Nanoparticle display of neuraminidase elicits enhanced antibody responses and protection against influenza A virus challenge.

Current Influenza virus vaccines primarily induce antibody responses against variable epitopes in hemagglutinin (HA), necessitating frequent updates. However, antibodies against neuraminidase (NA) can also confer protection against influenza, making NA an attractive target for the development of novel vaccines. In this study, we aimed to enhance the immunogenicity of recombinant NA antigens by presenting them multivalently on a nanoparticle carrier. Soluble tetrameric NA antigens of the N1 and N2 subtypes, confirmed to be correctly folded by cryo-electron microscopy structural analysis, were conjugated to Mi3 self-assembling protein nanoparticles using the SpyTag-SpyCatcher system. Immunization of mice with NA-Mi3 nanoparticles induced higher titers of NA-binding and -inhibiting antibodies and improved protection against a lethal challenge compared to unconjugated NA. Additionally, we explored the co-presentation of N1 and N2 antigens on the same Mi3 particles to create a mosaic vaccine candidate. These mosaic nanoparticles elicited antibody titers that were similar or superior to the homotypic nanoparticles and effectively protected against H1N1 and H3N2 challenge viruses. The NA-Mi3 nanoparticles represent a promising vaccine candidate that could complement HA-directed approaches for enhanced potency and broadened protection against influenza A virus.

The Origin, Dynamic Morphology, and PI4P-Independent Formation of Encephalomyocarditis Virus Replication Organelles.

Picornaviruses induce dramatic rearrangements of endomembranes in the cells that they infect to produce dedicated platforms for viral replication. These structures, termed replication organelles (ROs), have been well characterized for the Enterovirus genus of the Picornaviridae However, it is unknown whether the diverse RO morphologies associated with enterovirus infection are conserved among other picornaviruses. Here, we use serial electron tomography at different stages of infection to assess the three-dimensional architecture of ROs induced by encephalomyocarditis virus (EMCV), a member of the Cardiovirus genus of the family of picornaviruses that is distantly related. Ultrastructural analyses revealed connections between early single-membrane EMCV ROs and the endoplasmic reticulum (ER), establishing the ER as a likely donor organelle for their formation. These early single-membrane ROs appear to transform into double-membrane vesicles (DMVs) as infection progresses. Both single- and double-membrane structures were found to support viral RNA synthesis, and progeny viruses accumulated in close proximity, suggesting a spatial association between RNA synthesis and virus assembly. Further, we explored the role of phosphatidylinositol 4-phosphate (PI4P), a critical host factor for both enterovirus and cardiovirus replication that has been recently found to expedite enterovirus RO formation rather than being strictly required. By exploiting an EMCV escape mutant, we found that low-PI4P conditions could also be overcome for the formation of cardiovirus ROs. Collectively, our data show that despite differences in the membrane source, there are striking similarities in the biogenesis, morphology, and transformation of cardiovirus and enterovirus ROs, which may well extend to other picornaviruses.IMPORTANCE Like all positive-sense RNA viruses, picornaviruses induce the rearrangement of host cell membranes to form unique structures, or replication organelles (ROs), that support viral RNA synthesis. Here, we investigate the architecture and biogenesis of cardiovirus ROs and compare them with those induced by enteroviruses, members of the well-characterized picornavirus genus Enterovirus The origins and dynamic morphologies of cardiovirus ROs are revealed using electron tomography, which points to the endoplasmic reticulum as the donor organelle usurped to produce single-membrane tubules and vesicles that transform into double-membrane vesicles. We show that PI4P, a critical lipid for cardiovirus and enterovirus replication, is not strictly required for the formation of cardiovirus ROs, as functional ROs with typical morphologies are formed under phosphatidylinositol 4-kinase type III alpha (PI4KA) inhibition in cells infected with an escape mutant. Our data show that the transformation from single-membrane structures to double-membrane vesicles is a conserved feature of cardiovirus and enterovirus infections that likely extends to other picornavirus genera.

Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein.

Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles [ROs]) with a unique protein and lipid composition on which they multiply their viral genome. Suitable tools for live-cell imaging of enterovirus ROs are currently unavailable, as recombinant enteroviruses that carry genes that encode RO-anchored viral proteins tagged with fluorescent reporters have not been reported thus far. To overcome this limitation, we used a split green fluorescent protein (split-GFP) system, comprising a large fragment [strands 1 to 10; GFP(S1-10)] and a small fragment [strand 11; GFP(S11)] of only 16 residues. The GFP(S11) (GFP with S11 fragment) fragment was inserted into the 3A protein of the enterovirus coxsackievirus B3 (CVB3), while the large fragment was supplied by transient or stable expression in cells. The introduction of GFP(S11) did not affect the known functions of 3A when expressed in isolation. Using correlative light electron microscopy (CLEM), we showed that GFP fluorescence was detected at ROs, whose morphologies are essentially identical to those previously observed for wild-type CVB3, indicating that GFP(S11)-tagged 3A proteins assemble with GFP(S1-10) to form GFP for illumination of bona fide ROs. It is well established that enterovirus infection leads to Golgi disintegration. Through live-cell imaging of infected cells expressing an mCherry-tagged Golgi marker, we monitored RO development and revealed the dynamics of Golgi disassembly in real time. Having demonstrated the suitability of this virus for imaging ROs, we constructed a CVB3 encoding GFP(S1-10) and GFP(S11)-tagged 3A to bypass the need to express GFP(S1-10) prior to infection. These tools will have multiple applications in future studies on the origin, location, and function of enterovirus ROs. IMPORTANCE Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition specialized for genome replication. Electron microscopy has revealed the morphology of enterovirus ROs, and immunofluorescence studies have been conducted to investigate their origin and formation. Yet, immunofluorescence analysis of fixed cells results in a rather static view of RO formation, and the results may be compromised by immunolabeling artifacts. While live-cell imaging of ROs would be preferred, enteroviruses encoding a membrane-anchored viral protein fused to a large fluorescent reporter have thus far not been described. Here, we tackled this constraint by introducing a small tag from a split-GFP system into an RO-resident enterovirus protein. This new tool bridges a methodological gap by circumventing the need for immunolabeling fixed cells and allows the study of the dynamics and formation of enterovirus ROs in living cells.

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.

Antiviral activity of the zinc ionophores pyrithione and hinokitiol against picornavirus infections.

We have discovered two metal ion binding compounds, pyrithione (PT) and hinokitiol (HK), that efficiently inhibit human rhinovirus, coxsackievirus, and mengovirus multiplication. Early stages of virus infection are unaffected by these compounds. However, the cleavage of the cellular eukaryotic translation initiation factor eIF4GI by the rhinoviral 2A protease was abolished in the presence of PT and HK. We further show that these compounds inhibit picornavirus replication by interfering with proper processing of the viral polyprotein. In addition, we provide evidence that these structurally unrelated compounds lead to a rapid import of extracellular zinc ions into cells. Imported Zn(2+) was found to be localized in punctate structures, as well as in mitochondria. The observed elevated level of zinc ions was reversible when the compounds were removed. As the antiviral activity of these compounds requires the continuous presence of the zinc ionophore PT, HK, or pyrrolidine-dithiocarbamate, the requirement for zinc ions for the antiviral activity is further substantiated. Therefore, an increase in intracellular zinc levels provides the basis for a new antipicornavirus mechanism.

Increased virus replication in mammalian cells by blocking intracellular innate defense responses.

The mammalian innate immune system senses viral infection by recognizing viral signatures and activates potent antiviral responses. Besides the interferon (IFN) response, there is accumulating evidence that RNA silencing or RNA interference (RNAi) serves as an antiviral mechanism in mammalian cells. Mammalian viruses encode IFN antagonists to counteract the IFN response in infected cells. A number of IFN antagonists are also capable of blocking RNAi in infected cells and therefore serve as RNA-silencing suppressors. Virus replication in infected cells is restricted by these innate antiviral mechanisms, which may kick in earlier than the viral antagonistic or suppressor protein can accumulate. The yield of virus vaccines and viral gene delivery vectors produced in mammalian producer cells may therefore be suboptimal. To investigate whether blocking of the innate antiviral responses in mammalian cells leads to increased viral vector production, we expressed a number of immunity suppressors derived from plant and mammalian viruses in human cells. We measured that the yield of infectious human immunodeficiency virus-1 particles produced in these cells was increased 5- to 10-fold. In addition, the production of lentiviral and adenoviral vector particles was increased 5- to 10-fold, whereas Sindbis virus particle production was increased approximately 100-fold. These results can be employed for improving the production of viral gene transfer vectors and viral vaccine strains.

