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A growing number of studies report that viruses can spread in groups in so-called collective infectious units. By increasing the cellular multiplicity of infection, collective dispersal may allow for social-like interactions, such as cooperation or cheating. Yet, little is known about how such interactions evolve. In previous work with vesicular stomatitis virus, we showed that virion aggregation accelerates early infection stages in most cell types, providing a short-term fitness benefit to the virus. Here, we examine the effects of virion aggregation over several infection cycles. Flow cytometry, deep sequencing, infectivity assays, reverse transcription-quantitative PCR, and electron microscopy revealed that virion aggregation rapidly promotes the emergence of defective interfering particles. Therefore, virion aggregation provides immediate fitness benefits to the virus but incurs fitness costs after a few viral generations. This suggests that an optimal strategy for the virus is to undergo virion aggregation only episodically, for instance, during interhost transmission. IMPORTANCE Recent insights have revealed that viruses use a highly diverse set of strategies to release multiple viral genomes into the same target cells, allowing the emergence of beneficial, but also detrimental, interactions among viruses inside infected cells. This has prompted interest among microbial ecologists and evolutionary biologists in studying how collective dispersal impacts the outcome of viral infections. Here, we have used vesicular stomatitis virus as a model system to study the evolutionary implications of collective dissemination mediated by viral aggregates, since this virus can spontaneously aggregate in the presence of saliva. We find that saliva-driven aggregation has a dual effect on viral fitness; whereas aggregation tends to increase infectivity in the very short term, virion aggregates are highly susceptible to invasion by noncooperative defective variants after a few viral generations., (Copyright © 2020 Andreu-Moreno and Sanjuán.)

Defective prophages, which are found in the genomes of many bacteria, are unable to complete a viral replication cycle and propagate in their hosts as healthy prophages. They package random DNA fragments derived from various sites of the host chromosome instead of their own genomes. In this study, we characterized a defective phage, PBP180, which was induced from Bacillus pumilus AB94180 by treatment with mitomycin C. Electron microscopy showed that the PBP180 particle has a head with a hexagonal outline of ~40 nm in diameter and a long tail. The DNA packaged in the PBP180 head consists of 8-kb DNA fragments from random portions of the host chromosome. The head and tail proteins of the PBP180 particle consist of four major proteins of approximately 49, 33, 16 and 14 kDa. The protein profile of PBP180 is different from that of PBSX, a well-known defective phage induced from Bacillus subtilis 168. A killing activity test against two susceptible strains each of B. subtilis and B. pumilus showed that the defective particles of PBP180 killed three strains other than its own host, B. pumilus AB94180, differing from the host-killing ranges of the defective phages PBSX, PBSZ (induced from B. subtilis W23), and PBSX4 (induced from B. pumilus AB94044). The genome of the PBP180 prophage, which is integrated in the B. pumilus AB94180 chromosome, is 28,205 bp in length, with 40 predicted open reading frames (ORFs). Further genomic comparison of prophages PBP180, PBSX, PBSZ and other PBSX-like prophage elements in B. pumilus strains revealed that their overall architectures are similar, but significant low homology exists in ORF29-ORF38, which presumably encode tail fiber proteins involved in recognition and killing of susceptible strains.

The use of adenovirus type 35 (Ad35) as a vector in vaccine and gene therapy studies is promising due to its broad cell tropism and low seroprevalence in humans. However, to date, a simple and effective system for producing recombinant Ad35 (rAd35) has not been well developed. This report describes a two-plasmid Ad35-Easy system to facilitate the production of recombinant Ad35 (rAd35). The system employed the pAd35-shuttle vector for foreign gene transfer and the pAd35-backbone vector to provide the Ad35 genomic backbone. A 293-Ad35E1B cell line was used to trans-complement rAd35 replication. rAd35 plasmids were obtained through homologous recombination following co-transformation of E. coli BJ5183 cells with recombinant pAd35-shuttle vectors harboring foreign genes. rAd35 viruses were obtained directly by transfecting 293-Ad35E1B cells with foreign gene-containing rAd35 plasmids and the pAd35-backbone vector. The production of E1 deficient rAd35 was evaluated by transfecting the 293-Ad35E1B cells with the rAd35 plasmid containing the enhanced green fluorescent protein (EGFP) gene. The virus grew effectively at a yield comparable to that of wild type Ad35 in HEp2 cells, indicating that the Ad35-Easy system is an efficient method for rapid production of rAd35 in sufficient quantities for vaccine development or gene therapy., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Replication-competent forms of herpes simplex virus 1 (HSV-1) defective in the viral neurovirulence factor infected cell protein 34.5 (ICP34.5) are under investigation for use in the therapeutic treatment of cancer. In mouse models, intratumoral injection of ICP34.5-defective oncolytic HSVs (oHSVs) has resulted in the infection and lysis of tumor cells, an associated decrease in tumor size, and increased survival times. The ability of these oHSVs to infect and lyse cells is frequently characterized as exclusive to or selective for tumor cells. However, the extent to which ICP34.5-deficient HSV-1 replicates in and may be neurotoxic to normal brain cell types in vivo is poorly understood. Here we report that HSV-1 defective in ICP34.5 expression is capable of establishing a productive infection in at least one normal mouse brain cell type. We show that γ34.5 deletion viruses replicate productively in and induce cellular damage in infected ependymal cells. Further evaluation of the effects of oHSVs on normal brain cells in animal models is needed to enhance our understanding of the risks associated with the use of current and future oHSVs in the brains of clinical trial subjects and to provide information that can be used to create improved oHSVs for future use.

