Plaque-forming Ability Research Articles

Antiviral and synergistic effects of photo-energy with acyclovir on herpes simplex virus type 1 infection

This experimental study aimed to evaluate the antiviral and synergistic effects of photoenergy irradiation on human herpes simplex virus type I (HSV-1) infection. We assessed viral replication, plaque formation, and relevant viral gene expression to examine the antiviral and synergistic effects of blue light (BL) with acyclovir treatment. Our results showed that daily BL (10 J/cm2) irradiation inhibited plaque-forming ability and decreased viral copy numbers in HSV-1-infected monkey kidney epithelial Vero cells and primary human oral keratinocyte (HOK) cells. Combined treatment with the antiviral agent acyclovir and BL irradiation increased anti-viral activity, reducing viral titers and copy numbers. In particular, accumulated BL irradiation suppressed characteristic viral genes including UL19 and US6, and viral DNA replication-essential genes including UL9, UL30, UL42, and UL52 in HOK cells. Our results suggest that BL irradiation has anti-viral and synergistic properties, making it a promising therapeutic candidate for suppressing viral infections in clinical trials.

Construction and efficacy of a new live chimeric C-strain vaccine with DIVA characteristics against classical swine fever.

To develop the new classical swine fever (CSF) vaccine candidate with differentiating infected vaccinated animals (DIVA) characteristics, a chimeric CSF virus (CSFV) was constructed based on an infectious cDNA clone of the CSF vaccine C-strain. The 5'- and 3'-untranslated regions (UTRs) and partial E2 region (residues 690-860) of the C-strain were substituted with the corresponding regions of bovine viral diarrhoea virus (BVDV) to construct the chimeric cDNA clone pC/bUTRs-tE2. The chimeric virus rC/bUTRs-tE2 was generated by several passages of pC/bUTRs-tE2-transfected PK15 cells. Stable growth and genetic properties of rC/bUTRs-tE2 were obtained after 30 serial passages. Compared to parental rC/bUTRs-tE2 (1st passage), two residue mutations (M834K and M979K) located in E2 in rC/bUTRs-tE2 P30 were observed. Compared to the C-strain, rC/bUTRs-tE2 exhibited unchanged cell tropism and decreased plaque-forming ability. Substituting the C-strain UTRs with the BVDV UTRs resulted in significantly increased viral replication in PK15 cells. Compared to CSFV Erns-positive and BVDV tE2-negative antibody responses induced by the CSF vaccine C-strain, immunization of rabbits and piglets with rC/bUTRs-tE2 resulted in serological profiles of CSFV Erns- and BVDV tE2-positive antibodies, which are used to serologically discriminate pigs that are clinically infected and vaccinated. Vaccination of piglets with rC/bUTRs-tE2 conferred complete protection against lethal CSFV challenge. Our results suggest that rC/bUTRs-tE2 is a promising new CSF marker vaccine candidate.

Phenotypic characterization of phage vB_vcM_Kuja.

Bacteriophage therapy targeting the increasingly resistant Vibrio cholerae is highly needed. Hence, studying the phenotypic behavior of potential phages under different conditions is a prerequisite to delivering the phage in an active infective form. The objective of this study was to characterize phage VP4 (vB_vcM_Kuja), an environmental vibriophage isolated from River Kuja in Migori County, Kenya in 2015. The phenotypic characteristics of the phage were determined using a one-step growth curve, restriction digestion profile, pH, and temperature stability tests. The results revealed that the phage is stable through a wide range of temperatures (20-50°C) and maintains its plaque-forming ability at pH ranging from 6 to 12. The one-step growth curve showed a latent period falling between 40 and 60 min, while burst size ranged from 23 to 30 plaque-forming units/10 µl at the same host strain. The restriction digestion pattern using EcoRI, SalI, HindIII, and XhoI enzymes showed that HindIII could cut the phage genome. The phage DNA could not be restricted by the other three enzymes. The findings of this study can be used in future studies to determine phage-host interactions.

