Plaquing Efficiency Research Articles

An explosive antisense RNA strategy for inhibition of a lactococcal bacteriophage.

The coding regions of six putative open reading frames (ORFs) identified near the phage phi31 late promoter and the right cohesive end (cos) of lactococcal bacteriophage phi31 were used to develop antisense constructs to inhibit the proliferation of phage phi31. Two middle-expressed ORFs (ORF 1 and ORF 2) and four late-expressed ORFs (ORF 3 through ORF 6) were cloned individually between the strong Lactobacillus P6 promoter and the T7 terminator (T(T7)) to yield a series of antisense RNA transcripts. When expressed on a high-copy-number vector from a strong promoter, the constructs had no effect on the efficiency of plaquing (EOP) or the plaque size of phage phi31. To increase the ratio of antisense RNA to the targeted sense mRNA appearing during a phage infection, the antisense cassettes containing the late-expressed ORFs (ORF 3 through ORF 6) were subcloned to pTRK360, a low-copy-number vector containing the phage phi31 origin of replication, ori31. ori31 allows for explosive amplification of the low-copy-number vector upon phage infection, thereby increasing levels of antisense RNA transcripts later in the lytic cycle. In addition, the presence of ori31 also lowers the burst size of phage phi31 fourfold, resulting in fewer sense, target mRNAs being expressed from the phage genome. The combination of ori31 and P6::anti-ORF 4H::T(T7) resulted in a threefold decrease in the EOP of phage phi31 (EOP = 0.11 +/- 0.03 [mean +/- standard deviation]) compared to the presence of ori31 alone (EOP = 0.36). One-step growth curves showed that expression of anti-ORF 4H RNA decreased the percentage of successful centers of infection (75 to 80% for ori31 compared to 35 to 45% for ori31 plus anti-ORF 4H), with no further reduction in burst size. Growth curves performed in the presence of varying levels of phage phi31 showed that ori31 plus anti-ORF 4H offered significant protection to Lactococcus lactis, even at multiplicities of infection of 0.01 and 0.1. These results illustrate a successful application of an antisense strategy to inhibit phage replication in the wake of recent unsuccessful reports.

Improvement of plasmid encoded lactococcal bacteriophage resistance by mutator strain Epicurian coli XL1-Red

A random mutation strategy using mutator strain, Epicurian coli XL1-Red, was applied to a plasmid, pND018, constructed by inserting a Lactococcus lacis bacteriophage resistance gene (abiI) into a L. lactis/E. coli shuttle vector (pDL278), to introduce random mutations throughout the plasmid. Following transformation of the mutated plasmid library to a plasmid free and phage sensitive strain of L. lactis (LM0230), mutated plasmids were screened by cross-streaking and efficiency of plaquing (EOP) assays. Two strains with enhanced resistance were obtained, as well as several phage sensitive strains. Repeated transformation of the mutated plasmids to LM0230 confirmed that the observed phenotypes were caused by mutations located on the plasmids. The EOP values and plaque morphology of two enhanced phage resistance mutants were characterized at 30 °C and 37 °C. These results indicate that this simple procedure can be applied to generate modified plasmids with improved phage resistance, which may be of commercial value.

Performance of Lactobacillus helveticus Spontaneous Phage-resistant Mutants in Hard Cheese Production

Abstract From the strain Lactobacillus helveticus ATCC 15807, a total of 66 clones were isolated using the lytic phages hv and ATCC 15807-B1. Phenotypic parameters related to their phage resistance capacity (efficiency of plaquing, phage resistance stability, lysogeny and adsorption rates) were determined. The morphological, biochemical (sugar fermentation patterns) and technological (acidifying power, proteolytic activity and slime production) characteristics of the isolates were also studied. Phage resistance stability was a variable property among the isolates but a high level of resistance was exhibited as quantified by the efficiency of plaquing. Furthermore, a total absence of acquired lysogeny was demonstrated. The phage-resistant variants were completely or partially unable to bind phages and did not show differences with Lb. helveticus ATCC 15807 in their biochemical characteristics. However, the technological properties (acidifying powers and proteolytic activities) were heterogeneously distributed among the mutants. The variant RB1-28 (isolated under selective pressure of the phage ATCC 15807-B1) was evaluated for the technological performance in Sardo cheese production in comparison with Lb. helveticus ATCC 15807. There were no significant differences between the two types of cheeses, taking in consideration physicochemical parameters (proteolysis level, dry matter, total and soluble proteins, moisture), microbiological counts or sensory analysis. These phage-resistant variants could be employed in starter strain rotation programs for cheesemaking.