Mitochondrial research has influenced concepts in anthropology, human physiology and pathophysiology. We present here direct evidence that human recombinant viruses can localize in mitochondria to disrupt their integrity. This finding, while opening new perspectives in viral gene therapy, may provide new insights into the pathogenesis, prevention and treatment of viral diseases. In addition, it may advance the current understanding of cell evolution., ((c) 2008 S. Karger AG, Basel)

The clinical outcome of advanced gastrointestinal (GI) cancers (especially pancreatic and oesophageal cancers) is dismal, despite the advance of conventional therapeutic strategies. Cancer gene therapy is a category of new therapeutics, among which conditionally replicative adenovirus (CRAd) is one promising strategy to overcome existing obstacles of cancer gene therapy. Various CRAds have been developed for GI cancer treatment by taking advantage of the replication biology of adenovirus. Some CRAds have already been tested in clinical trials, but have fallen short of initial expectations. Concerns for clinical applicability include therapeutic potency, replication selectivity and interval end points in clinical trials. In addition, improvement of experimental animal models is needed for a deeper understanding of CRAd biology. Despite these obstacles, CRAds continue to be an exciting area of investigation with great potential for clinical utility. Further virological and oncological research will eventually lead to full realisation of the therapeutic potential of CRAds in the field of GI cancers.

A field emission scanning electron microscopy (FESEM) method was developed to assess the stability of a recombinant adenovirus (rAd). This method was designed to simultaneously sort, count, and size the total number of rAd viral species observed within an image field. To test the method, a preparation of p53 transgene-expressing recombinant adenovirus (rAd/p53) was incubated at 37 degrees C and the viral particles were evaluated by number, structure, and degree of aggregation as a function of time. Transmission electron microscopy (TEM) was also used to obtain ultrastructural detail. In addition, the infectious activity of the incubated rAd/p53 samples was determined using flow cytometry. FESEM image-analysis revealed that incubation at 37 degrees C resulted in a time-dependent decrease in the total number of detectable single rAd/p53 virus particles and an increase in apparent aggregates composed of more than three adenovirus particles. There was also an observed decrease in both the diameter and perimeter of the single rAd/p53 viral particles. TEM further revealed the accumulation of damaged single particles with time at 37 degrees C. The results of this study demonstrate that FESEM, coupled with sophisticated image analysis, may be an important tool in quantifying the distribution of aggregated species and assessing the overall stability of rAd samples.

The electron microscopic study of several Erwinia carotovora strains showed that the SOS-induced cells of this pectolytic phytopathogenic bacterium produce particular phage parts (tails, heads, and baseplates) but do not assemble them into fully functional phage particles. E. carotovora cells produced several times greater amounts of phage tails in response to induction by mitomycin C than in response to induction by nalidixic acid. The tails were 128-192 nm in length and 13-21 nm in diameter. Phage heads were characterized by four discrete ranges of diameters: 18, 55-59, 66-75, and 92-98 nm. The diameters of phage baseplates varied from 39 to 53 nm, depending on the particular strain. It was shown that cells of the same species may contain several different types of phage tails and heads. The structural organization of phage tails and baseplates in the nalidixic acid-induced lysate of E. carotovora J2 was studied in more detail. The data obtained suggest that pectolytic phytopathogenic erwinia are characterized by defective polylysogeny.

While baculovirus expression of Gag proteins from numerous retroviruses has led reliably to production of virus-like particles (VLPs), we observed that expression of Rous sarcoma virus Gag failed to produce VLPs. Transmission and scanning electron microscopy analysis revealed that the Gag protein reached the plasma membrane but was unable to correctly form particles. Addition of a myristylation signal had no effect on the budding defect, but deletion of the PR domain of Gag restored normal budding. The resulting VLPs were morphologically distinct from human immunodeficiency virus type 1 VLPs expressed in parallel.

Fluorescence imaging of cells is a powerful tool for exploring the dynamics of organelles, proteins, and viruses. Fluorescent adenoviruses are a model system for cargo transport from the cell surface to the nucleus. Here, we describe a procedure to quantitate adenovirus-associated fluorescence in different subcellular regions. CCD camera-captured fluorescence sections across entire cells were deblurred by a fast Fourier transformation, the background was subtracted images merged, and virus fluorescence quantitated. The validity of the deblurring routine was verified by confocal laser scanning microscopy, demonstrating that objects were neither generated nor deleted. Instead, the homogeneity of both the average intensity and the size of fluorescent particles was increased, facilitating automated quantification. We found that nuclear fluorescence of wt adenovirus, but not of a virus mutant ts1, which fails to escape from endosomes, was maximal at 90 min postinfection (p.i.). Surprisingly, nuclear fluorescence decreased at 120 min, but increased again at 240 min p.i., suggesting that wt virus targeting to the nucleus may be multiphasic and regulated. Interestingly, only the first nuclear transport period of wt but not ts1 virus coincided with a significant increase of the peripheral and decrease of the cytoplasmic regions, indicative of signal-dependent cell contraction., (Copyright 2000 Academic Press.)

