Virus Passage through Track-Etch Membranes Modified by Salinity and a Nonionic Surfactant

Why do viruses sometimes not pass through larger pores in track-etch filters? Increasing the salinity (0.8 to 160 mM Na⁺) decreased X174 and PRD1 passage through track-etch polycarbonate membranes (sodium dodecyl sulfate coated but not polyvinylpyrrolidone coated) and PRD1 passage through polyester membranes. Undiminished passage when 0.1% Tween 80 was added implied that nonionic virus adsorption occurred and indicated that high levels of salinity decreased virus passage by decreasing electrostatic repulsion that prevented adsorption.     

The IncP plasmid-encoded cell envelope-associated DNA transfer complex increases cell permeability.

IncP-type plasmids are broad-host-range conjugative plasmids. DNA translocation requires DNA transfer-replication functions and additional factors required for mating pair formation (Mpf). The Mpf system is located in the cell membranes and is responsible for DNA transport from the donor to the recipient. The Mpf complex acts as a receptor for IncP-specific phages such as PRD1. In this investigation, we quantify the Mpf complexes on the cell surface by a phage receptor saturation technique. Electrochemical measurements are used to show that the Mpf complex increases cell envelope permeability to lipophilic compounds and ATP. In addition it reduces the ability of the cells to accumulate K+. However, the Mpf complex does not dissipate the membrane voltage. The Mpf complex is rapidly disassembled when intracellular ATP concentration is decreased, as measured by a PRD1 adsorption assay.     

Minimized virus binding for tests of barrier materials.

Viruses are used to test the barrier properties of materials. Binding of virus particles during passage through holes in the material may yield misleading test results. The choices of challenge virus and suspending medium may be important for minimizing confounding effects that might arise from such binding. In this study, different surrogate viruses, as well as different support media, were evaluated to determine optimal test parameters. Two membranes with high-binding properties (nitrocellulose and cationic polysulfone) were used as filters to compare binding activities of different surrogate challenge viruses (MS2, phi X174, T7, PRD1, and phi 6) in different media. The media consisted of buffered saline with surfactants, serum, or culture broth as additives. In addition, elution rates of viruses that bound to the membranes were determined. The results suggest that viruses can bind by hydrophobic and electrostatic interactions, with phi X174 displaying the lowest level of binding by either process. The nonionic detergents Triton X-100 and Tween 80 (0.1%) equally minimized hydrophobic interactions. Neither anionic nor cationic surfactants were as effective at nontoxic levels. Serum was effective at reducing both hydrophobic and electrostatic binding, with 2% being sufficient for eliminating binding under our test conditions. Thus, phi X174 remains the best choice as a surrogate virus to test barrier materials, and Triton X-100 (0.1%) remains a good choice for reducing hydrophobic binding. In addition, binding of viruses by barrier materials is unlikely to prevent passage of blood-borne pathogens.     

Membrane-assisted viral DNA ejection

Genome packaging and delivery are fundamental steps in the replication cycle of all viruses. Icosahedral viruses with linear double-stranded DNA (dsDNA) usually package their genome into a preformed, rigid procapsid using the power generated by a virus-encoded packaging ATPase. The pressure and stored energy due to this confinement of DNA at a high density is assumed to drive the initial stages of genome ejection. Membrane-containing icosahedral viruses, such as bacteriophage PRD1, present an additional architectural complexity by enclosing their genome within an internal membrane vesicle. Upon adsorption to a host cell, the PRD1 membrane remodels into a proteo-lipidic tube that provides a conduit for passage of the ejected linear dsDNA through the cell envelope. Based on volume analyses of PRD1 membrane vesicles captured by cryo-electron tomography and modeling of the elastic properties of the vesicle, we propose that the internal membrane makes a crucial and active contribution during infection by maintaining the driving force for DNA ejection and countering the internal turgor pressure of the host. These novel functions extend the role of the PRD1 viral membrane beyond tube formation or the mere physical confinement of the genome. The presence and assistance of an internal membrane might constitute a biological advantage that extends also to other viruses that package their linear dsDNA to high density within an internal vesicle.     