AbiQ, an abortive infection mechanism from Lactococcus lactis.

Lactococcus lactis W-37 is highly resistant to phage infection. The cryptic plasmids from this strain were coelectroporated, along with the shuttle vector pSA3, into the plasmid-free host L. lactis LM0230. In addition to pSA3, erythromycin- and phage-resistant isolates carried pSRQ900, an 11-kb plasmid from L. lactis W-37. This plasmid made the host bacteria highly resistant (efficiency of plaquing <10(-8)) to c2- and 936-like phages. pSRQ900 did not confer any resistance to phages of the P335 species. Adsorption, cell survival, and endonucleolytic activity assays showed that pSRQ900 encodes an abortive infection mechanism. The phage resistance mechanism is limited to a 2.2-kb EcoRV/BclI fragment. Sequence analysis of this fragment revealed a complete open reading frame (abiQ), which encodes a putative protein of 183 amino acids. A frameshift mutation within abiQ completely abolished the resistant phenotype. The predicted peptide has a high content of positively charged residues (pI = 10.5) and is, in all likelihood, a cytosolic protein. AbiQ has no homology to known or deduced proteins in the databases. DNA replication assays showed that phage c21 (c2-like) and phage p2 (936-like) can still replicate in cells harboring AbiQ. However, phage DNA accumulated in its concatenated form in the infected AbiQ+ cells, whereas the AbiQ- cells contained processed (mature) phage DNA in addition to the concatenated form. The production of the major capsid protein of phage c21 was not hindered in the cells harboring AbiQ.

Characterization of Lactobacillus helveticus phage resistant mutants by RAPD fingerprints and phenotypic parameters

Spontaneous phage resistant mutants of Lactobacillus helveticus ATCC 15807 were isolated using the lytic phages hv and ATCC 15807-B1. Their genetic diversity (RAPD fingerprints and DNA extrachromosomal content) and phenotypic parameters related to their phage resistance capacity (efficiency of plaquing, phage resistance stability, lysogeny and adsorption rates) were investigated to characterize them and to identify potential genetic differences with the parent strain. Phage resistance stability turned out to be a variable property among the isolates, but a high level of resistance was exhibited by them as quantified by the efficiency of plaquing. Furthermore, a total absence of acquired lysogeny was demonstrated. The mutants were completely or partially unable to bind phages, showing the existence of a widespread resistance mechanism based on adsorption interference. No substantial differences in plasmid profiles were observed between the phage resistant variants and the sensitive strain. The RAPD fingerprints obtained with three arbitrary primers gave a cluster structure in which all clones appeared to be almost identical (similarity coefficient >75%) to the wild strain. RAPD appears as a useful tool to rapidly confirm the kindred between parent strains and their phage resistant derivatives.

Evidence of a Restriction/Modification system in lactobacillus delbrueckii subsp. lactis CNRZ 326

Lactobacillus delbrueckii subsp. lactis (Lb. lactis) CNRZ 326 is widely used in the propagation of Lb. delbrueckii bacteriophages. In this study, evidence is presented that this strain possesses a restriction-modification (R/M) system. The mitomycin C-induced temperate bacteriophage lb539 has a reduced efficiency of plaquing (EOP) on CNRZ 326 cells (EOP = 10(-3)), but after several passages on this strain, or on the indicator strain Lb. lactis LKT, the recovered phages (phages lb539.326 and lb539.LKT) have an EOP equal to 1. Restrictive development on CNRZ 326 was also observed after phage lb539.326 was propagated on the strain Lb. lactis CRL 934. The R/M system was also active against the virulent Lb. delbrueckii phage ll-h. Plasmid DNA was not detected in CNRZ 326, which suggests that the R/M system described is chromosomally encoded.