The role of the N-myristoylation of the human immunodeficiency virus type 1 (HIV-1) gag protein in ACH-2 cells was studied. The infectivity of HIV-1 from the cells stimulated with phorbol 12-myristate 13-acetate (PMA) was suppressed by pretreatment with N-myristoyl glycinal diethylacetal (N-Myr-GOA), a potent N-myristoylation inhibitor, and the blockage of myristoylation resulted in accumulation of immature gag precursors. The viral particles which budded from the non-N-Myr-GOA-treated ACH-2 cells stimulated with PMA exhibited a typical viral phenotype, whereas those which budded from the N-Myr-GOA-treated ACH-2 cells stimulated with PMA were twisted, as observed electron microscopically. In electron microscopic analyses with gold-labeled monoclonal antibodies to gag and env, gag and env were detected adjacent to each other in the PMA-stimulated ACH-2, but no env was detected in the cells treated with N-Myr-GOA. Taken together, the results suggest that the myristoylation of HIV-1 gag seems to be responsible for both maturation of gag and acquisition of HIV-1 infectivity.

The vaccinia virus D4R open reading frame, encoding the essential DNA repair enzyme uracil DNA glycosylase, was expressed in two permanent cell lines, the rabbit kidney cell line RK13 and the human fibroblast cell line 293. The temperature-sensitive vaccinia virus mutant ts4149, which maps within D4R, was able to grow under restrictive conditions in both of these transformed cell lines. Cell clones complemented D4R function to various degrees, demonstrating complementation of an essential vaccinia virus gene by a cell line constitutively expressing the essential function. Thus, the complementing host cells allowed the rescue of a virus defective in the D4R gene, demonstrating that this system may be used for the propagation of defective cytoplasmic DNA viruses. The defective virus grew to high yields only in the engineered cell lines. The data support the hypothesis that early gene products, such as uracil DNA glycosylase, supplied in trans can fully complement essential viral functions.

The ultrastructure and morphogenesis of human immunodeficiency virus (HIV) were elucidated by observation with several techniques including immunoelectron microscopy and cryo-microscopy. The virus particle consists of an envelope, a core and matrix. The virus particles were observed extracellularly as having one of three profiles: (1) a centric or an eccentric electron-dense core, (2) rod-shaped electron-dense core, and (3) doughnut-shaped. HIV-1 particles in the hydrated state were observed by high resolution electron cryo-microscopy to be globular, and the lipid membrane was clearly resolved as a bilayer. Many projections around the circumference were seen to be knob-like. The shapes and sizes of the projections, especially head parts, were found to vary in each projection. By isolation with Nonidet P40 and glutaraldehyde, HIV-1 cores were confirmed to consist of p24 protein by immunogold labeling. When the virus enters the cell, two entry modes were found: membrane fusion and endocytosis. No structures resembling virus particles could be seen in the cytoplasm after viral entry. In HIV-1-infected cells, positive reactions by immuno-labeling suggest that HIV-1 Gag may be produced in membrane-bound structures and transported to the cell surface by cytoskeletons. Then a crescent electron-dense layer was first formed underneath the cell membrane. Finally, the virus particle was released from the cell surface. Several cell clones producing defective particles were isolated from MT-4/HIV-1 cells. Among them, doughnut-shaped or teardrop-shaped particles were seen to be produced in the extracellular space. In the doughnut-shaped particles, Gag p17 and p24 proteins faced each other against the inner electron dense ring, suggesting that the inner ring consists of a precursor Gag protein.

Previous studies have shown that the protein encoded by herpes simplex virus type 1 (HSV-1) gene UL6 is required for processing and packaging of replicated viral DNA and is a minor component of virions and capsids. In this report, we describe the construction of UL6- HSV-1 mutants with a disrupted UL6 gene using complementing cells and show that they fail to synthesize the UL6 protein or produce infectious virus in noncomplementing cells. The mutants synthesized but failed to process and encapsidate viral DNA and accumulated only immature capsids which lacked the UL6 protein. Immunofluorescence analysis showed that the UL6 protein, when expressed transiently in transfected cells in the absence of other HSV-1 proteins, is localized exclusively to the nucleus. We also investigated an HSV-1 mutant with a defect in gene UL33, the product of which is also thought to be involved in viral DNA processing and packaging. The phenotype of this mutant on noncomplementing cells with regard to failure to process and encapsidate viral DNA, accumulation of immature capsids, and inability to produce infectious virus was the same as that of UL6- viruses. This mutant, however, produced capsids containing the UL6 protein, indicating that association of the UL6 protein with the capsid is independent of the UL33 protein.

One of the major obstacles to pulmonary-directed gene therapy using adenoviral vectors is the induction of inflammation. We investigated whether the adenoviral particles that constitute the initial inoculum can serve as an inflammatory stimulus, independent of their ability to express genes that they contain. Viral particles were prepared that are defective in gene expression by (i) isolating particles that have incomplete genomes by selecting those that have buoyant densities on CsCl density gradients lighter than complete viruses; and (ii) cross-linking viral DNA by exposure to ultraviolet light in the presence of 8-methoxypsoralen. The defective particles retained their icosahedral appearance when viewed by electron microscopy but lost their plaque-forming ability on 293 cells. High doses of intact, incomplete, or inactivated viral particles were instilled intratracheally into CBA/J mice, and after 6 days the amount of inflammation was quantified by counting inflammatory cells contained within lung tissue. We found that the inflammatory responses induced by the incomplete or inactivated viral vectors were quantitatively similar to those caused by intact, competent viral vectors. We conclude that high doses of adenoviral vectors that are used for gene therapy can induce pulmonary inflammation, independent of expressing the genes they contain.

