Complete Replication Cycle Research Articles

Immortalization of caprine fibroblasts permissive for replication of small ruminant lentiviruses

Abstract Objective To establish immortalized caprine fibroblastic cell lines permissive for replication of small ruminant lentiviruses. Animals Carpal synovial membrane explants collected aseptically from a surgically delivered fetus of a lentivirus-seronegative goat. Procedure Immortalization of goat embryonic fibroblasts was performed by DNA transfection with plasmids coding for simian virus 40 large T antigen. The generated cell lines were phenotypically characterized. Cytogenetics, growth pattern, and sensitivity to viral infection were studied. Results 3 cell lines, designated TIGEF, mMTSV-54, and mMTSV-93, were generated. They had a more rapid doubling time than did fibroblasts from which they were derived, and retained morphologic and phenotypic fibroblastic characteristics. They were immortalized but not transformed because tumor formation was not promoted after their SC injection into athymic nude mice. The 3 cell lines were susceptible to caprine arthritisencephalitis virus and visna-maedi virus infections, and supported a complete virus replication cycle. Conclusions Cultured caprine fibroblastic cells were immortalized, using simian virus 40 large T antigen. The TIGEF, mMTSV-54, and mMTSV-93 immortalized cell lines were permissive to in vitro small ruminant lentivirus replication. Clinical Relevance Because lentivirus detection, as well as detailed studies of host-lentivirus interactions, are hampered by differences in viral susceptibility of each primary culture, permanent cell lines are essential tools for such analysis. (Am J Vet Res 1997;58:579–584)

In vitro study using leukemia cell line panel to demonstrate rapid thymic T cell death due to contact with HIV-1 carrier cell clones.

A rapid (within 1 h) and profound cytotoxic cell death of immature TdT+CD4+CD8+ and/or TdT+CD4+ thymic T cell type leukemia cell lines, and of normal thymocyte populations rich in TdT+CD4+CD8+ cells was induced by contact with some human immunodeficiency virus type-1 (HIV-1) carrier T cell clones. This cytotoxic reaction, without requiring a complete viral replication cycle in the thymic T cells, did not occur in any mature CD4+CD8+ and/or CD4+ T cells which are otherwise permissive for virus infection. Although it was not an antigen-specific cytotoxic reaction, the rapid and profound thymic T cell destruction was shown, at the individual clonal level, to be triggered specifically by the binding of CD4 molecules on thymic T cells with gp120/gp160 on HIV-1 carrier clones. The present study suggesting direct elimination of immature T cells by contact with some HIV-1-infected T cells, may provide a novel insight into the mechanism responsible for the mature CD4+ T cell depletion in HIV-1 infection.

Replication of poliovirus in Xenopus oocytes requires two human factors.

We described a novel system to study poliovirus replication in Xenopus oocytes. Poliovirus RNA microinjected into Xenopus oocyte initiates a complete cycle of viral replication, yielding a high level of infectious viruses. Two distinct HeLa cell activities are required, one involved in initiation of translation and the other in RNA synthesis. The translation factor is a large cytoplasmic protein or complex, which is specifically used for initiation of poliovirus translation. The replication factor is required at early stages of RNA synthesis. Formation of infectious poliovirus is highly temperature dependent. At temperatures below 27 degrees C, capsid assembly appears to be impaired. The oocyte system described here could be useful in identifying and characterizing viral and cellular factors involved in virus replication.

Tissue Macrophages Are Infected by Human Cytomegalovirus In Vivo

On the basis of in vitro experiments, it has been suggested that cells of hematopoietic origin play a major role in the pathogenesis and latency of human cytomegalovirus (HCMV). To elucidate the in vivo importance of hematopoietic cells in acute HCMV infection, tissue sections from various infected organs were investigated by immunohistochemical double-labeling analyses. Monoclonal antibodies directed against distinct viral and cellular antigens were used to identify infected macrophages, polymorphonuclear cells, and lymphocytes. Macrophages and polymorphonuclear cells were targets for HCMV infection in different tissues. Viral proteins representing all stages of permissive HCMV infection were detected in macrophages, suggesting that these cells support the complete viral replication cycle. In polymorphonuclear cells, viral gene expression was restricted to the immediate early phase, indicating that these cells are abortively infected. These findings suggest that macrophages play an important role in the hematogenous spread of HCMV into solid organs.

RNA duplex unwinding activity of alfalfa mosaic virus RNA-dependent RNA polymerase

An RNA-dependent RNA polymerase (RdRp) purified from alfalfa mosaic virus-infected tobacco is capable of synthesizing in vitro full-size RNAs of minus and plus polarities. However, the enzyme is not able to perform a complete replication cycle in vitro. The products were found to be completely base-paired to their templates. The enzyme was able to use double-stranded RNA as a template for RNA synthesis if it could initiate from a single-stranded promoter. The inability (of most) of our enzyme preparations to create a single-stranded initiation site could explain why they could not perform a complete replication cycle in vitro. This is the first report on duplex RNA unwinding activities by a plant viral RdRp.