The Effect of Herpes Simplex Virus Type 1 L-Particles on Virus Entry, Replication, and the Infectivity of Naked Herpesvirus DNA

Herpes simplex virus type 1(HSV-1) L-particles are known to be composed mainly of envelope and tegument proteins, to lack the nucleocapsid, and to be noninfectious. Thus L-particles represent interesting vaccine candidates. L-particles at >1000/cell interfered with HSV-1 virion adsorption and penetration. While L-particles did not affect HSV-1 growth kinetics in resting or nonresting BHK cultures infected with purified virions, treatment with L-particles before, or after, transfection with HSV-1 DNA resulted in a progressive increase in plaque numbers (five- to sixfold at 1000 L-particles/cell). Transfection assays using HSV-1tsmutant DNA (ts1201) revealed that enhancement was due to induction of otherwise nonreplicating genomes. The enhancement obtained with L-particles produced by WT HSV-1 or by mutants that are either deleted, or defective, in certain gene products was compared. Most important were the Vmw110 (ICP0) and Vmw65 (α-TIF) proteins, but VP11/12, VP13/14, and vhs also have a role. The L-particle-associated Vmw175 (ICP 4) protein did not appear be involved. The effect of homologous and heterologous combinations of pseudorabies virus, equineherpesvirus-1, and HSV-1 DNA's and L-particles was investigated in transfection assays. The L-particles of each virus, to varying extent, enhanced the plaquing efficiency of their own DNA but were also effective in heterologous combinations.

Bacteriophage-triggered defense systems: phage adaptation and design improvements.

A novel bacteriophage defense system, based on an inducible suicide gene, was challenged with a lactococcal bacteriophage to investigate the potential for phage adaptation. The defense system was encoded by pTRK414H, a high-copy-number replicon encoding a tightly regulated phi 31p trigger promoter fused to the lethal LlaIR+ restriction endonuclease cassette. Repeated transfers of Lactococcus lactis NCK690(pTRK414H) in the presence of phi 31 selected for phage phi 31 derivatives which were markedly less sensitive to phi 31p-LlaIR(+)-encoded restriction than the parental phage, phi 31. The efficiency of plaquing (EOP) on L. lactis NCK690(pTRK414H) was 10(-4) for phi 31 versus 0.4 for the derived phages. The mutant phages remained fully sensitive to LlaIR+ restriction, suggesting an alteration in the recognition or firing of the phi 31p promoter. Sequencing over the promoter region in four mutant phages revealed the identical C-to-A transversion, generating a Phe-to-Leu substitution, in a transcriptional activator of the phi 31p promoter, designated ORF2. The mutant phages were analyzed for their ability to induce the native phi 31p promoter element fused to a lacZst reporter gene. Compared to the parental phage, phi 31, lower levels of beta-galactosidase activity were induced throughout the lytic cycle, indicating that the strength at which the mutant phages activated the phi 31p promoter was altered. Based on these observations, improvements were made in promoter strength and restriction activity in an attempt to elevate the effectiveness of the phage-triggered suicide system. When the phi 31p-LlaIR+ cassette was paired with other abortive defense systems, Per31 and AbiA, the EOP of phi 31 was reduced to < 10(-10) and the level of phage in the culture was lowered below the detection limits of the assay.

A triggered-suicide system designed as a defense against bacteriophages.

A novel bacteriophage protection system for Lactococcus lactis based on a genetic trap, in which a strictly phage-inducible promoter isolated from the lytic phage phi31 is used to activate a bacterial suicide system after infection, was developed. The lethal gene of the suicide system consists of the three-gene restriction cassette LlaIR+, which is lethal across a wide range of gram-positive bacteria. The phage-inducible trigger promoter (phi31P) and the LlaIR+ restriction cassette were cloned in Escherichia coli on a high-copy-number replicon to generate pTRK414H. Restriction activity was not apparent in E. coli or L. lactis prior to phage infection. In phage challenges of L. lactis(pTRK414H) with phi31, the efficiency of plaquing was lowered to 10(-4) and accompanied by a fourfold reduction in burst size. Center-of-infection assays revealed that only 15% of infected cells released progeny phage. In addition to phage phi31, the phi31P/LlaIR+ suicide cassette also inhibited four phi31-derived recombinant phages at levels at least 10-fold greater than that of phi31. The phi31P/LlaIR+-based suicide system is a genetically engineered form of abortive infection that traps and eliminates phages potentially evolving in fermentation environments by destroying the phage genome and killing the propagation host. This type of phage-triggered suicide system could be designed for any bacterium-phage combination, given a universal lethal gene and an inducible promoter which is triggered by the infecting bacteriophage.