We have characterized a temperature-sensitive mutant of vaccinia virus, ts16, originally isolated by Condit et al. (Virology 128:429-443, 1983), at the permissive and nonpermissive temperatures. In a previous study by Kane and Shuman (J. Virol 67:2689-2698, 1993), the mutation of ts16 was mapped to the I7 gene, encoding a 47-kDa protein that shows partial homology to the type II topoisomerase of Saccharomyces cerevisiae. The present study extends previous electron microscopy analysis, showing that in BSC40 cells infected with ts16 at the restrictive temperature (40 degrees C), the assembly was arrested at a stage between the spherical immature virus and the intracellular mature virus (IMV). In thawed cryosections, a number of the major proteins normally found in the IMV were subsequently localized to these mutant particles. By using sucrose density gradients, the ts16 particles were purified from cells infected at the permissive and nonpermissive temperatures. These were analyzed by immunogold labelling and negative-staining electron microscopy, and their protein composition was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. While the ts16 virus particles made at the permissive temperature appeared to have a protein pattern identical to that of wild-type IMV, in the mutant particles the three core proteins, p4a, p4b, and 28K, were not proteolytically processed. Consistent with previous data the sucrose-purified particles could be labelled with [3H]thymidine. In addition, anti-DNA labelling on thawed cryosections suggested that most of the mutant particles had taken up DNA. On thawed cryosections of cells infected at the permissive temperature, antibodies to I7 labelled the virus factories, the immature viruses, and the IMVs, while under restrictive conditions these structures were labelled much less, if at all. Surprisingly, however, by Western blotting (immunoblotting) the I7 protein was present in similar amounts in the defective particles and in the IMVs isolated at the permissive temperature. Finally, our data suggest that at the nonpermissive temperature the assembly of ts16 is irreversibly arrested in a stage at which the DNA is in the process of entering but before the particle has completely sealed, as monitored by protease experiments.

The integration of a DNA copy of the human immunodeficiency virus type 1 (HIV-1) genome into a chromosome of an infected cell is a pivotal step in virus replication. Integration requires the activity of the virus-encoded integrase, which enters the cell as a component of the virion. Results of numerous mutagenesis studies have identified amino acid residues and protein domains of HIV-1 integrase critical for in vitro activity, but only a few of these mutants have been studied for their effects on HIV replication. We have introduced site-directed changes into an infectious DNA clone of HIV-1 and show that integrase mutations can affect virus replication at a variety of steps. We identified mutations that altered virion morphology, levels of particle-associated integrase and reverse transcriptase, and viral DNA synthesis. One replication-defective mutant virus which had normal morphology and protein composition displayed increased levels of circular viral DNA following infection of a T-cell line. This virus also had a significant titer in a CD4-positive indicator cell assay, which requires the viral Tat protein. Although unintegrated viral DNA can serve as a template for Tat expression in infected indicator cells, this level of expression is insufficient to support a spreading viral infection in CD4-positive lymphocytes.

The assembly of infectious poliovirus virions requires a proteolytic cleavage between an asparagine-serine amino acid pair (the maturation cleavage site) in VP0 after encapsidation of the genomic RNA. In this study, we have investigated the effects that mutations in the maturation cleavage site have on P1 polyprotein processing, assembly of subviral intermediates, and encapsidation of the viral genomic RNA. We have made mutations in the maturation cleavage site which change the asparagine-serine amino acid pair to either glutamine-glycine or threonine-serine. The mutations were created by site-directed mutagenesis of P1 cDNAs which were recombined into wild-type vaccinia virus to generate recombinant vaccinia viruses. The P1 polyproteins expressed from the recombinant vaccinia viruses were analyzed for proteolytic processing and assembly defects in cells coinfected with a recombinant vaccinia virus (VV-P3) that expresses the poliovirus 3CD protease. A trans complementation system using a defective poliovirus genome was utilized to assess the capacity of the mutant P1 proteins to encapsidate genomic RNA (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 67:3684-3690, 1993). The mutant P1 proteins containing the glutamine-glycine amino acid pair (VP4-QG) and the threonine-serine pair (VP4-TS) were processed by 3CD provided in trans from VV-P3. The processed capsid proteins VP0, VP3, and VP1 derived from the mutant precursor VP4-QG were unstable and failed to assemble into subviral structures in cells coinfected with VV-P3. However, the capsid proteins derived from VP4-QG did assemble into empty-capsid-like structures in the presence of the defective poliovirus genome. In contrast, the capsid proteins derived from processing of the VP4-TS mutant assembled into subviral intermediates both in the presence and in the absence of the defective genome RNA. By a sedimentation analysis, we determined that the capsid proteins derived from the VP4-TS precursor encapsidated the defective genome RNA. However, the cleavage of VP0 to VP4 and VP2 was delayed, resulting in the accumulation of provirions. The maturation cleavage of the VP0 protein containing the VP4-TS mutation was accelerated by incubation of the provirions at 37 degrees C. The results of these studies demonstrate that mutations in the maturation cleavage site have profound effects on the subsequent capability of the capsid proteins to assemble and provide evidence for the existence of the provirion as an assembly intermediate.