Nanoparticles Influence Lytic Phage T4-like Performance In Vitro.

Little is known about interactions of non-filamentous, complex-structured lytic phages and free, non-ordered nanoparticles. Emerging questions about their possible bio-sanitization co-applications or predictions of possible contact effects in the environment require testing. Therefore, we revealed the influence of various nanoparticles (NPs; SiO2, TiO2-SiO2, TiO2, Fe3O4, Fe3O4-SiO2 and SiO2-Fe3O4-TiO2) on a T4-like phage. In great detail, we investigated phage plaque-forming ability, phage lytic performance, phage progeny burst times and titers by the eclipse phase determinations. Additionally, it was proved that TEM micrographs and results of NP zeta potentials (ZP) were crucial to explain the obtained microbiological data. We propose that the mere presence of the nanoparticle charge is not sufficient for the phage to attach specifically to the NPs, consequently influencing the phage performance. The zeta potential values in the NPs are of the greatest influence. The threshold values were established at ZP < −35 (mV) for phage tail binding, and ZP > 35 (mV) for phage head binding. When NPs do not meet these requirements, phage–nanoparticle physical interaction becomes nonspecific. We also showed that NPs altered the phage lytic activity, regardless of the used NP concentration. Most of the tested nanoparticles positively influenced the phage lytic performance, except for SiO2 and Fe3O4-SiO2, with a ZP lower than −35 (mV), binding with the phage infective part—the tail.

Effects of HA2 154 deglycosylation and NA V202I mutation on biological property of H5N6 subtype avian influenza virus

In recent years, clade 2.3.4.4 H5N6 subtype avian influenza virus (AIV) has been predominantly prevalent in poultry flocks in China. During our AIV surveillance in 2018–2019, 6 circulating strains of H5N6 that possess the natural loss of glycosylation site 154 due to N154D mutation in HA2 protein were isolated. In particular, 5 strains simultaneously carried the V202I mutation in NA protein. Based on the paired backbone H5N6 viruses Y6 and RY6, which just diverged in the glycosylation status at site 158 in HA1 protein, 8 reassortants of rY6-154 N/202 V, rY6-154D/202 V, rY6-154 N/202I and rY6-154D/202I plus rRY6-154 N/202 V, rRY6-154D/202 V, rRY6-154 N/202I and rRY6-154D/202I were constructed with different variation patterns at site 154 in HA2 and site 202 in NA. By determining those reassortants in growth performance on cells, plaque-forming ability, heat and low pH stability, and pathogenicity in mammals, the results showed that HA2 N154D and NA V202I could singly or jointly change the viral biological properties both in vitro and in vivo. Additionally, the effect of HA mutation was significantly more robust than that of NA, and the resulting increasing or reducing impact was closely related to the glycosylation at HA1 site 158. The present study provided a reference for further parsing the relevant mechanism of the functional match between HA and NA proteins of the influenza virus.

Biosynthesis of Calipyridone A Represents a Fungal 2-Pyridone Formation without Ring Expansion in Aspergillus californicus.

A chemical investigation of the filamentous fungus Aspergillus californicus led to the isolation of a polyketide-nonribosomal peptide hybrid, calipyridone A (1). A putative biosynthetic gene cluster cpd for production of 1 was next identified by genome mining. The role of the cpd cluster in the production of 1 was confirmed by multiple gene deletion experiments in the host strain as well as by heterologous expression of the hybrid gene cpdA inAspergillus oryzae. Moreover, chemical analyses of the mutant strains allowed the biosynthesis of 1 to be elucidated. The results indicate that the generation of the 2-pyridone moiety of 1 via nucleophilic attack of the iminol nitrogen to the carbonyl carbon is different from the biosynthesis of other fungal 2-pyridone products through P450-catalyzed tetramic acid ring expansions. In addition, two biogenetic intermediates, calipyridones B and C, showed modest inhibition effects on the plaque-forming ability of SARS-CoV-2.

Deciphering the Role of Holin in Mycobacteriophage D29 Physiology.

