Improved bactericidal efficacy and thermostability of Staphylococcus aureus-specific bacteriophage SA3821 by repeated sodium pyrophosphate challenges

As antibiotic resistance is being a threat to public health worldwide, bacteriophages are re-highlighted as alternative antimicrobials to fight with pathogens. Various wild-type phages isolated from diverse sources have been tested, but potential mutant phages generated by genome engineering or random mutagenesis are drawing increasing attention. Here, we applied a chelating agent, sodium pyrophosphate, to the staphylococcal temperate Siphoviridae phage SA3821 to introduce random mutations. Through 30 sequential sodium pyrophosphate challenges and random selections, the suspected mutant phage SA3821M was isolated. SA3821M maintained an intact virion morphology, but exhibited better bactericidal activity against its host Staphylococcous aureus CCARM 3821 for up to 17 h and thermostability than its parent, SA3821. Sodium pyrophosphate-mediated mutations in SA3821M were absent in lysogenic development genes but concentrated (83.9%) in genes related to the phage tail, particularly in the tail tape measure protein, indicating that changes in the tail module might have been responsible for the altered traits. This intentional random mutagenesis through controlled treatments with sodium pyrophosphate could be applied to other phages as a simple but potent method to improve their traits as alternative antimicrobials.

Engineering viable foot-and-mouth disease viruses with increased acid stability facilitate the development of improved vaccines

Foot-and-mouth disease virus (FMDV), the most acid-unstable virus among picornaviruses, tends to disassemble into pentamers at pH values slightly below neutrality. However, the structural integrity of intact virion is one of the most important factors that influence the induction of a protective antibody response. Thus, improving the acid stability of FMDV is required for the efficacy of vaccine preparations. According to the previous studies, a single substitution or double amino acid substitutions (VP1 N17D, VP2 H145Y, VP2 D86H, VP3 H142D, VP3 H142G, and VP1 N17D + VP2 H145Y) in the capsid were introduced into the full-length infectious clone of type O FMDV vaccine strain O/HN/CHN/93 to develop seed FMDV with improved acid stability. After the transfection into BSR/T7 cells of constructed plasmids, substitution VP1 N17D or VP2 D86H resulted in viable and genetically stable FMDVs, respectively. However, substitution VP2 H145Y or VP1 N17D + VP2 H145Y showed reverse mutation and additional mutations, and substitution VP3 H141G or VP3 H141D prevented viral viability. We found that substitution VP1 N17D or VP2 D86H could confer increased acid resistance, alkali stability, and thermostability on FMDV O/HN/CHN/93, whereas substitution VP1 N17D was observed to lead to a decreased replication ability in BHK-21 cells and mildly impaired virulence in suckling mice. In contrast, substitution VP2 D86H had no negative effect on viral infectivity. These results indicated that the mutant rD86H carrying substitution VP2 D86H firstly reported by us could be more adequate for the development of inactivated FMD vaccines with enhanced acid stability.

Translational Cross-Activation of the Encapsidated RNA of Potexviruses

We had shown the genomic RNA of potexviruses potato virus X and the alternanthera mosaic virus to be inaccessible in vitro to ribosomes while in intact virion form, but the RNAs can be translationally activated following the binding of movement protein 1 (MP1) to virus particles. Here, we present the results of the follow-up study targeting two more potexvirus species - the Narcissus mosaic virus and the Potato aucuba mosaic virus. We found encapsidated potexviral RNA to share common translational features in vitro and the MP1 to be potent over homological virions of its own species and over heterological virions of other species, as well exhibiting selective specificity. Reciprocal cross-activation is observed among viral species phylogenetically either close or distant. There is direct evidence that MP1 binding to the end of the virion is necessary, but not sufficient, for translational activation of encapsidated RNA.

Cell-Penetrating Peptides Derived from Viral Capsid Proteins

Cell-penetrating peptides (CPP) can translocate across the cell membrane and have been extensively studied for the delivery of proteins, nucleic acids, and therapeutics in mammalian cells. However, characterizations of CPP in plants have only recently been initiated. We showed that the intact virion and a recombinant capsid protein (CaP) from a plant-infecting nonenveloped icosahedral RNA virus, Brome mosaic virus (BMV), can penetrate the membranes of plant protoplasts but are trapped by the extracellular matrix. Furthermore, a 22-residue peptide derived from the N-terminal region of the CaP (CPNT) can enter barley protoplasts and cells of intact barley and Arabidopsis roots. An inhibitor of the macropinocytosis reduced CPNT entry, while treatment with NiCl2 changed the cellular localization of CPNT. CPNT increased uptake of the green flourescent protein (GFP) into the cell when covalently fused to GFP or when present in trans of GFP. The BMV CPNT overlaps with the sequence known to bind BMV RNA, and it can deliver BMV RNAs into cells, resulting in viral replication, as well as deliver double-stranded RNAs that can induce gene silencing.