Murine acquired immunodeficiency syndrome (MAIDS) induced by defective LP-BM5 murine leukemia virus is a disease with many similarities to human AIDS. Previous studies indicated that the depressed hematopoiesis observed in LP-BM5-infected marrow cultures may be attributable to a defect of hematopoietic stroma. We report here the generation of permanent stromal cell lines from noninfected and LP-BM5-infected marrow cultures. Retrovirus infection was confirmed by polymerase chain reaction for viral genome. The ability of these cell lines to support in vitro hematopoiesis was studied. Results indicated that, when cocultured with normal or infected nonadherent mononuclear cells, noninfected cell lines efficiently supported the production of hematopoietic precursors, whereas viral-infected cell lines induced suppression of both normal and viral-infected progenitors. Expression of cytokine genes in stromal cell lines was also examined. All cell lines expressed equivalent levels of transcripts for stem cell factor and tumor necrosis factor alpha. However, infection was associated with higher levels of interleukin-4 and transforming growth factor beta 1 transcript expression. These findings suggest that infected stromal cell lines exhibit a defective hematopoietic microenvironment that produced altered cytokine expression resulting in faulty hematopoiesis. Further characterization of the defective cell lines should prove valuable for studies of the pathogenesis of murine AIDS.

Prophage kappa in V. cholerae el tor strain SLH22(J) could be induced spontaneously or by treatment with nitrofurantoin, though the efficiency of induction was very low (not more than 0.8%). V. cholerae el tor cells were found to release many different aberrant structures of the temperate phage, kappa. These aberrant structures were characterized by density gradient centrifugation and electron microscopy.

Temperate coliphage P2 and satellite phage P4 have icosahedral capsids and contractile tails with side tail fibers. Because P4 requires all the capsid, tail, and lysis genes (late genes) of P2, the genomes of these phages are in constant communication during P4 development. The P4 genome (11,624 bp) and the P2 genome (33.8 kb) share homologous cos sites of 55 bp which are essential for generating 19-bp cohesive ends but are otherwise dissimilar. P4 turns on the expression of helper phage late genes by two mechanisms: derepression of P2 prophage and transactivation of P2 late-gene promoters. P4 also exploits the morphopoietic pathway of P2 by controlling the capsid size to fit its smaller genome. The P4 sid gene product is responsible for capsid size determination, and the P2 capsid gene product, gpN, is used to build both sizes. The P2 capsid contains 420 capsid protein subunits, and P4 contains 240 subunits. The size reduction appears to involve a major change of the whole hexamer complex. The P4 particles are less stable to heat inactivation, unless their capsids are coated with a P4-encoded decoration protein (the psu gene product). P4 uses a small RNA molecule as its immunity factor. Expression of P4 replication functions is prevented by premature transcription termination effected by this small RNA molecule, which contains a sequence that is complementary to a sequence in the transcript that it terminates.

In addition to its polarity-suppressing activity, the Psu protein of bacteriophage P4 also serves to stabilize the capsid against heat treatment and binds externally to the phage capsid. However, the heat stability is lost upon purification of the virus, indicating a loss of Psu protein from the capsid. By using three-dimensional reconstruction from cryo-electron micrographs of P4 psu1 amber mutants lacking Psu, and of P4 virions, which have been saturated with Psu protein to regain heat stability, we have determined the position of this protein on the virus surface. Our results are consistent with the hypothesis that the function of Psu is to stabilize the hexameric capsomer assembly.

An alternative approach to structure-function analysis of vesicular stomatitis virus (VSV) gene products and their interactions with one another during each phase of the viral life cycle is described. We showed previously by using the vaccinia virus-T7 RNA polymerase expression system that when cells expressing the nucleocapsid protein (N), the phosphoprotein (NS), and the large polymerase protein (L) of VSV were superinfected with defective interfering (DI) particles, rapid and efficient replication and amplification of (DI) particle RNA occurred. Here, we demonstrate that all five VSV proteins can be expressed simultaneously when cells are contransfected with plasmids containing the matrix protein (M) gene and the glycoprotein (G) gene of VSV in addition to plasmids containing the genes for the N, NS, and L proteins. When cells coexpressing all five VSV proteins were superinfected with DI particles, which because of their defectiveness are unable to express any viral proteins or to replicate, DI particle replication, assembly, and budding were observed and infectious DI particles were released into the culture fluids. Omission of either the M or G protein expression resulted in no DI particle budding. The vector-supported DI particles were similar in size and morphology to the authentic DI particles generated from cells coinfected with DI particles and helper VSV and their infectivity could be blocked by anti-VSV or anti-G antiserum. The successful replication, assembly, and budding of DI particles from cells expressing all five VSV proteins from cloned cDNAs provide a powerful approach for detailed structure-function analysis of the VSV gene products in each step of the replicative cycle of the virus.