In the era of antibiotic resistance, phage therapy is gaining attention for the treatment of pathogenic organisms such as Mycobacterium tuberculosis. The selection of phages for therapeutic purposes depends upon several factors such as the host range that a phage can infect, which can be narrow or broad, time required for the host cell lysis, and the burst size. Mycobacteriophage D29 is a virulent phage that has the ability to infect and kill several slow- and fast-growing mycobacterial species including the pathogenic M. tuberculosis. It, therefore, has the potential to be used in phage therapy against M. tuberculosis. D29 lytic cassette encodes three proteins viz. peptidoglycan hydrolase (LysA), mycolylarabinogalactan esterase (LysB), and holin, which together ensure host cell lysis in a timely manner. In this work, we have scrutinized the importance of holin in mycobacteriophage D29 physiology. Bacteriophage Recombineering of Electroporated DNA (BRED) approach was used to generate D29 holin knockout (D29Δgp11), which was further confirmed by the Deletion amplification detection assay (DADA)-PCR. Our results show that D29Δgp11 is viable and retains plaque-forming ability, although with reduced plaque size. Additionally, the host cell lysis governed by the mutant phage is significantly delayed as compared to the wild-type D29. In the absence of holin, D29 shows increased latent period and reduced burst size. Thus, our experiments show that while holin is dispensable for phage viability, it is essential for the optimal phage-mediated host cell lysis and phage propagation, which further points to the significance of the “clock” function of holin. Taken together, we show the importance of holin in governing timely and efficient host cell lysis for efficient progeny phage release, which further dictates its critical role in phage biology.

Development of high-growth influenza H7N9 prepandemic candidate vaccine viruses in suspension MDCK cells

BackgroundInfluenza vaccine manufacturers traditionally use egg-derived candidate vaccine viruses (CVVs) to produce high-yield influenza viruses for seasonal or pandemic vaccines; however, these egg-derived CVVs need an adaptation process for the virus to grow in mammalian cells. The low yields of cell-based manufacturing systems using egg-derived CVVs remain an unsolved issue. This study aimed to develop high-growth cell-derived CVVs for MDCK cell-based vaccine manufacturing platforms.MethodsFour H7N9 CVVs were generated in characterized Vero and adherent MDCK (aMDCK) cells. Furthermore, reassortant viruses were amplified in adherent MDCK (aMDCK) cells with certification, and their growth characteristics were detected in aMDCK cells and new suspension MDCK (sMDCK) cells. Finally, the plaque-forming ability, biosafety, and immunogenicity of H7N9 reassortant viruses were evaluated.ResultsThe HA titers of these CVVs produced in proprietary suspension MDCK (sMDCK) cells and chicken embryos were 2- to 8-fold higher than those in aMDCK cells. All H7N9 CVVs showed attenuated characteristics by trypsin-dependent plaque assay and chicken embryo lethality test. The alum-adjuvanted NHRI-RG5 (derived from the fifth wave H7N9 virus A/Guangdong/SP440/2017) vaccine had the highest immunogenicity and cross-reactivity among the four H7N9 CVVs. Finally, we found that AddaVax adjuvant improved the cross-reactivity of low pathogenic H7N9 virus against highly pathogenic H7N9 viruses.ConclusionsOur study indicates that cell-derived H7N9 CVVs possessed high growth rate in new sMDCK cells and low pathogenicity in chicken embryo, and that CVVs generated by this platform are also suitable for both cell- and egg-based prepandemic vaccine production.