PDTC inhibits picornavirus polyprotein processing and RNA replication by transporting zinc ions into cells.

Previously, it was shown that pyrrolidine dithiocarbamate (PDTC) inhibits proteolytic polyprotein processing and replication of human rhinovirus by transporting metal ions into cells. Here, it is shown that PDTC also inhibits replication of two other picornaviruses: coxsackievirus B3 (CVB3), a closely related virus that belongs to the genus Enterovirus, and mengovirus, an encephalomyocarditis virus strain that belongs to the genus Cardiovirus, and that this inhibition is due to the dithiocarbamate moiety of the compound. Making use of subgenomic replicons, evidence is provided that PDTC inhibits replication of these two viruses by disturbing viral RNA synthesis. Furthermore, it is shown that PDTC transports zinc ions into cells and that these zinc ions play an important role in the antiviral activity mediated by PDTC. Finally, it is shown that PDTC interferes with proteolytic processing of the polyproteins of both CVB3 and mengovirus, but that the underlying mechanism between these two viruses differs. In CVB3-infected cells, PDTC interferes strongly with the proteolytic activity of 3CD(pro), as shown by the impaired production of the mature capsid proteins as well as the autocleavage of 3CD(pro) into 3C(pro) and 3D(pol). In mengovirus-infected cells, however, PDTC had no effect on the proteolytic production of capsid proteins or the autocleavage of 3CD(pro). Instead, PDTC caused the accumulation of a high-molecular-mass precursor protein, due to an impairment in the primary 'break' that normally occurs at the 2A-2B junction. Thus, PDTC disturbs polyprotein processing and replication of two groups of picornaviruses, enteroviruses and cardioviruses, but the underlying mechanism is different.

Inhibition of polyprotein processing and RNA replication of human rhinovirus by pyrrolidine dithiocarbamate involves metal ions.

Pyrrolidine dithiocarbamate (PDTC) is an antiviral compound that was shown to inhibit the replication of human rhinoviruses (HRVs), poliovirus, and influenza virus. To elucidate the mechanism of PDTC, the effects on the individual steps of the infection cycle of HRV were investigated. PDTC did not interfere with receptor binding or internalization by receptor mediated endocytosis of HRV2 particles into HeLa cells. But we demonstrate that the processing of the viral polyprotein was prevented by PDTC treatment in HeLa cells infected with HRV2. Furthermore, PDTC inhibited the replication of the viral RNA, even when added four hours post infection. As PDTC is described as a metal ion binding agent, we investigated the effect of other metal chelators on the multiplication of HRV2. We show that EDTA, omicron-phenanthroline, and bathocuproine disulfonic acid do not exhibit any antiviral properties. Surprisingly, these substances, coadministered with PDTC, abolished the antiviral effect of PDTC, suggesting that metal ions play a pivotal role in the inhibition of virus multiplication. These results suggest that PDTC inhibits the activity of the viral proteases in a metal ion dependent way.

Manipulation of disulfide bonds differentially affects the intracellular transport, sorting, and processing of neuroendocrine secretory proteins.