The antigenic relationships among different hepatitis B virus (HBV) isolates were investigated by using monoclonal antibodies (MAbs) specific for HBs, preS2 (pHSA binding site), and preS1 (hepatocyte receptor-binding site) epitopes in a double immunoradiometric assay. In order to define possible functional differences resulting from structural and antigenic differences in the HBV env protein, the HBV isolates were compared in an in vitro cell-binding assay based on the attachment of 125I-labeled HBV to human hepatoma HepG2 cells. We provided evidence for a variability of the expression of preS1 and preS2 specificities in the peplomer (glyco)protein of HBV depending on dly subtype of HBsAg, which could affect the viral infectivity. We showed that the integrity of the HBV envelope structure associated with a large expression of preS1(21-47) epitopes is an essential factor for effective binding to HepG2 cells. Interestingly, the HBs-specific MAbs directed to disulfide-bond-dependent epitopes were found to be the best inhibitors of the preS1-HepG2 cell interaction (greater than 50%, at the final concentration of 0.5 micrograms/ml). The MAb F35.25 directed to the preS1(21-47) sequence corresponding to the hepatocyte receptor recognition site was, however, also found to inhibit binding. Thus, our results demonstrate the abilities of both anti-HBs and anti-preS(21-41) to block the attachment of complete HBV particles to HepG2 cells, suggesting that these antibodies should be virus neutralizing and would be expected to confer protection against reinfection.

A total of 81 cell clones persistently infected with the LAV-1 or HTLV-IIIB strain of human immunodeficiency virus type 1 (HIV-1) was isolated from cells which were obtained by serial passage of some proliferating MT-4 cells after a drastic cytolysis of most cells by HIV-1-infection. These cell clones were classified into 8 types (I to VIII) in terms of the expression of HIV-1 antigens, syncytium formation capacity, and reverse transcriptase activity and infectivity of virus particles in the culture fluid. Type I cell clones were producers of infectious HIV-1 particles, while types II to VIII cell clones did not produce infectious HIV-1 or were producers of uninfectious defective HIV-1 particles. Immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis (PAGE) showed that the gag precursor protein in L-2 cell clone (type IV) was not cleaved to mature gag proteins, while the env precursor protein on L-3 cell clone (type III) was not cleaved to mature env protein. H-7 cell clone (type VIII) did not express any HIV-1 antigen. All these cell clones after the superinfection with infectious HIV-1 synthesized intact gag and env proteins, which were, at least in part, related to the HIV-1 genome persistently present in the cell clones before the superinfection, resulting in production of infectious HIV-1. For example, it was found that L-2 cell clone contained a single copy of the LAV-1 genome per haploid cell and produced doughnut-shaped particles. On the other hand, the cell clone isolated from the L-2 cell clone superinfected with infectious HTLV-IIIB contained the integrated HTLV-IIIB genome in addition to the LAV-1 genome present before the superinfection, and produced intact HIV-1 particles in addition to doughnut-shaped particles from a single cell. These results indicate that complementation and/or genetic recombination events in the superinfected cells may account for the production of infectious intact HIV-1 virions.

We examined further the physical structure of the simian virus 40 (SV40) and bacteriophage lambda DNA sequences in an SV40-lambda hybrid that had been propagated in monkey kidney cells. The SV40 vector portion of the hybrid, which was a small fragment isolated from a reiteration mutant of SV40, contained the site for initiation of SV40 DNA replication. Electron microscope heteroduplex and restriction endonuclease analyses revealed a tandem duplication of the SV40 vector segment linked to a 2,300-base pair portion (lambda map units 71 to 76) of the lambda immunity region. The defective hybrid genome thus harbors two origins for SV40 DNA replication in addition to the leftward operator and the N gene of lambda.

The wild-type New Jersey serotype of vesicular stomatitis virus generated two types of defective interfering T-particles. The physical properties of these particles and the wild-type virion were determined by laser light scattering spectroscopy, sedimentation measurements, and electron microscopy.

This paper describes Q beta noninfectious particles produced at 41 degrees C in a streptomycin-resistant Escherichia coli mutant which is temperature sensitive for suppression of a nonsense codon. The noninfectious particles resembled Q beta under the electron microscope and contained coat protein molecules in an amount similar to the amount in Q beta. However, they did not adsorb to F-piliated bacteria, and they were deficient in both minor capsid proteins of Q beta, maturation (IIa) and read-through (IIb). Proteins IIa and IIb were not produced in Qbeta-infected mutant cells at 41 degrees C. In addition, instead of the 30S RNA of Q beta, a shorter RNA, which sedimented mainly at 23 S, was found in the defective particles. The results are discussed in relation to the roles of proteins IIa and IIb of Q beta.

Aquaspirillum itersonii spontaneously produces rhapidosomes. These structures, which are found throughout the cell cycle, are compared with the defective bacteriophage particles that can be induced by mitomycin C treatment (0.5 microgram/mL). The rhapidosomes are composed of three proteins (MW 63 000, 23 500, and 15 000). The defective bacteriophage particles are composed of two proteins (MW 50 000 and 17 500). The defective bacteriophage particles do not react with anti-rhapidosomal protein antiserum. These results indicate that the rhapidosome are not polysheaths of the defective bacteriophage particles inducible in this organism as earlier proposed in the literature. It is concluded that the rhapidosomes of A. itersonii are an assembly of three proteins that are distributed in the cytosol after synthesis and are present at all times in the growth cycle.