Redox-tuning of oxidizing disulfide oxidoreductase generates a potent disulfide isomerase

Oxidative folding of extracellular proteins is pivotal for the biogenesis of bacterial virulence factors. Escherichia coli DsbA catalyzes disulfide bond formation in extracellular proteins and in multicomponent architectures on the cell surface. The present study assessed the significance of the redox properties of DsbA by exploiting the plaque-forming ability of bacteriophage M13, which specifically recognizes F-pili during infection of the host cell. A library of mutant dsbA genes was constructed by randomizing the dipeptide XX sequence in the active-site redox motif CXXC and then screened for mutants that altered plaque yield and appearance. In total, 24 dsbA mutant alleles produced substantially different degrees of complementation, and one mutant dsbA gene that encodes a CDIC sequence produced over 40-fold more clear plaques than wild type dsbA. The redox potential of purified DsbA [CDIC] was −172 mV, representing a less-oxidizing catalysis than the wild type DsbA (−122 mV), but one that is closer to yeast protein disulfide isomerase (−175 mV). DsbA [CDIC] exhibited a greater ability to refold fully denatured glutathionylated ribonuclease A than the wild type enzyme and a DsbA [CRIC] mutant, which has the same redox potential of −172 mV. Homology modeling and molecular dynamics simulation suggest that the CDIC mutant may have an enlarged substrate-binding cleft near the redox center, which confers kinetic advantages when acting on protein substrates.

Enhanced intracellular survival and epithelial cell adherence abilities of Burkholderia pseudomallei morphotypes are dependent on differential expression of virulence-associated proteins during mid-logarithmic growth phase.

Comparative analyses were performed on the ability of wild-type and small colony variant of B. pseudomallei to adhere, invade and form plaques on human epithelial cells. In addition, proteomic analysis of these different colony morphotypes was also carried out. This research provides insights into the virulence and pathogenesis attributes of B. pseudomallei and contributes to better understand the pathogenesis of melioidosis.

Surfactant-modified nanoclay exhibits an antiviral activity with high potency and broad spectrum.

Nanomaterials have the characteristics associated with high surface-to-volume ratios and have been explored for their antiviral activity. Despite some success, cytotoxicity has been an issue in nanomaterial-based antiviral strategies. We previously developed a novel method to fully exfoliate montmorillonite clay to generate the most fundamental units of nanoscale silicate platelet (NSP). We further modified NSP by capping with various surfactants and found that the surfactant-modified NSP (NSQ) was less cytotoxic. In this study, we tested the antiviral potentials of a series of natural-clay-derived nanomaterials. Among the derivatives, NSP modified with anionic sodium dodecyl sulfate (NSQc), but not the pristine clay, unmodified NSP, a silver nanoparticle-NSP hybrid, NSP modified with cationic n-octadecanylamine hydrochloride salt, or NSP modified with nonionic Triton X-100, significantly suppressed the plaque-forming ability of Japanese encephalitis virus (JEV) at noncytotoxic concentrations. NSQc also blocked infection with dengue virus (DEN) and influenza A virus. Regarding the antiviral mechanism, NSQc interfered with viral binding through electrostatic interaction, since its antiviral activity can be neutralized by Polybrene, a cationic polymer. Furthermore, NSQc reduced the lethality of JEV and DEN infection in mouse challenge models. Thus, the surfactant-modified exfoliated nanoclay NSQc may be a novel nanomaterial with broad and potent antiviral activity. Nanomaterials have being investigated as antimicrobial agents, yet their antiviral potential is overshadowed by their cytotoxicity. By using a novel method, we fully exfoliated montmorillonite clay to generate the most fundamental units of nanoscale silicate platelet (NSP). Here, we show that the surfactant-modified NSP (NSQ) is less cytotoxic and that NSQc (NSP modified with sodium dodecyl sulfate) could potently block infection by dengue virus (DEN), Japanese encephalitis virus (JEV), and influenza A virus at noncytotoxic concentrations. For the antiviral mechanism, we find that the electrostatic interaction between the negatively charged NSQc and the positively charged virus particles blocks viral binding. Furthermore, we used mouse challenge models of JEV and DEN to demonstrate the in vivo antiviral potential of NSQc. Thus, NSQc may function as a potent and safe antiviral nanohybrid against several viruses, and our success in synthesizing surfactant-modified NSP with antiviral activity may shed some light on future antiviral development.