To investigate if the prevention of disulfide bond formation affects the intracellular transport, sorting, and processing of a distinct set of neuroendocrine proteins in the regulated secretory pathway, we have treated Xenopus intermediate pituitaries with the thiol-reducing agent dithiothreitol. Pulse-chase incubations in combination with immunoprecipitation analysis were used to monitor the fates of the prohormone proopiomelanocortin (POMC), prohormone convertase PC2 and its helper protein 7B2, as well as secretogranin III. Manipulation of the disulfide bonds in POMC and proPC2 blocked their transport to the trans-Golgi network and strongly inhibited their processing. Reduction of the single disulfide bond in 7B2 did not disturb its transport and cleavage, but caused its missorting to the constitutive secretory pathway. Moreover, the liaison between proPC2 and 7B2 was prevented. Dithiothreitol did not affect transport, sorting, and cleavage of secretogranin III, which lacks disulfide bonds. When the reducing agent was washed away, POMC processing, proPC2 maturation, and the association between proPC2 and 7B2 were reestablished. Collectively, our findings indicate that manipulation of disulfide bonds differentially affects the fates of neuroendocrine proteins during their transit through the secretory pathway.

Genetic analysis of mengovirus protein 2A: its function in polyprotein processing and virus reproduction.

To examine the functional requirements of mengovirus 2A for virus reproduction, a series of mutants with overlapping deletions within the 2A region of mengovirus, and two chimeric constructs in which 2A is replaced either by Theiler's murine encephalomyelitis virus (TMEV) 2A or by coxsackie B3 virus (CBV3) 2Apro were generated. In vitro polyprotein synthesis showed that in both deletion mutants and the TMEV 2A chimeric construct, viral 3C protease (3Cpro)-mediated cleavage at the VP1-2A junction was disturbed, which resulted in decreased formation of mature capsid proteins and accumulation of the P1-2A precursor. 2Apro-mediated processing of the chimeric VP1-2Apro junction was highly efficient. Although the resulting L-P1 precursor was cleaved at the L-VP4 junction, further processing of the P1 precursor was abrogated. Two deletion mutant viruses and a TMEV 2A chimeric virus were obtained after transfection. The CBV 2Apro construct did not result in viable virus. Deletion mutant virus production was less than 3% compared to wild-type virus production, whereas chimeric virus production was reduced to 25%. Although inhibition of host-cell translation was identical in wild-type and mutant virus-infected cells, viral protein and RNA synthesis were reduced in cells infected with mutant virus, independently of the impaired P1-2A processing. It is concluded that mengovirus 2A may play a functional role in either virus translation or replication, and that the functional aspects of mengovirus and TMEV 2A cannot be exchanged. The results also confirm that the processing cascade of L-P1-2A occurs sequentially and is probably regulated by subsequent conformational transitions of the cleavage products after each proteolytic event. The sequential release of L and 2A may be essential in the context of their function in virus replication.

Chimeric coxsackie B3 virus genomes that express hybrid coxsackievirus-poliovirus 2B proteins: functional dissection of structural domains involved in RNA replication.

The 2B proteins of coxsackievirus and poliovirus (PV) share significant structural similarity and exhibit similar biochemical activities, namely inhibition of protein secretion and modification of membrane permeability. Both proteins contain two hydrophobic domains in the carboxy-terminal two-thirds of their sequence, of which one has the potential to form a cationic amphipathic alpha-helix. To gain more insight into the structural requirements of enterovirus protein 2B for its functioning in viral RNA replication, a chimeric cDNA approach was used. Chimeric coxsackie B3 virus (CBV3) genomes were constructed that expressed either the entire PV 2B protein or hybrid proteins in which specific segments of CBV3 2B were substituted by their corresponding PV counterparts. In vitro synthesis and processing of the chimeric polyproteins showed no abnormalities. CBV3 genomes carrying the entire PV 2B gene failed to replicate. A chimeric genome that expressed a hybrid 2B protein consisting of the amino-terminal one-third of PV and the remainder of CBV3 yielded viable viruses. In contrast, a 2B protein consisting of the amino-terminal one-third of CBV3 and the remainder of PV failed to drive replication. These data imply that a sequence-specific interaction with another viral protein is required to drive RNA replication and suggest that the proposed sites of contact reside in the carboxy-terminal two-thirds of 2B. Hybrid genomes in which either the amphipathic alpha-helix or the other hydrophobic domain was replaced failed to replicate. The potential contribution of these domains to the structure and functioning of protein 2B are discussed.