A novel envelope protein involved in White spot syndrome virus infection

One open reading frame (designated vp76) from the White spot syndrome virus (WSSV) genome has the motif of a cytokine I receptor and has been identified as a structural protein. In this paper, vp76 was expressed in Escherichia coli and used to prepare a specific antibody to determine the location of the corresponding protein in the intact virion, the nucleocapsids and the envelope of WSSV. Western blotting with the VP76 antiserum confirmed that VP76 was an envelope protein of WSSV. To investigate the function of the VP76, WSSV was neutralized with the VP76-specific antiserum at different concentrations and injected intramuscularly into crayfish. The mortality curves showed that the VP76 antiserum could partially attenuate infection with WSSV, suggesting that VP76 is an envelope protein involved in WSSV infection.

Development and Characterization of Novel Empty Adenovirus Capsids and Their Impact on Cellular Gene Expression

ABSTRACT Adenovirus (Ad) has been extensively studied as a eukaryotic viral vector. As these vectors have evolved from first-generation vectors to vectors that contain either very few or no viral genes (“gutless” Ad), significant reductions in the host innate immune response upon infection have been observed. Regardless of these vector improvements an unknown amount of toxicity has been associated with the virion structural proteins. Here we demonstrate the ability to generate high particle numbers (1011 to 1012) of Ad empty virions based on a modification of Cre/lox gutless Ad vectors. Using a battery of analyses (electron microscopy, atomic force microscopy, confocal images, and competition assays) we characterized this reagent and determined that it (i) makes intact virion particles, (ii) competes for receptor binding with wild-type Ad, and (iii) enters the cell proficiently, demonstrating an ability to carry out essential steps of viral entry. To further study the biological impact of these Ad empty virions on infected cells, we carried out DNA microarray analysis. Compared to that for recombinant Ad, the number of mRNAs modulated upon infection was significantly reduced but the expression signatures were similar. This reagent provides a valuable tool for studies of Ad in that researchers can examine the effect of infection in the presence of the virion capsid alone.

Genetic Evidence for a Structural Interaction between the Carboxy Termini of the Membrane and Nucleocapsid Proteins of Mouse Hepatitis Virus

ABSTRACT The coronavirus membrane (M) protein is the most abundant virion protein and the key component in viral assembly and morphogenesis. The M protein of mouse hepatitis virus (MHV) is an integral membrane protein with a short ectodomain, three transmembrane segments, and a large carboxy-terminal endodomain facing the interior of the viral envelope. The carboxy terminus of MHV M has previously been shown to be extremely sensitive to mutation, both in a virus-like particle expression system and in the intact virion. We have constructed a mutant, MΔ2, containing a two-amino-acid truncation of the M protein that was previously thought to be lethal. This mutant was isolated by means of targeted RNA recombination with a powerful host range-based selection allowed by the interspecies chimeric virus fMHV (MHV containing the ectodomain of the feline infectious peritonitis virus S protein). Analysis of multiple second-site revertants of the MΔ2 mutant has revealed changes in regions of both the M protein and the nucleocapsid (N) protein that can compensate for the loss of the last two residues of the M protein. Our data thus provide the first genetic evidence for a structural interaction between the carboxy termini of the M and N proteins of MHV. In addition, this work demonstrates the efficacy of targeted recombination with fMHV for the systematic genetic analysis of coronavirus structural protein interactions.

The glycoprotein isolated from vesicular stomatitis virus is mitogenic for mouse B lymphocytes.

The glycoprotein (G protein) of VSV was purified from the intact virion by Triton X-100 extraction. The isolated G protein has been shown to be a T cell-independent, B lymphocyte mitogen and polyclonal activator. Neither G protein nor the intact virion are stimulatory for murine T lymphocytes. The greater the density of G protein in lipid vesicles or the degree of aggregation of isolated G protein, the more highly stimulatory it is for murine splenocytes. As G protein is spread out in artificial vesicles, it becomes less mitogenic. It is probable that other viral components are also stimulatory since the Triton-insoluble pellet and VSV from which the G protein has been enzymatically removed retain mitogenic activity. To out knowledge, this is the first time a purified viral component has been demonstrated to be lymphocyte mitogen.