Herpes simplex virus type 1 glycoprotein K is not essential for infectious virus production in actively replicating cells but is required for efficient envelopment and translocation of infectious virions from the cytoplasm to the extracellular space.

We characterized the glycoprotein K (gK)-null herpes simplex virus type 1 [HSV-1] (KOS) delta gK and compared it to the gK-null virus HSV-1 F-gKbeta (L. Hutchinson et al., J. Virol. 69:5401-5413, 1995). delta gK and F-gKbeta mutant viruses produced small plaques on Vero cell monolayers at 48 h postinfection. F-gKbeta caused extensive fusion of 143TK cells that was sensitive to melittin, a specific inhibitor of gK-induced cell fusion, while delta gK virus did not fuse 143TK cells. A recombinant plasmid containing the truncated gK gene specified by F-gKbeta failed to rescue the ICP27-null virus KOS (d27-1), while a plasmid with the delta gK deletion rescued the d27-1 virus efficiently. delta gK virus yield was approximately 100,000-fold lower in stationary cells than in actively replicating Vero cells. The plaquing efficiencies of delta gK and F-gKbeta virus stocks on VK302 cells were similar, while the plaquing efficiency of F-gKbeta virus stocks on Vero cells was reduced nearly 10,000-fold in comparison to that of delta gK virus. Mutant delta gK and F-gKbeta infectious virions accumulated within Vero and HEp-2 cells but failed to translocate to extracellular spaces. delta gK capsids accumulated in the nuclei of Vero but not HEp-2 cells. Enveloped delta gK virions were visualized in the cytoplasms of both Vero and HEp-2 cells, and viral capsids were found in the cytoplasm of HEp-2 cells within vesicles. Glycoproteins B, C, D, and H were expressed on the surface of delta gK-infected Vero cells in amounts similar to those for KOS-infected Vero cells. These results indicate that gK is involved in nucleocapsid envelopment, and more importantly in the translocation of infectious virions from the cytoplasm to the extracellular spaces, and that actively replicating cells can partially compensate for the envelopment but not for the cellular egress deficiency of the delta gK virus. Comparison of delta gK and F-gKbeta viruses suggests that the inefficient viral replication and plaquing efficiency of F-gKbeta virus in Vero cells and its syncytial phenotype in 143TK- cells are most likely due to expression of a truncated gK.

Ribotypes and virulence gene polymorphisms suggest three distinct Listeria monocytogenes lineages with differences in pathogenic potential.

A total of 133 Listeria monocytogenes isolates were characterized by ribotyping and allelic analysis of the virulence genes hly, actA, and inlA to uncover linkages between independent phylogenetic and specific virulence markers. PCR-restriction fragment length polymorphisms revealed 8 hly, 11 inl4, and 2 actA alleles. The combination of these virulence gene alleles and ribotype patterns separated L. monocytogenes into three distinct lineages. While distinct hly and inlA alleles were generally found to cluster into these three lineages, actA alleles segregated independently. These three phylogenetic lineages were confirmed when 22 partial actA DNA sequences were analyzed. The clinical history of the L. monocytogenes strains showed evidence for differences in pathogenic potential among the three lineages. Lineage I contains all strains isolated during epidemic outbreaks of listeriosis, while no human isolates were found in lineage III. Animal isolates were found in all three lineages. We found evidence that isolates from lineages I and III have a higher plaquing efficiency than lineage II strains in a cell culture assay. Strains from lineage III also seem to form larger plaques than strains from lineage II. A distinctive ribotype fragment and unique 16S rRNA gene sequences furthermore suggest that lineage III might represent a L. monocytogenes subspecies. None of the 20 human isolates available but 11% of our animal isolates were grouped in this lineage, indicating that strains in this lineage might have reduced virulence for humans.

Transcriptional targeting of herpes simplex virus for cell-specific replication.