Human cytomegalovirus immediate-early gene expression is restricted by the nuclear domain 10 component Sp100

Nuclear domains 10 (ND10s) are discrete subnuclear structures that contain the three major protein components promyelocytic leukaemia protein (PML), hDaxx and Sp100. Previous studies identified the ND10-components PML and hDaxx as cellular restriction factors that independently counteract human cytomegalovirus (HCMV) infection via the repression of viral immediate-early (IE) gene expression. Consequently, we asked whether Sp100 is likewise involved in this repressive activity. Infection of Sp100 knockdown (kd) cells with HCMV resulted in a significantly increased plaque-forming ability. In addition, ablation of Sp100 led to a considerable increase in the number of IE1-expressing cells, indicating that Sp100 suppresses the initiation of viral gene expression. Next, double-kd cells, lacking either Sp100/hDaxx or Sp100/PML, were generated. Here, infection resulted in an additional enhancement in HCMV replication efficacy compared with the single-kd cells. Thus, our results further strengthen the concept that the three major ND10-components independently contribute to the cellular restriction of HCMV replication.

Study of the relationship between bacteriophage and selective host cells according to different conditions of UV-C irradiation

We investigate the potential for reactivation to restore infectivity to UV-damaged bacteriophage 2MPØ isolated from wastewater. The virus belongs to the Myoviridae family of the tailed phages. This Phage was chosen for detailed analysis, and was inspected by electron microscopy, host range determination. In addition, we explore the dynamics of bacteriophage host interactions in different conditions of UV-C irradiation. The inactivation kinetic or dose-response relationship was described by the method of plaque-forming ability. The UV-inactivation kinetic was represented in the exponential curve according to the model of Chick–Watson and Series-event kinetic model. In a 99.99% inactivation of 2MPØ bacteriophage which corresponded to 720 mW s/cm2 of UV-dose, high correlation was observed between the loss of infectivity and the increase of UV-C dose. The restore of infectivity of UVexposed bacteriophage was also determined under conditions which either activated or repressed the light-dependent photolyase enzyme in host cells, in order to examine the damage-dependent response of this bacterial repair system. The results of reactivation indicated that light-dependent repair (photoreactivation) was much more efficient than dark repair in restoring infectivity to bacteriophage damaged by UV-C radiation.

Screening for and characterization of Lactococcus lactis bacteriophages with high thermal resistance

Fifty-six Lactococcus lactis phage isolates collected from different German dairies and obtained from a starter culture manufacturer were tested for their heat resistance. About 40% of these isolates resisted treatment at 80 °C for 5 min when they were heated in milk. The most resistant phage isolate, P1532, was collected from sour cream. Plaque-formation was still detectable even after heating at 97 °C for 5 min. The second heat-resistant one, P680, showed some plaque-forming ability after heating at 95 °C for 5 min. Kinetic parameters for the thermal inactivation of these two resistant phages were determined for temperatures ranging from 70 to 97 °C. The inactivation of phage P1532 in skim milk and in buffer medium were found to follow first-order kinetics and did not exhibit tailing, whereas in the inactivation curves of phage P680 tailing was observed. The D-value of P1532 at pasteurization temperature of 72 °C was calculated as 112 min.

Evaluation of chicken-origin (DF-1) and quail-origin (QT-6) fibroblast cell lines for replication of avian influenza viruses

Avian influenza viruses (AIVs) are isolated routinely and propagated in specific pathogen free embryonated chicken eggs (ECE) and mammalian origin Madin-Darby Canine Kidney (MDCK) cell line. Continuous avian cell lines offer advantages for propagation of AIVs over MDCK cells because they maintain species specificity, and lower recurring costs compared to ECE. In this study, the characteristics of two avian fibroblast cell lines were evaluated, DF-1 (chicken-origin) and QT-6 (quail-origin), and their ability to support the growth of AIVs ( n = 19) belonging to nine different hemagglutinin subtypes from a variety of avian species. The replication efficiency of the AIVs in QT-6 and DF-1 cells was comparable to those in primary chicken embryo fibroblast (CEF) and MDCK cells. Receptor distribution analysis demonstrated high prevalence of SA α2,3-gal linked receptors in QT-6 and DF-1 cells which support a high growth of AIVs in these cell lines. Furthermore, the QT-6 and DF-1 cells supported high plaque-forming ability of representative highly pathogenic Eurasian H5N1 and H7N1 subtype AIVs. These two avian cell lines, especially QT-6 cells, also showed high transfection efficiency and could be useful for reverse genetics based rescue of AIVs. This study indicates that the DF-1 and QT-6 cell lines may be useful as a substitute for primary CEF and MDCK cells for AIV research in the areas of in vitro host range, molecular pathobiology and molecular genetics.