Coxsackievirus protein 2B modifies endoplasmic reticulum membrane and plasma membrane permeability and facilitates virus release.

Digital-imaging microscopy was performed to study the effect of Coxsackie B3 virus infection on the cytosolic free Ca2+ concentration and the Ca2+ content of the endoplasmic reticulum (ER). During the course of infection a gradual increase in the cytosolic free Ca2+ concentration was observed, due to the influx of extracellular Ca2+. The Ca2+ content of the ER decreased in time with kinetics inversely proportional to those of viral protein synthesis. Individual expression of protein 2B was sufficient to induce the influx of extracellular Ca2+ and to release Ca2+ from ER stores. Analysis of mutant 2B proteins showed that both a cationic amphipathic alpha-helix and a second hydrophobic domain in 2B were required for these activities. Consistent with a presumed ability of protein 2B to increase membrane permeability, viruses carrying a mutant 2B protein exhibited a defect in virus release. We propose that 2B gradually enhances membrane permeability, thereby disrupting the intracellular Ca2+ homeostasis and ultimately causing the membrane lesions that allow release of virus progeny.

Structure-function analysis of coxsackie B3 virus protein 2B.

Expression of poliovirus protein 2B in mammalian cells inhibits protein secretion and increases the susceptibility of the cells to hygromycin B, consistent with the increase in plasma membrane permeability seen during poliovirus infection (J. R. Doedens and K. Kirkegaard, EMBO J. 14, 894-907, 1995). We report here that expression of protein 2B of the closely related coxsackie B3 virus (CBV3) leads to the same biochemical alterations. Analysis of several mutant CBV3 2B proteins that contain mutations in a predicted cationic amphipathic alpha-helix (F. J. M. van Kuppeveld, J. M. D. Galama, J. Zoll, P. J. J. C. van den Hurk, and W. J. G. Melchers, J. Virol. 70, 3876-3886, 1996) demonstrated that the integrity of this domain is crucial for both biochemical functions of 2B. Mutations in a second hydrophobic domain (F. J. M. van Kuppeveld, J. M. D. Galama, J. Zoll, and W. J. G. Melchers, J. Virol. 69, 7782-7790, 1995), on the other hand, are more disruptive to the ability of CBV3 2B to inhibit protein secretion than to increase membrane permeability. Therefore, inhibition of protein secretion is not merely a consequence of the membrane changes that increase uptake of hygromycin B. The existence of mutations that interfere with virus growth but do not impair the ability of 2B to inhibit protein secretion or increase membrane permeability argues for additional functions of protein 2B.

Intracellular transport, sorting, and proteolytic processing of regulated secretory proteins does not require protein sulfation.

The biological significance of protein sulfation is poorly understood. To study a possible role of protein sulfation in the regulated secretory pathway, neurointermediate lobes (NIL) of the pituitary of South-African clawed toads (Xenopus laevis) were treated with chlorate, a potent in vivo inhibitor of protein sulfation. We monitored the fates of newly synthesized proopiomelanocortin (POMC), prohormone convertase (PC2), and secretogranin III (SgIII), which are sulfated and regulated secretory proteins. Inhibition of protein sulfation had no effect on the proteolytic processing of these precursor proteins and the kinetics of release of their cleavage products. The release was sensitive to apomorphine, a drug that blocks the release of proteins via the regulated secretory pathway, indicating that no missorting to the constitutive pathway had occurred. Our results suggest that protein sulfation is not required for the intracellular transport, sorting, and proteolytic processing of regulated secretory proteins.

Mutagenesis of the coxsackie B3 virus 2B/2C cleavage site: determinants of processing efficiency and effects on viral replication.