Tissue- or cell-specific targeting of vectors is critical to the success of gene therapy. We describe a novel approach to virus-mediated gene therapy, where viral replication and associated cytotoxicity are limited to a specific cell type by the regulated expression of an essential immediate-early viral gene product. This is illustrated with a herpes simplex virus type 1 (HSV-1) vector (G92A) whose growth is restricted to albumin-expressing cells. G92A was constructed by inserting an albumin enhancer/promoter-ICP4 transgene into the thymidine kinase gene of mutant HSV-1 d120, deleted for both copies of the ICP4 gene. This vector also contains the Escherichia coli lacZ gene under control of the thymidine kinase promoter, a viral early promoter, to permit easy detection of infected cells containing replicating vector. In the adult, albumin is expressed uniquely in the liver and in hepatocellular carcinoma and is transcriptionally regulated. The plaquing efficiency of G92A is > 10(3) times higher on human hepatoma cells than on non-albumin-expressing human cells. The growth kinetics of G92A in albumin-expressing cells is delayed compared with that of wild-type HSV-1, likely due to aberrant expression of ICP4 protein. Cells undergoing a productive infection expressed detectable levels of ICP4 protein, as well as the reporter gene product beta-galactosidase. Confining a productive, cytotoxic viral infection to a specific cell type should be useful for tumor therapy and the ablation of specific cell types for the generation of animal models of disease.

Assessment of the Ability of Ratite-Origin Influenza Viruses to Infect and Produce Disease in Rheas and Chickens

Pathobiologic characteristics were determined for three mildly pathogenic (MP) ratite-origin avian influenza viruses (AIVs). Ratite-origin AIVs produced respiratory disease in rheas, and virus was reisolated from oropharyngeal and cloacal swabs on days 2-6 postinoculation. Inoculation of two ratite-origin AIVs in the upper respiratory tract of chickens resulted in viral infections, but the mean chicken infectious dose (CID50) for A/emu/Texas/39924/93 (H5N2) (Emu/Texas) virus was 500-fold lower than the CID50 for the A/rhea/North Carolina/39482/93 (H7N1) virus. In ovo and in vivo passage of the MP parent Emu/Texas isolate resulted in emergence of a highly pathogenic (HP) variant that had high plaquing efficiency in chicken embryo fibroblast cultures and was highly lethal in chicken pathotyping tests. This variant virus produced gross lesions in chickens similar to those reported for other HP AIVs. These findings demonstrated that ratite-origin AIVs can produce significant clinical disease in rheas and have a realistic potential for interspecies transmission to domestic poultry. Furthermore, HP variants can emerge from MP H5 ratite-origin AIVs if introduced and allowed to circulate in chicken populations.

Isolation and classification of temperature-sensitive mutants of influenza B virus.

We isolated 25 temperature-sensitive mutants of B/Kanagawa/73 strain generated by mutagenesis with 5-fluorouracil and classified them into seven recombination groups by pair-wise crosses. All mutants showed a ratio of plaquing efficiency at the nonpermissive temperature (37.5 C) to the permissive temperature (32 C) of 10(-4) or less. At 37.5 C most of group I, II, and III mutants did not produce appreciable amounts of protein, but all other group mutants were protein synthesis-positive. A group VII mutant produced active hemagglutinin (HA) and neuraminidase (NA) at the nonpermissive temperature, but Group V mutants produced only active NA and were defective in the HA molecule. The other group mutants, including group IV mutants with mutation only in the NA gene (8, 10), lacked both activities at the nonpermissive temperature. One of nine influenza B virus isolates in 1989 had EOP 37.5/32 of 1/3 x 10(-2) and belonged to recombination group VII.

Phenotypic Consequences of Altering the Copy Number of abiA , a Gene Responsible for Aborting Bacteriophage Infections in Lactococcus lactis

The abiA gene (formerly hsp) encodes an abortive phage infection mechanism which inhibits phage DNA replication. To analyze the effects of varying the abiA gene dosage on bacteriophage resistance in Lactococcus lactis, various genetic constructions were made. An IS946-based integration vector, pTRK75, was used to integrate a single copy of abiA into the chromosomes of two lactococcal strains, MG1363 and NCK203. In both strains, a single copy of abiA did not confer any significant phage resistance on the host except for one of the MG1363 integrants, NCK625, which exhibited a slightly higher level of resistance to phages sk1 and p2. Hybridization of the total cellular RNA from NCK625 to an abiA-specific probe indicated that the integration took place downstream of a promoter causing stronger expression of abiA in this integrant. Three abiA-containing plasmids of various copy numbers were introduced into both strains, and the recombinants were evaluated for resistance to phages c2, p2, sk1, and phi31. Plasmid pTRK18 has a copy number of approximately six (cn = 6) and caused a decreased plaque size for all phages evaluated. Integration of pTRK75 into a native plasmid of NCK203 generated pTRK362 (cn = 13), which caused a reduced efficiency of plaquing (EOP = 10) and reduced plaque size. A high-copy-number abiA plasmid (pTRK363), based on the pAMbeta1 origin of replication, was also constructed (cn = 100). Plasmid pTRK363 caused a significant reduction in EOP (10 to 10) and plaque size for all phages tested, although in some cases, this plasmid caused the evolution of AbiA-resistant phage derivatives. Altering the gene dosage or expression level of abiA significantly affects the phage resistance levels.