Isolation and characterization of a novelEnterococcus faecalisbacteriophage φEF24C as a therapeutic candidate

Vancomycin-resistant Enterococcus faecalis (VRE) has become a significant threat in nosocomial settings. Bacteriophage (phage) therapy is frequently proposed as a potential alternative therapy for infections caused by this bacterium. To search for candidate therapeutic phages against Enterococcus faecalis infections, 30 Enterococcus faecalis phages were isolated from the environment. One of these, virulent phage phiEF24C, which has a broad host range, was selected for analysis. The plaque-forming ability of phiEF24C was virtually unaffected by differences in the clinical host strains. Furthermore, the phage had a shorter latent period and a larger burst size than ordinary tailed phages, indicating that phiEF24C has effective lytic activity against many Enterococcus faecalis strains, including VRE. Morphological and genomic analyses revealed that phiEF24C is a large myovirus (classified as family Myoviridae morphotype A1) with a linear double-stranded DNA genome of c. 143 kbp. Analyses of the N-terminal amino acid sequences of the virion proteins, together with the morphology and the genome size, speculated that phiEF24C is closely related to other myoviruses of Gram-positive bacteria that have been used experimentally or practically for therapy or prophylaxis. Considering these results, phiEF24C may be a potential candidate therapeutic phage against Enterococcus faecalis infections.

Coliphage N4 N-Acetylmuramidase Defines a New Family of Murein Hydrolases

Escherichia coli phage N4 infection leads to delayed host cell lysis, 3000 particles per infected bacterium and a small plaque phenotype. We show that bacteriophage N4 encodes a murein hydrolase (gp61) that is essential for N4 plaque-forming ability. gp61 has a high level of sequence similarity to hypothetical proteobacterial proteins, and Vibrio harveyi phage VHML ORF 19. Nano-electrospray ionization (nESI) quadrupole ion trap (QIT) mass spectrometry (MS) analysis of muropeptides from purified gp61 digestion of E. coli peptidoglycan indicates that gp61 is an N-acetylmuramidase. The EGGY motif present near the N terminus of gp61 and its homologs contains the glutamic acid residue essential for enzymatic activity. These results provide evidence that N4 gp61 and its homologs define a new family of N-acetylmuramidases (pfam05838.4, DUF847, COG3926). In contrast to its homologs, gp61 contains an N-terminal signal sequence. When expressed at levels present during phage infection, gp61 localizes primarily to the cell inner membrane; in contrast, over-expression of recombinant N4 gp61 is sufficient for rapid cell lysis. Overproduction of the recombinant Salmonella typhimurium (STM0016) homolog is sufficient for cell lysis only when fused to the gp61 N-terminal signal sequence. The results of subcellular localization and of mutagenesis of the gp61 N-terminal signal sequence indicate that gp61 must be released from the inner membrane to be catalytically active.