The enterovirus 2B/2C cleavage site differs from the common cleavage site motif AxxQ/G by the occurrence of either polar residues at the P1' position or large aliphatic residues at the P4 position. To study (i) the putative contribution of these aberrant residues to the stability of precursor protein 2BC, (ii) the determinants of cleavage site specificity and efficiency of 3Cpro, and (iii) the importance of efficient cleavage at this site for viral replication, a mutational analysis of the coxsackie B3 virus (CBV3) 2B/2C cleavage site (AxxQ/N) was performed. Neither replacement of the P1' asparagine with a serine or a glycine nor replacement of the P4 alanine with a valine significantly affected 2B/2C cleavage efficiency, RNA replication, or virus growth. The introduction of a P4 asparagine, as can be found at the CBV3 3C/3D cleavage site, caused a severe reduction in 2B/2C cleavage and abolished virus growth. These data support the idea that a P4 asparagine is an unfavorable residue that contributes to a slow turnover of precursor protein 3CD but argue that it is unlikely that the aberrant 2B/2C cleavage site motifs serve to regulate 2B/2C processing efficiency and protein 2BC stability. The viability of a double mutant containing a P4 asparagine and a P1' glycine demonstrated that a P1' residue can compensate for the adverse effects of an unfavorable P4 residue. Poliovirus (or poliovirus-like) 2B/2C cleavage site motifs were correctly processed by CBV 3Cpro, albeit with a reduced efficiency, and yielded viable viruses. Analysis of in vivo protein synthesis showed that mutant viruses containing poorly processed 2B/2C cleavage sites were unable to completely shut off cellular protein synthesis. The failure to inhibit host translation coincided with a reduced ability to modify membrane permeability, as measured by the sensitivity to the unpermeant translation inhibitor hygromycin B. These data suggest that a critical level of protein 2B or 2C, or both, may be required to alter membrane permeability and, possibly as a consequence, to shut off host cell translation.

Mengovirus leader is involved in the inhibition of host cell protein synthesis.

The presence of a leader peptide in picornaviruses is restricted to the Cardiovirus and Aphthovirus genera. However, the leader peptides of these two genera are structurally and functionally unrelated. The aphthovirus leader is a protease involved in viral polyprotein processing and host cell translation shutoff. The function of the cardiovirus leader peptide is still unknown. To gain an insight into the function of the cardiovirus leader peptide, a mengovirus leader peptide deletion mutant was constructed. The deletion mutant was able to grow at a reduced rate in baby hamster kidney cells (BHK-21). Mutant virus production in mouse fibroblasts (L929 cells), however, could be demonstrated only after inoculation of BHK-21 cells with the transfected L929 cells. Analysis of cellular and viral protein synthesis in mutant virus-infected cells showed a delayed inhibition of host cell protein synthesis and a reduced production of viral proteins. In a single-cycle infection, mutant virus produced only 1% of wild-type virus yield at 8 h postinfection. Host cell translation shutoff in L929 cells infected with mutant virus was restored by the addition of the kinase inhibitor 2-aminopurine. Mutant virus production in 2-aminopurine-treated L929 cells was increased to 60% of wild-type virus yield at 8 h postinfection. Our results suggest that the cardiovirus leader peptide is involved in the inhibition of host cell protein synthesis.

Coxsackie B3 virus protein 2B contains cationic amphipathic helix that is required for viral RNA replication.

Enterovirus protein 2B has been shown to increase plasma membrane permeability. We have identified a conserved putative amphipathic alpha-helix with a narrow hydrophilic face and an arrangement of cationic residues that is typical for the so-called lytic polypeptides. To examine the functional and structural roles of this putative amphipathic alpha-helix, we have constructed nine coxsackie B3 virus mutants by site-directed mutagenesis of an infectious cDNA clone. Six mutants contained substitutions of the charged residues in the hydrophilic face of the alpha-helix. Three mutants contained insertions of leucine residues between the charged residues, causing a disturbance of the amphipathic character of the alpha-helix. The effect of the mutations on virus viability was assayed by transfection of cells with copy RNA transcripts. The effect on positive-strand RNA replication was examined by introduction of the mutations in a subgenomic luciferase replicon and analysis of luciferase accumulation following the transfection of BGM cells with RNA transcripts. It is shown that both the amphipathy of the domain and the presence of cationic residues in the hydrophilic face of the alpha-helix are required for virus growth. Mutations that disturbed either one of these features caused defects in viral RNA synthesis. In vitro translation reactions and the analysis of viral protein synthesis in vivo demonstrated that the mutations did not affect synthesis and processing of the viral polyprotein. These results suggest that a cationic amphipathic alpha-helix is a major determinant for a function of protein 2B, and possibly its precursor 2BC, in viral RNA synthesis. The potential role of the amphipathic alpha-helix in the permeabilization of cellular membranes is discussed.