Polysheaths were spontaneously formed inside cells of the hydrogen bacterium Alcaligenes eutrophus H 16. These particles are long tube-like structures of 24 nm diam. belonging to the phage tail-like defective bacteriophages (Lotz, 1976). In mid log-phase fermenter-grown cells, polysheaths were observed in about 20% of all cells sectioned. Evidence is provided for an inhibition of cell fission by polysheaths. Polysheaths were isolated by differential centrifugation and precipitation techniques using PEG and antibodies. The morphology of polysheaths was investigated electron microscopically by negative staining, ultrathin sectioning and metal shadowing. A surface lattice of the polysheath was derived from light optical diffraction data. The particles were also characterized by their biochemical and biophysical features: mol. wt. of the subunit determined by SDS-gel electrophoresis (58 000), amino acid composition, isoelectric point (4.4), u.v. absorbance spectrum indicating the absence of nucleic acid, buoyant density (1.258), and stability against denaturants and proteolytic enzymes.

PK-15 cells infected with pig and lamb rotavirus strains which were not adapted to serial growth in cell cultures were examined by electron microscopy. A major difference between virus morphogenesis in the initial passage in PK-15 cells and in intestinal epithelial cells was the generation of large numbers of coreless virus particles in PK-15 cells. The numbers of coreless particles increased with increasing multiplicity of infection. Infectious virus was synthesized in PK-15 cells, but a variable decrease in infectivity titre occurred between 12 and 24 hours after infection. It is suggested that synthesis of defective interfering particles or an inhibitory substance such as interferon might account for this decrease.

Widespread defective lysogeny was detected in Alcaligenes eutrophus by electron microscopic analysis of cultures. Mitomycin C treatment of the cultures resulted in the production of defective (inco-) particles. Polysheaths were produced both with and without induction. With the simultaneous isolation technique six phages were isolated for hydrogen-oxidizing strains of the new species Pseudomonas pseudoflava. The phages were able to replicate under autotrophic conditions and were found to have a very restricted host range. Electron microscopic analysis allowed classification into two structural groups. Group I contained phages with contractile tails; group II contained phages with flexible, noncontractile tails. All but one (gb) of the new phages were shown to be temperate by isolation of lysogens and induction with mitomycin C.

Sendai virus defective interfering nucleocapsids, isolated from infected cell cytoplasm by equilibrium banding in CsCl gradients, contain only the viral N protein. Neither end of the genomic RNA within these nucleocapsids is accessible to RNase digestion.

In a population of influenza virus, virions have different degree of the functional capacities to produce infectious virus, to induce neuraminidase production and to interfere with infectious virus. Fractions of virus were found which had a poor capacity for production of infectious progeny and a relatively high induction of neuraminidase synthesis as well as induction of heterogenous light fractions of RNA in cells. In the population of defective virus a fraction of particles possessing marked interfering activity in the absence of infectivity was found.

The titre of defective interfering (DI) influenza virus measured by an assay based on the inhibition of cytopathology caused by A/WSN (H1N1) influenza virus in MDCK cells was 320,000-fold greater than titres measured by inhibition of infectious centre formation. Interference was less in other types of cell. By electron microscopy, we have shown that the ratio between physical particles and DI units in preparations of the DI virus was approximately unity, which suggested that one or few DI particles is/are required to confer resistance of a MDCK cell to viral cytopathology. This human DI virus interfered heterotypically with an avian H7N1 influenza virus.

Satellite bacteriophage P4 requires all morphogenic gene products provided by a helper phage, such as coliphage P2, to assemble its own capsid, which is one-third the volume of the larger helper capsid. We have isolated a satellite phage P4 sid (size determination) mutant that is unable to direct the assembly of the small wild-type-size P4 capsid. Instead, this mutant produces P4 plaque-forming units with large P2-size capsids which contain two or three copies of the P4 sid1 genome. P4 sid1 is evidently mutated in a protein that is specifically responsible for determining the precise size and symmetry of the structure into which the helper P2 gene products will assemble. In addition, we have found that the physical size of the genome does not appear to play an essential role in the proper assembly of the icosahedral capsid, since the majority of the P4 sid1 plaque-forming units do not contain a complete capsidful of DNA.

72 strains of 24 Bacillus species were induced with mitomycin C. The lysates were examined for the presence of defective phages resembling PBS X in morphology. All strains tested of B. amyloliquefaciens. B, licheniformis, B. pumilus and B. subtilis contained such phages. Five morphological types of defective, PBS X-like phage could be distinguished, differing in their tail lengths and in the number of cross-striations on the tail. The quaternary structure of the tail, the molecular weight of the main tail protein and the antigenic properties of the phages were identical. The killing ranges of the defective phages have been determined and their possible use in taxonomy discussed.