Accumulation and Fate of Microorganisms and Microspheres in Biofilms Formed in a Pilot-Scale Water Distribution System

The accumulation and fate of model microbial "pathogens" within a drinking-water distribution system was investigated in naturally grown biofilms formed in a novel pilot-scale water distribution system provided with chlorinated and UV-treated water. Biofilms were exposed to 1-mum hydrophilic and hydrophobic microspheres, Salmonella bacteriophages 28B, and Legionella pneumophila bacteria, and their fate was monitored over a 38-day period. The accumulation of model pathogens was generally independent of the biofilm cell density and was shown to be dependent on particle surface properties, where hydrophilic spheres accumulated to a larger extent than hydrophobic ones. A higher accumulation of culturable legionellae was measured in the chlorinated system compared to the UV-treated system with increasing residence time. The fate of spheres and fluorescence in situ hybridization-positive legionellae was similar and independent of the primary disinfectant applied and water residence time. The more rapid loss of culturable legionellae compared to the fluorescence in situ hybridization-positive legionellae was attributed to a loss in culturability rather than physical desorption. Loss of bacteriophage 28B plaque-forming ability together with erosion may have affected their fate within biofilms in the pilot-scale distribution system. The current study has demonstrated that desorption was one of the primary mechanisms affecting the loss of microspheres, legionellae, and bacteriophage from biofilms within a pilot-scale distribution system as well as disinfection and biological grazing. In general, two primary disinfection regimens (chlorination and UV treatment) were not shown to have a measurable impact on the accumulation and fate of model microbial pathogens within a water distribution system.

Existence of transdominant and potentiating mutants of UL9, the herpes simplex virus type 1 origin-binding protein, suggests that levels of UL9 protein may be regulated during infection.

UL9 is a multifunctional protein required for herpes simplex virus type 1 (HSV-1) replication in vivo. UL9 is a member of the superfamily II helicases and exhibits helicase and origin-binding activities. We have previously shown that mutations in the conserved helicase motifs of UL9 can have either a transdominant or potentiating effect on the plaque-forming ability of infectious DNA from wild-type virus (A. J. Malik and S. K. Weller, J. Virol. 70:7859-7866, 1996). In this paper, the mechanisms of transdominance and potentiation are explored. We show that the motif V mutant protein containing a G to A substitution at residue 354 is unstable when expressed by transfection and is either processed to a 38-kDa N-terminal fragment or degraded completely. The overexpression of the MV mutant protein is able to influence the steady-state protein levels of wild-type UL9 and to override the inhibitory effects of wild-type UL9. Potentiation correlates with the ability of the UL9 variants containing the G354A mutation to be processed or degraded to the 38-kDa form. We propose that the MV mutant protein is able to interact with full-length UL9 and that this interaction results in a decrease in the steady-state levels of UL9, which in turn leads to enhanced viral infection. Furthermore, we demonstrate that inhibition of HSV-1 infection can be obtained by overexpression of full-length UL9, the C-terminal third of the protein containing the origin-binding domain, or the N-terminal two-thirds of UL9 containing the conserved helicase motifs and the putative dimerization domain. Our results suggest that transdominance can be mediated by overexpression, origin-binding activity, and dimerization, whereas potentiation is most likely caused by the ability of the UL9 MV mutant to influence the steady-state levels of wild-type UL9. Taken together, the results presented in this paper suggest that the regulation of steady-state levels of UL9 may play an important role in controlling viral infection.

Isolation of a peptide inhibitor of human rhinovirus

Cell culture-based transdominant genetic techniques provide new methods for discovering peptide/RNA modulators of cellular pathways. We applied this technology to isolate a peptide inhibitor of human rhinovirus. A green fluorescent protein (GFP)-scaffolded library of cDNA fragments was expressed in HeLa cells from a retroviral vector and screened for inhibitors of rhinovirus-mediated cell killing. A DNA clone, I421, increased cell survival in an HRV14 challenge assay from less than 0.5% to greater than 60%. It encodes a 53-amino-acid C-terminal extension of the GFP scaffold. Particular subclones of Hela cells expressing I421 (exemplified by I421dp3) show a delay in virus production and a 50-fold decrease in viral RNA levels at 6–8 h postinfection. HRV2, HRV14, and HRV16 show a dramatic decrease in plaque-forming ability on I421dp3 while Coxsackievirus B3 showed a small reduction. Levels of ICAM-1, the receptor for the main rhinovirus serotype, are not altered in I421dp3.