Demonstration of Mycoplasma contamination in cell cultures by a Mycoplasma group- specific polymerase chain reaction.

Cell cultures are widely used in both medical and biotechnical research centers, industries, and also as diagnostic tools in a clinical setting It has been reported that up to 50% of cell cultures are contaminated with mycoplasmas (1). Mycoplasma contamination may alter cellular growth characteristics, enzyme patterns, and cell membrane composition, and can induce chromosomal abnormalities and cytopathogenic changes (2-5). In experimental results being published, it is now becoming standard practice to show that mycoplasma-free cell cultures have been used. For industrial production of biological materials derived from mammalian cells and intended for diagnostic or therapeutic use in humans, regulatory guidelines are emerging that are intended to guarantee the quality and safety of these products. Obligatory testing for mycoplasmas is therefore increasing (6).

Genetic analysis of a hydrophobic domain of coxsackie B3 virus protein 2B: a moderate degree of hydrophobicity is required for a cis-acting function in viral RNA synthesis.

Coxsackie B virus protein 2B contains near its C terminus a hydrophobic domain with an amino acid composition that is characteristic for transmembrane regions. A molecular genetic approach was followed to define the role of this domain in virus reproduction and to study the structural and hydrophobic requirements of the domain. Nine substitution mutations were introduced in an infectious cDNA clone of coxsackie B3 virus. The effects of the mutations were studied in vivo by transfection of Buffalo green monkey cells with copy RNA transcripts. The results reported here suggest that a critical degree of hydrophobicity of the domain is essential for virus growth. The mutations S77M, C75M, I64S, and V66S, which caused either a small increase or decrease in mean hydrophobicity, yielded viable viruses. The double mutations S77M/C75M and I64S/V6-6S, which caused a more pronounced increase or decrease in hydrophobicity, were nonviable. Negatively charged residues (mutations A71E, I73E, and A71E/I73E) abolished virus growth. The mutations had no effect on the synthesis and processing of the viral polyprotein. Replication and complementation were studied by using a subgenomic coxsackievirus replicon containing the luciferase gene in place of the capsid coding region. Analysis of luciferase accumulation demonstrated that the mutations cause primary defects in viral RNA synthesis that cannot be complemented by wild-type protein 2B provided in trans. The hydrophobic domain is predicted by computer analysis to form a multimeric transmembrane helix. The proposed interaction with the membrane and the implications of the mutations on this interaction are discussed.

16S rRNA based polymerase chain reaction compared with culture and serological methods for diagnosis of Mycoplasma pneumoniae infection.

The use of a 16S rRNA based polymerase chain reaction (PCR) for the detection of Mycoplasma pneumoniae infection was investigated. Sputum samples from 34 patients with respiratory illness and evidence of pneumonia as judged by chest X-ray were analyzed by PCR and microbiological culture. Throat swabs from 14 healthy individuals were used as controls. For serology, an enzyme immunoassay for the detection of immunoglobulin M antibodies and a complement fixation assay were performed. Evidence of Mycoplasma pneumoniae infection was obtained in ten patients (29%), eight of whom were found positive by both PCR and serology. Two of the sputum samples from these eight patients were negative by culture. Of the remaining two patients positive for Mycoplasma pneumoniae, one was positive by PCR and culture but negative by serology, and one was found positive by serology but negative by PCR and culture. Thirteen of the 14 controls were negative by both PCR and serology. One control, however, was negative by serology but positive by PCR, which was probably due to asymptomatic carriage of Mycoplasma pneumoniae. The results of this study indicate the suitability of the PCR for the detection of Mycoplasma pneumoniae in clinical samples as well as its potential value as an additional tool for the diagnosis of infection.