Entry of herpes simplex virus 1 in BJ cells that constitutively express viral glycoprotein D is by endocytosis and results in degradation of the virus.

The BJ cell line which constitutively expresses herpes simplex virus 1 glycoprotein D is resistant to infection with herpes simplex viruses. Analysis of clonal lines indicated that resistance to superinfecting virus correlates with the expression of glycoprotein D. Resistance was not due to a failure of attachment to cells, since the superinfecting virus absorbed to the BJ cells. Electron microscopic studies showed that the virions are juxtaposed to coated pits and are then taken up into endocytic vesicles. The virus particles contained in the vesicles were in various stages of degradation. Viral DNA that reached the nucleus was present in fewer copies per BJ cell than that in the parental BHKtk- cells infected at the same multiplicity. Moreover, unlike the viral DNA in BHKtk- cells which was amplified, that in BJ cells decreased in copy number. The results suggest that the glycoprotein D expressed in the BJ cell line interfered with fusion of the virion envelope with the plasma membrane but not with the adsorption of the virus to cells and that the viral proteins that mediate adsorption to and fusion of membranes appear to be distinct.     

Efficient Filtration and Sizing of Viruses with Membrane Filters

Untreated membrane filters retain viruses by adsorption, as well as by physical restriction which occurs when the pore diameter of the filter is smaller than that of the virus particle. As originally recommended by Elford, membranes had to be pretreated with proteinaceous material to preclude virus adsorption. However, coating materials that prevent adsorption of certain viruses do not necessarily prevent adsorption of other viruses. In contrast to proteins, salts enhance virus adsorption. Viruses treated with sodium lauryl sulfate to reduce the surface tension, or purified viruses in distilled water, are not adsorbed to membranes. A procedure is recommended by which viruses may be passed through membranes with a porosity twice the diameter of the virus. Such filtrates, which contain 50 to 100% of the initial virus concentration, should be used for sizing viruses by subsequent filtration through smaller pores. The determination of virus size would then be based on the major population of particles in the virus suspension. In the past, as little as 0.1 to 0.001% of the initial virus population was the basis for size determination, because more than 99.9% of the virus was often lost by adsorption to membranes during the clarifying procedures.     

Interactions between protein molecules and the virus removal membrane surface: Effects of immunoglobulin G adsorption and conformational changes on filter performance

Membrane fouling commonly occurs in all filter types during virus filtration in protein‐based biopharmaceutical manufacturing. Mechanisms of decline in virus filter performance due to membrane fouling were investigated using a cellulose‐based virus filter as a model membrane. Filter performance was critically dependent on solution conditions; specifically, ionic strength. To understand the interaction between immunoglobulin G (IgG) and cellulose, sensors coated with cellulose were fabricated for surface plasmon resonance and quartz crystal microbalance with energy dissipation measurements. The primary cause of flux decline appeared to be irreversible IgG adsorption on the surface of the virus filter membrane. In particular, post‐adsorption conformational changes in the IgG molecules promoted further irreversible IgG adsorption, a finding that could not be adequately explained by DLVO theory. Analyses of adsorption and desorption and conformational changes in IgG molecules on cellulose surfaces mimicking cellulose‐based virus removal membranes provide an effective approach for identifying ways of optimizing solution conditions to maximize virus filter performance. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:379–386, 2018     

Stable Packaging of Phage PRD1 DNA Requires Adsorption Protein P2, Which Binds to the IncP Plasmid-Encoded Conjugative Transfer Complex