Identification of viral determinants of macrophage tropism for simian immunodeficiency virus SIVmac

Simian immunodeficiency virus (SIV), a lymphocytopathic lentivirus, induces an AIDS-like disease in rhesus macaques (Macaca mulatta). A pathogenic molecular clone of rhesus macaque SIV (SIVmac), SIVmac-239, replicates and induces cytopathology in T lymphocytes but is restricted for replication in macrophages. In contrast, a nonpathogenic molecular clone of SIVmac, SIVmac-1A11, replicates and induces syncytia (multinucleated giant cells) in cultures of both T lymphocytes and macrophages. SIVmac-1A11 does not cause disease in macaques. To map the viral determinants of macrophage tropism, reciprocal recombinant genomes were constructed between molecular clones of SIVmac-239 and SIVmac-1A11. Infectious recombinant viruses were rescued by transfection of cloned viral genomes into permissive lymphoid cells. Analysis of one pair of reciprocal recombinants revealed that an internal 6.2-kb DNA fragment of SIVmac-1A11 was necessary and sufficient for both syncytium formation and efficient replication in macrophages. This region includes the coding sequences for a portion of the gag gene, all of the pol, vif, vpr, and vpx genes, the first coding exons of tat and rev, and the external env glycoprotein gp130. Thus, the transmembrane glycoprotein of env, the nef gene, the second coding exons of tat and rev, and the long terminal repeats are not essential for in vitro macrophage tropism. Analysis of additional recombinants revealed that syncytium formation, but not virus production, was controlled by a 1.4-kb viral DNA fragment in SIVmac-1A11 encoding only the external env glycoprotein gp130. Thus, gp130 env of SIVmac-1A11 is necessary for entry of virus into macrophages but is not sufficient for a complete viral replication cycle in this cell type. We therefore conclude that gp130 env and one or more genetic elements (exclusive of the long terminal repeats, transmembrane glycoprotein of env, and second coding exons of tat and rev, and nef) are essential for a complete replication cycle of SIVmac in rhesus macaque macrophages.

Characterization of cloned chicken anemia virus DNA that contains all elements for the infectious replication cycle

Circular double-stranded replication intermediates were identified in low-molecular-weight DNA of cells of the avian leukemia virus-induced lymphoblastoid cell line 1104-X-5 infected with chicken anemia virus (CAV). To characterize the genome of CAV, we cloned linearized CAV DNA into the vector pIC20H. Transfection of the circularized cloned insert into chicken cell lines caused a cytopathogenic effect, which was arrested when a chicken serum with neutralizing antibodies directed against CAV was added. Chickens inoculated at 1 day of age with CAV collected from cell lines transfected with cloned CAV DNA developed clinical signs of CAV. The 2,319-bp cloned CAV DNA contained all the genetic information needed for the complete replication cycle of CAV. The CAV DNA sequence has three partially overlapping major reading frames coding for putative peptides of 51.6, 24.0, and 13.6 kDa. The CAV genome probably contains only one promoter region and only one poly(A) addition signal. Southern blot analysis using oligomers derived from the CAV DNA sequence showed that infected cells contained double- and single-stranded CAV DNAs, whereas purified virus contained only the minus strand. It is the first time that the genome of one of the three known single-stranded circular DNA viruses has been completely analyzed.

Direct procedure for the determination of the number of replication forks and the reinitiation fraction in bacteria.

A direct method for calculating the average number of replication forks per chromosome in an exponentially growing bacterial culture and the fraction of reinitiation after an inductive treatment of the initiation step is presented. This method has allowed the development of REPLICON, a computer program designed for the resolution of the algorithm and simulation of the bacterial chromosome replication. Using REPLICON the following parameters can be obtained: average number of replication forks per chromosome, time required for the complete replication cycle, average amount of DNA per nucleotide, gene frequency of any chromosomal locus and reinitiation fraction. The use of this analysis also permits the determination of the uni- or bidirectionality of replication.