Bacillus brevis strains contains defective prophage. In strain ATCC 10068 this prophage is responsible for the production of particles resembling phage tails and the induction of events leading to cell lysis. There is a slow rate of spontaneous production of particles, which is greatly increased by treating growing cells with mitomycin C, ultraviolet light, N-methyl-N-nitroso-N'-nitroguanidine, and acridine orange or by changing the growth medium. There is no conversion of host DNA to a unique, phage-size DNA entity associated with induction. It was not possible to detect strains that did not produce particles on treatment with mitomycin C. The particles themselves had no recognised biological activity. The molecular weight of the major sheath protein was 49 000 and at least four other protein components were detected in partially purified preparations. DNA and RNA were not associated with the particles but a small amount of carbohydrate was detected.

Four Agrobacterium tumefaciens temperate phages (PB2A, PB6(omega), PV-1(LV-1) and PS8), were shown to have the same genome size. Moreover hybridization experiments by the heteroduplex method and electron microscopy showed a 100% homology between these four phage genomes. Indications for lysogeny were found by direct means for the Agrobacterum timefaciens strain 396, Agrobacterium radiobacter strain 8149 and Agrobacterium species 0362 and by the electron microscope negative staining technique for the A.tumefaciens strains b6-806,b6-6,b6s3,b2as,cv-1,4452,11156,11158,396, and 925; for A.radiobacter strains tr-1 and 8149, the latter being bi-lysogenic, and for the A. species 0362. These isolated phage particles, most of which appear to be defective, could be grouped into different classes. No particles could be detected in the lysates of A. tumefaciens RV3, A. radiobacter strains 4718 and S1005, and A. species 0363. Further characterization by genome size was carried out for the defective temperate phages PB6-806, P4452,P8149 and P0362. No evidence for homology between PB6-806 and PB6 omega could be found. The defective phages PB6-806 and P4452 showed the same morphology but a different genome size, whereas the two phages P0362 and P8149 had a very different morphology and genome size.

Myxococcus coralloides D was lysogenic for a defective prophage. The particles of the defective bacteriophage could be induced by ultraviolet light and mitomycin C, but the particles did not appear in the supernatants, unless the cells were lysed with chloroform. The phage tails were purified by using a two-phase separation method, ultracentrifugation, chromatography through Sepharose 4B, treatment with chloroform, dialysis and centrifugation on a sucrose gradient. The chemical analysis of the purified samples revealed that the phage tails contained only proteins, neither DNA nor RNA. The different parts of the phage tails (sheath, core and baseplate) did not have the same sensitivity to the chemical and physical agents which were assayed.

Five minority populations of aberrant, varicella-zoster virus (VZV)-derived genomes were identified among the encapsidated DNAs obtained from the nuclear and cytoplasmic fractions of an in vitro infection initiated with a lyophilized sample of the BIKEN VZV vaccine (strain Oka). These were (i) VZV genomes, present within nuclear but not cytoplasmic viral capsids, which had been cleaved at a specific site within the short segment and which were, therefore, 3.15 megadaltons (approximately 4% of the VZV genome length) short of full length; (ii) highly deleted, repetitive VZV genomes which contained the errant cleavage site but not the usual VZV genome terminal sequences; (iii) VZV genomes into which multiples of 1 through 5 defective genome repeat units had been inserted into a homologous site; (iv) VZV genomes with additions of 0.1 or 0.18 megadaltons of DNA at both the terminal and internal ends of the short segment; and (v) VZV DNA which had lost the HindIII restriction site at map position 0.11.

High multiplicity, undiluted passage of equine herpesvirus type 1 (EHV-1) in L-M cells resulted in the rapid production of virus particles whose genome was genetically less complex, contained more reiterated DNA sequences and exhibited a greater buoyant density (rho = 1.724 g/ml) than the DNA (rho = 1.716 g/ml) of standard virus. These data and the finding that these particles inhibited the replication of standard virus in interference assays confirmed that these were defective interfering (DI) particles (Henry et al. 1979). Additional evidence for this has been obtained from the pattern of cyclic fluctuation in infectious virus titre through 17 serial passages as well as from the pronounced variation in the particle to plaque ratio for each passage. Total particle production was markedly reduced in cells infected with virus preparations containing DI particles and quantification of major cell-associated EHV-1 capsid species by electron microscopy and analysis in Renografin density gradients indicated that this reduction occurred at the level of capsid assembly. Although total capsid production was reduced in cells infected with DI particle preparations, the synthesis of I (immature) capsids increased relative to that of L (empty) capsids and these alterations in the assembly of capsid species could be related to changes in the synthesis of capsid proteins. In cells infected with EHV-1 preparations rich in DI particles, the synthesis of major capsid protein 150000 was greatly reduced, whereas core protein 46000, a major component of I capsids, was overproduced as compared to standard virus infection. Capsids produced in cells infected with virus preparations rich in DI particles were identical in polypeptide composition to those made in standard virus infection.

A method for obtaining large quantities of defective interfering (DI) rabies virus particles that fulfill all the criteria delineated by Huang and Baltimore (1970) is described. The purified rabies DI virion was found to be much shorter (60 to 80 nm) than the complete virion (180 nm) and to have a viral genome of about half the size of normal rabies RNA but with all of the structural proteins of standard virions. Rabies DI virions were noninfectious for both cells in culture and for animals. As determined by in vitro and in vivo techniques, interference with the replication of standard virus was specific to rabies virus. The possible role of rabies DI virion in the pathogenicity of rabies virus infection and in the establishment of attenuated strains for use as live rabies vaccines is discussed.