The double-stranded DNA bacteriophage PRD1 uses an IncP plasmid-encoded conjugal transfer complex as a receptor. Plasmid functions in the PRD1 life cycle are restricted to phage adsorption and DNA entry. A single phage structural protein, P2, located at the fivefold capsid vertices, is responsible for PRD1 attachment to its host. The purified recombinant adsorption protein was judged to be monomeric by gel filtration, rate zonal centrifugation, analytical ultracentrifugation, and chemical cross-linking. It binds to its receptor with an apparent K(d) of 0.20 nM, and this binding prevents phage adsorption. P2-deficient particles are unstable and spontaneously release the DNA with concomitant formation of the tail-like structure originating from the phage membrane. We envisage the DNA to be packaged through one vertex, but the presence of P2 on the other vertices suggests a mechanism whereby the injection vertex is determined by P2 binding to the receptor.     

Membrane-containing viruses with icosahedrally symmetric capsids

Viruses with an icosahedrally symmetric protein capsid and a membrane infect hosts from all three domains of life. Similar architectural principles are shared by different viral families, as exemplified by double-stranded DNA viruses such as PRD1 and STIV. During virus assembly, the membrane lipids are selectively acquired from the host cell. The X-ray structure of bacteriophage PRD1 revealed that the lipids are asymmetrically distributed between the two leaflets and facet length is controlled by a tape-measure protein. In most membrane-containing viruses, viral and host membranes fuse during viral entry. In the best-understood systems of the alphaviruses, flaviviruses and herpes viruses, fusion is mediated by viral glycoproteins. Recent structural advances reveal how very different protein architectures can be used to form trimeric extensions that extend into the target cell membrane and then fold back to mediate fusion of the target and viral membranes.     

Salinity affects intracellular calcium in corn root protoplasts

Previous work with the fluorescent Ca probe chlorotetracycline (CTC) showed that salinity displaces Ca from membranes of root cells. Using a variety of indirect approaches, we studied whether salinity displaces Ca from the cell surface or from internal membranes of corn (Zea mays L. cv Pioneer 3377) root protoplasts. Preloading the cells with supplemental Ca counteracted subsequent NaCl effects on CTC fluorescence. CTC quenching by exogenous EGTA was not competitive with CTC quenching by NaCl. The Ca channel reagent (+)-202-791 had significant interactions with the effect of NaCl on CTC fluorescence. The effect of NaCl on CTC fluorescence was attenuated by pretreatment with Li, but was restored by inositol. Salinity increased Na influx, decreased Ca influx, and increased Ca efflux from the cells. Fluorescence anisotropy indicated that NaCl decreased the fluidity of the external face of the plasmalemma but increased the fluidity of cell membranes in general. Our results suggest that salinity displaces Ca associated with intracellular membranes through activation of the phosphoinositide system and depletion of intracellular Ca pools.     

Application of a quinohaemoprotein alcohol dehydrogenase in bio-electrocatalysis: Immobilization of the enzyme and mediated electron transfer between the enzyme and an electrode

Summary Quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni was immobilized on polypyrrole-coated track-etch and microporous membranes. On the track-etch membrane, 3.4 to 4.8 × 10^–3 Units of enzyme/cm² was immobilized whilst on the microporous membrane 0.05 U/cm² was immobilized. The track-etch membrane was then used in electrochemical studies using ferricyanide as a redox mediator giving a maximum catalytic current of 0.022 mA/cm² membrane with 1-pentanol as the substrate. The kinetic parameters (K_m and V_max) of the immobilized enzyme are of the same order of magnitude as those of the free enzyme.     

Regulation of helper cell activity by specifically adsorbable T lymphocytes.

