively in their expression of HE and produce either the wild-type protein (HE+), an enzymatically inactive HE protein (HE0), or no HE at all. HE expression or the lack thereof did not lead to gross differences in in vitro growth properties. Yet the expression of HE was rapidly lost during serial cell culture passaging. Competition experiments with mixed infections revealed that this was not due to the enzymatic activity: MHVs expressing HE+ or HE0 propagated with equal efficiencies. During the propagation of recombinant MHV-HE+, two types of spontaneous mutants accumulated. One produced an anchorless HE, while the other had a Gly-to-Trp substitution at the predicted C-terminal residue of the HE signal peptide. Neither mutant incorporated HE into virion particles, suggesting that wild-type HE reduces the in vitro propagation efficiency, either at the assembly stage or at a postassembly level. Our findings demonstrate that the expression of "luxury" proteins may come at a fitness penalty. Apparently, under natural conditions the costs of maintaining HE are outweighed by the benefits.

Soluble receptor-mediated targeting of mouse hepatitis coronavirus to the human epidermal growth factor receptor.

The mouse hepatitis coronavirus (MHV) infects murine cells by binding of its spike (S) protein to murine CEACAM1a. The N-terminal part of this cellular receptor (soR) is sufficient for S binding and for subsequent induction of the conformational changes required for virus-cell membrane fusion. Here we analyzed whether these characteristics can be used to redirect MHV to human cancer cells. To this end, the soR domain was coupled to single-chain monoclonal antibody 425, which is directed against the human epidermal growth factor receptor (EGFR), resulting in a bispecific adapter protein (soR-425). The soR and soR-425 proteins, both produced with the vaccinia virus system, were able to neutralize MHV infection of murine LR7 cells. However, only soR-425 was able to target MHV to human EGFR-expressing cancer cells. Interestingly, the targeted infections induced syncytium formation. Furthermore, the soR-425-mediated infections were blocked by heptad repeat-mimicking peptides, indicating that virus entry requires the regular S protein fusion process. We conclude that the specific spike-binding property of the CEACAM1a N-terminal fragment can be exploited to direct the virus to selected cells by linking it to a moiety able to bind a receptor on those cells. This approach might be useful in the development of tumor-targeted coronaviruses.

Envelope protein requirements for the assembly of infectious virions of porcine reproductive and respiratory syndrome virus.

Virions of porcine reproductive and respiratory syndrome virus (PRRSV) contain six membrane proteins: the major proteins GP5 and M and the minor proteins GP2a, E, GP3, and GP4. Here, we studied the envelope protein requirements for PRRSV particle formation and infectivity using full-length cDNA clones in which the genes encoding the membrane proteins were disrupted by site-directed mutagenesis. By transfection of RNAs transcribed from these cDNAs into BHK-21 cells and analysis of the culture medium using ultracentrifugation, radioimmunoprecipitation, and real-time reverse transcription-PCR, we observed that the production of viral particles is dependent on both major envelope proteins; no particles were released when either the GP5 or the M protein was absent. In contrast, particle production was not dependent on the minor envelope proteins. Remarkably, in the absence of any one of the latter proteins, the incorporation of all other minor envelope proteins was affected, indicating that these proteins interact with each other and are assembled into virions as a multimeric complex. Independent evidence for such complexes was obtained by coexpression of the minor envelope proteins in BHK-21 cells using a Semliki Forest virus expression system. By analyzing the maturation of their N-linked oligosaccharides, we found that the glycoproteins were each retained in the endoplasmic reticulum unless expressed together, in which case they were collectively transported through the Golgi complex to the plasma membrane and were even detected in the extracellular medium. As the PRRSV particles lacking the minor envelope proteins are not infectious, we hypothesize that the virion surface structures formed by these proteins function in viral entry by mediating receptor binding and/or virus-cell fusion.

Targeting non-human coronaviruses to human cancer cells using a bispecific single-chain antibody.

To explore the potential of using non-human coronaviruses for cancer therapy, we first established their ability to kill human tumor cells. We found that the feline infectious peritonitis virus (FIPV) and a felinized murine hepatitis virus (fMHV), both normally incapable of infecting human cells, could rapidly and effectively kill human cancer cells artificially expressing the feline coronavirus receptor aminopeptidase N. Also 3-D multilayer tumor spheroids established from such cells were effectively eradicated. Next, we investigated whether FIPV and fMHV could be targeted to human cancer cells by constructing a bispecific single-chain antibody directed on the one hand against the feline coronavirus spike protein--responsible for receptor binding and subsequent cell entry through virus-cell membrane fusion--and on the other hand against the human epidermal growth factor receptor (EGFR). The targeting antibody mediated specific infection of EGFR-expressing human cancer cells by both coronaviruses. Furthermore, in the presence of the targeting antibody, infected cancer cells formed syncytia typical of productive coronavirus infection. By their potent cytotoxicity, the selective targeting of non-human coronaviruses to human cancer cells provides a rationale for further investigations into the use of these viruses as anticancer agents.