Mutations in the nsP1 Coding Sequence of Sindbis Virus Which Restrict Viral Replication in Secondary Cultures of Chick Embryo Fibroblasts Prepared from Aged Primary Cultures

SVMPA, a mutant of Sindbis virus (SV), which is able to replicate in Aedes albopictus cells treated with mycophenolic acid (MPA) or ribavirin, also has a host range phenotype. This phenotype is most clearly demonstrated by means of efficiency of plaquing (EOP) assays on secondary chick embryo fibroblasts (CEF) prepared from aged primary CEF. For example, in one experiment in which standard SV (SVSTD) had a relative EOP (EOP on primary CEF ÷ EOP on secondary CEF) of 1.6 the corresponding value for SVMPA was 2340. The host restriction of SVMPA was also seen with similarly prepared secondary cultures of duck embryo fibroblasts, but not with established lines of quail cells. The finding that the accumulation of viral RNA was much lower in SVMPA-infected secondary CEF than in SVSTD-infected CEF indicated that the replication of SVMPA in these cultures was blocked at an early step. Revertants of SVMPA were isolated which were no longer host-restricted but had retained their resistance to MPA. Of the three mutations [nucleotide (nt) 120, 127, and 963] in the nsP1 coding sequence of SVMPA, which lead to amino acid changes, these revertants had retained the nt 127 and nt 963 mutations but had lost the nt 120 mutation. This, along with earlier findings, indicated that only the nt 127 and nt 963 mutations are required for resistance to MPA. This result also associated the nt 120 mutation with the host restriction phenotype. In other experiments derivatives of pToto1101 (a plasmid from which infectious Sindbis virus RNA can be transcribed) were constructed by site-directed mutagenesis and used to test the effect of specific mutations on the viral phenotype. Although we were unable to obtain viable virus with the nt 120 mutation alone, virus with the nt 120 and nt 127 mutations was viable and host-restricted. We suggest that the nt 120 mutation by itself is lethal and that the nt 127 mutation suppresses the lethal effect of the nt 120 mutation. The SVMPA mutation at nt 120, which is associated with the host range phenotype, changes Gln21 of nsP1 to Lys. When a more conservative change was engineered, i.e., to Asn, the virus was not host-restricted. Although the reason for the restriction of SVMPA replication in secondary CEF is not known, some possible explanations are discussed.

Herpes Simplex Virus (HSV) Glycoproteins B and K Inhibit Cell Fusion Induced by HSV Syncytial Mutants

Herpes simplex virus type 1 (HSV-1) glycoproteins K and B (gK and gB) are intimately involved in virus-induced fusion of cells. Certain mutations in the UL27 (gB) and UL53 (gK) genes confer a syncytial (syn) phenotype characterized by extensive fusion of infected cells and giving rise to multinucleated cells. We have used HSV-1 syn mutants in conjunction with transfected cells or adenovirus vectors to overexpress wild-type gK or gB, in order to study the role of these proteins in virus-induced membrane fusion. Transient expression of wild-type gK inhibited fusion induced by HSV-1 encoding a mutant form of gK (syn gK) but not by viruses encoding a mutant form of gB (syn gB). Stably transformed cells expressing relatively high levels of gK suppressed cell fusion induced by HSV-1 mutants with lesions in the gK gene but not an HSV-1 with a syn mutation in the gB gene. In addition, there were marked reductions in the plaquing efficiencies and yields of HSV-1 on these cell lines. Cell fusion caused by HSV-1 syn20, a mutant encoding syn gK, was suppressed when cells were coinfected with an Ad vector, AdgK, which expresses wild-type gK. AdgK did not suppress fusion induced by HSV-1 KTTS-1, which expresses syn gB. Conversely, cells coinfected with AdgB, an Ad vector expressing a wild-type form of gB and HSV-1 KTTS.1 (syn gB) were not fused, whereas cells coinfected with AdgB and HSV-1 syn20 (syn gK) were fused. Expression of a number of other HSV-1 glycoproteins using Ad vectors did not reduce membrane fusion induced by syn gK or syn gB. Together, these results support models in which gK and gB participate directly in the fusion of HSV-infected cells. Mutant forms of gB and gK apparently disregulate the fusion process, whereas wild-type forms of gB and gK can act to suppress membrane fusion induced by their mutant counterparts.