Mice immunized with soluble proteins such as human serum albumin (HSA) or ovalbumin (OA) develop in their spleens antigen-specific T and B lymphocytes. These populations of lymphocytes can be separated from each other by different means; e.g. treatment with anti-theta-antiserum and complement removes selectively T lymphocytes, whereas passage through glass bead columns coated with mouse immunoglobulin (Ig): anti-Ig complexes creates a relatively pure population of T lymphocytes. During the course of such separation studies it was observed that the helper capacity of HSA (or OA) immune mouse spleen cells after Ig:anti-Ig column passage frequently was higher than expected from the enrichment in theta-positive cells. In addition, after adsorption onto antigen coated Bio-Gel beads this effect was even more pronounced, i.e., and increase in the relative helper capacity of about 3 or 4 times compared with an increase in the content of theta-positive cells from about 30% to 40 to 50% after adsorption. The present results will demonstrate that the increased helper capacity was a specific phenomenon which was regulated by theta-positive cells. The regulatory cells specifically adsorbed onto antigen-coated Bio-Gel beads have not been successfully eluted by EDTA or excess-free antigen so far, and they were still adsorbed after pre-incubation with anti-Ig antibodies under conditions where specific B lymphocyte adsorption was almost prevented.     

Filtration sizes of human immunodeficiency virus type 1 and surrogate viruses used to test barrier materials.

Filters with well-defined holes were used to determine the effective diameters in buffer of human immunodeficiency virus type 1, herpes simplex virus type 1, and four bacteriophages (phi X174, T7, PRD1, and phi 6), which may serve as surrogate viruses for testing barrier materials. Bacteriophages phi 6 and PRD1 most closely model human immunodeficiency virus type 1 in filtration size.     

Structural study of the lipid-containing bacteriophage PRD1 and its capsid and DNA components by laser Raman spectroscopy

The Raman spectrum of a virus contains the structural signature of each of its molecular components (Thomas, 1987). We report the first Raman spectrum obtained from an intact, lipid-containing virus--the icosahedral bacteriophage PRD1--and show that this spectrum contains characteristic structure markers for the major capsid protein, the packaged double-stranded DNA genome, and the viral membrane which resides between the capsid and DNA. We find that the packaged genome of PRD1 exhibits Raman markers typical of the B-DNA secondary structure. Comparison of the Raman spectrum of the packaged DNA with that of protein-free DNA extracted from the virion shows further that the B-form secondary structure is not significantly perturbed by packaging in the virion. The Raman signature of the PRD1 membrane, monitored within the virion at 4 degrees C, is that of a phospholipid liquid-crystalline phase. The PRD1 capsid, which comprises several hundred copies of the major coat protein P3 (product of viral gene III) and a few copies of minor proteins, incorporates P3 capsomers predominantly in the beta-sheet conformation. The beta-sheet structure of P3 is maintained in the fully assembled PRD1 virion, as well as in the empty capsid. The present results demonstrate the feasibility of obtaining structural information from the three different classes of biomolecules--nucleic acid, protein, and lipid--which constitute a membrane-lined virus particle. Our results also demonstrate that the coat protein and double-stranded DNA components of a lipid-containing bacteriophage share many structural features in common with bacteriophage lacking a lipid membrane.     

Concentration of Enteroviruses on Membrane Filters

Enteroviruses can be made to adsorb or to pass through membrane filters by manipulation of the suspending medium. Salts facilitate virus adsorption, but membrane-coating components (MCC) interfere. Because cells release MCC into the culture medium during viral growth, MCC must be removed before virus can be adsorbed to membranes. Adsorbed virus can be eluted with diluents containing MCC (cell extracts or serum) or agents that reduce surface tension (sodium lauryl sulfate). By membrane adsorption and elution, enteroviruses can be readily concentrated and quantitatively recovered from crude virus harvests.     

Soil inactivation of DNA viruses in septic seepage

AIMS: To generate field-relevant inactivation data for incorporation into models to predict the likelihood of viral contamination of surface waters by septic seepage. METHODS AND RESULTS: Inactivation rates were determined for PRD1 bacteriophage and Adenovirus 2 in two catchment soils under a range of temperature, moisture and biotic status regimes. Inactivation rates presented for both viruses were significantly different at different temperatures and in different soil types (alpha = 0.05). Soil moisture generally did not significantly affect virus inactivation rate. Biotic status significantly affected inactivation rates of PRD1 in the loam soil but not the clay-loam soil. Adenovirus 2 was inactivated more rapidly in the loam soil than PRD1 bacteriophage. CONCLUSIONS: Virus inactivation rates incorporated into models should be appropriate for the climate/catchment in question with particular regard to soil type and temperature. Given that PRD1 is similar in size to adenoviruses, yet more conservative with regard to inactivation in soil, it may be a useful surrogate in studies of Adenovirus fate and transport. SIGNIFICANCE AND IMPACT OF THE STUDY: A better understanding of the factors that govern virus fate and transport in catchments would facilitate the design of barrier measures to prevent viral contamination of surface waters by septic seepage.     

Proline 78 is crucial for human immunodeficiency virus type 1 Nef to down-regulate class I human leukocyte antigen

Human immunodeficiency virus type 1 Nef down-regulates human leukocyte antigen class I (HLA-I) in T lymphocytes, and the down-regulation involves the Nef proline-rich domain (PRD) containing four prolines at positions 69, 72, 75, and 78. We used a Sendai virus vector with nef and examined regulation by Nef of HLA-I and CD4 in suspension cultures of cells such as T lymphocytes. Analyses of a series of PRD substitution mutants indicated that, because the substitution of Pro78 with Ala abolished down-regulation of HLA-I but not of CD4, Pro78 is important for HLA-I down-regulation in T lymphocytes.     

Filtration sizes of human immunodeficiency virus type 1 and surrogate viruses used to test barrier materials.

Filters with well-defined holes were used to determine the effective diameters in buffer of human immunodeficiency virus type 1, herpes simplex virus type 1, and four bacteriophages (phi X174, T7, PRD1, and phi 6), which may serve as surrogate viruses for testing barrier materials. Bacteriophages phi 6 and PRD1 most closely model human immunodeficiency virus type 1 in filtration size.     

Bacteriophage PRD1 contains a labile receptor-binding structure at each vertex.

Bacteriophage PRD1 is a membrane-containing virus with an unexpected similarity to adenovirus. We mutagenized unassigned PRD1 genes to identify minor capsid proteins that could be structural or functional analogs to adenovirus proteins.We report here the identification of an amber mutant, sus525, in an essential PRD1 gene XXXI. The gene was cloned and the gene product was overexpressed and purified to near homogeneity. Analytical ultracentrifugation and gel filtration showed that P31 is a homopentamer of about 70 kDa. The protein was shown to be accessible on the virion surface and its absence in the sus525 particles led to the deficiency of two other viral coat proteins, protein P5 and the adsorption protein P2. Cryo-electron microscopy and image reconstruction of the sus525 particles indicate that these proteins are located on the capsid vertices, because in these particles the entire vertex structure was missing along with the peripentonal major capsid protein P3 trimers. Sus525 particles package DNA effectively but loose it upon purification.All of the PRD1 vertex structures are labile and potentially capable of mediating DNA delivery; this is in contrast to other dsDNA phages which employ a single vertex for packaging and delivery. We propose that this arises from a symmetry mismatch between protein P2 and the pentameric P31 in analogy to that between the adenovirus penton base and the receptor-binding spike.     

Interaction of mycoplasma virus type 2 with cellular components of Acholeplasma laidlawii strain JA1.

Mycoplasma virus type 2 was shown to adsorb specifically to intact cells, membranes, and lipoglycan of Acholeplasma laidlawii strain JA1 but not to these components of Acholeplasma oculi. The oligosaccharide chain of the lipoglycan defined the specificity of the receptor site since deacylation not only did not reduce adsorption but increased it threefold. Actual adsorption of virus to lipoglycan was demonstrated by sucrose density gradient separation of the virus-lipoglycan complex. A strain of A. laidlawii, JA1r, resistant to infection with mycoplasma virus type 2, was incapable of adsorbing the virus and was devoid of lipoglycan.