Effect of Increasing the Copy Number of Bacteriophage Origins of Replication, in trans , on Incoming-Phage Proliferation

Bacteriophage resistance mechanisms which are derived from a bacteriophage genome are termed Per (phage-encoded resistance). When present in trans in Lactococcus lactis NCK203, Per50, the cloned origin of replication from phage phi50, interferes with phi50 replication. The per50 fragment was found to afford negligible protection to NCK203 against phi50 infection when present in a low-copy-number plasmid, pTRK325. A high-copy-number Per50 construct (pTRK323) dramatically affected phi50 infection, reducing the efficiency of plaquing (EOP) to 2.5 x 10 and the plaque size to pinhead proportions. This clone also afforded significant protection against other related small isometric phages. Per31 was cloned from phage phi31 and demonstrated to function as an origin of replication by enabling replication of per31-containing plasmids, in NCK203, on phi31 infection. A low-copy-number Per31 plasmid (pTRK360) reduced the EOP of phi31 on NCK203 to 0.3 and the plaque diameter from 1.5 to 0.5 mm. When this plasmid was cloned in high copy number, the EOP was further reduced to 7.2 x 10 but the plaques were large and contained Per31-resistant phages. Characterization of these "new" phages revealed at least two different types that were similar to phi31, except that DNA alterations were noted in the region containing the origin. This novel and powerful abortive phage resistance mechanism should prove useful when directed at specific, problematic phages.

Host RecJ is required for growth of P22 erf bacteriophage

Growth of bacteriophage P22 erf is known to require host RecA recombination function. We show that the RecA function is necessary but not sufficient to restore the plaque-forming ability of phage P22 erf; such mutant phage also requires host RecJ function. The residual efficiency of plaquing of P22 erf in a recJ background (0.03%) is completely abolished in recJ recB hosts (< 0.001%), suggesting that the RecBCD nuclease can provide an alternative function allowing phage growth. One tentative explanation is that circularization of P22 erf DNA mostly proceeds through the RecF pathway of recombination; however, less efficient circularization via the RecBCD pathway may also occur. In a recJ background, lysates obtained upon induction of an erf prophage show reduced yield (10%), suggesting that growth of P22 erf may require host RecJ in a step(s) other than circularization of phage DNA.

African swine fever virus-induced DNA polymerase is resistant to aphidicolin

African swine fever virus (ASFV) induces the synthesis of a virus-specific DNA polymerase, which is inhibited by phosphonoacetic acid and cytosine arabinoside. In contrast to all other a-like DNA polymerases of DNA viruses, ASFV-specific DNA polymerase is resistant to aphidicolin. Concentrations of the drug as high as 160 μM had no effect on virus production or plaquing efficiency. The resistance of ASFV DNA polymerase to aphidicolin was confirmed by analyzing the effect of the drug on viral DNA synthesis. A moderate inhibition of viral DNA synthesis was observed when aphidicolin was added immediately after virus adsorption but normal synthesis occurred, with a peak at 10 hr p.i., when the drug was added at 2 or 4 hr p.i. This suggests that a very early phase of ASFV DNA replication is sensitive to aphidicolin and is probably catalyzed by a different enzyme. An in vitro assay of DNA polymerase activity was used to assay the sensitivity of the virus-specific DNA polymerase to inhibitors. In correspondence to the results observed in vivo, phosphonoacetic acid strongly inhibited the enzyme activity, whereas aphidicolin had no effect. Resistance to aphidicolin was independent of the concentration of dCTP used in the assay. Three independent ASFV mutants resistant to phosphonoacetic acid showed the same resistance to aphidicolin as wild type virus.