Replication of adeno-associated virus in synchronized cells without the addition of a helper virus

We investigated the helper-independent replication of adeno-associated virus (AAV) in cells synchronized by pretreatment with hydroxyurea, reversal of polyamine depletion, or physical mitotic detachment. In Chinese hamster cells (OD4 line) treated with hydroxyurea prior to infection. AAV underwent a complete cycle of replication. Transfection of such cells with plasmid-cloned AAV DNAs also gave rise to infectious viral progeny. Synchronization of OD4 cells by reversal of polyamine depletion or mitotic detachment led to independent AAV DNA synthesis (and infectious viral progeny in the case of the former procedure), but these procedures were not as effective as hydroxyurea pretreatment. Independent AAV DNA synthesis was also detected in some other cell lines of Chinese hamster, human, and monkey origin treated with hydroxyurea prior to infection. The results demonstrate that, in contrast to previous notions, the AAV infectious process is not absolutely dependent upon the addition of a coinfecting helper virus.

Transfection of a rearranged viral DNA fragment, WZhet, stably converts latent Epstein-Barr viral infection to productive infection in lymphoid cells.

An Epstein-Barr viral gene (ZEBRA) is identified that, in human lymphoblastoid cells, activates a switch causing the virus to shift from the latent to the replicative phase of its life cycle. We have shown that a 2.7-kilobase-pair rearranged Epstein-Barr virus DNA fragment of this gene (BamHI fragment WZhet) induced transient expression of viral replicative antigens and polypeptides when it was transfected into a somatic cell hybrid, which was derived from the fusion of an epithelial line cell with a Burkitt lymphoma cell. We now show that this rearranged WZhet fragment, when introduced stably into lymphoblastoid cells, will activate expression of the complete viral replicative cycle in 1-10% of the lymphoblastoid cells, leading to production of biologically active virions that can immortalize primary lymphocytes. The transfected plasmid appears to be regulated in a manner analogous to the complete Epstein-Barr virus genome.

Complete enzymatic replication of plasmids containing the origin of the Escherichia coli chromosome.

During enzymatic replication of plasmids containing the origin of the Escherichia coli chromosome, oriC, formation of an active initiation complex consisting of dnaA, dnaB, dnaC, and HU proteins, requires a supercoiled DNA template. Relaxed covalently closed plasmids are active only if supercoiled by gyrase prior to initiation; nicked and linear DNAs are inactive. Semi-conservative replication proceeds via delta structure as intermediates. Daughter molecules include nicked intermediates. Daughter molecules include nicked monomers and catenated pairs. Elongation is rapid, but late replicative intermediates accumulate because the final elongation and termination steps are slow. Production of covalently closed circular daughter DNA molecules requires removal of ribonucleotide residues (primers) by DNA polymerase I, assisted by ribonuclease H, gap filling, and ligation of nascent strands by ligase. Reconstitution of a complete cycle of oriC plasmid replication, beginning and ending with supercoiled molecules, has been achieved with purified proteins.

Inhibition of a Complete Replication Cycle of Human Cytomegalovirus in Actinomycin Pre-treated Cells

The study of human cytomegalovirus (HCMV) in cultures of human embryo lung fibroblasts, pre-treated with actinomycin D, has shown that under these conditions the virus infection does not proceed beyond the 'early' events of the virus replication cycle. In the same experimental conditions the growth of poliovirus type I, vaccinia virus and herpes simplex type I virus, was completely unaffected. These results suggest that the complete HCMV replication cycle requires some cellular function(s) between early transcription of the input virus genome and virus DNA synthesis.

Isolation of host-range variants of mouse mammary tumor viruses that efficiently infect cells in vitro

Host-range variants of mouse mammary tumor virus (MMTV) have been isolated that have the ability to productively infect cells in vitro with high efficiency (at multiplicities of infection </=1) and with extremely short latent periods to the production of de novo virus (as short as 4 days after infection). These variants of the highly oncogenic MMTV of RIII, C3H, and GR mice were obtained by serial virus passage in feline cells. The resultant variant stocks react in group-specific radioimmunoassays for the MMTV major external glycoprotein (gp52) and major internal protein (p28), possess a protein profile similar to that of wild-type MMTV, and contain a virion-associated DNA polymerase with a magnesium cation preference. Addition of dexamethasone and insulin to culture media enhances the titer of de novo MMTV to levels of approximately 10(10) particles per 75-cm(2) flask (containing 5 x 10(6) cells) per 24 hr. Variant stocks exhibit no evidence of contamination with either murine or feline type C retroviruses, as assayed by various techniques. The variants of MMTV derived from C3H and RIII mice exhibit differential host ranges that include the ability to productively infect feline, canine, bat, mink, murine, and human cells. Use of these MMTV host-range variants now facilitates the study of the complete replicative cycle of MMTV as well as an elucidation of the interaction of MMTV with various hormones, physical or chemical carcinogens, and tumor promoters in the initiation and promotion of mammary neoplasia.

The mediator of cellular immunity. XII. Inhibition of activated T cells by Newcastle disease virus.

Newcastle disease virus (NDV) can interact in at least two ways with rat T cells. By adsorbing to circulating lymphocytes, the virus can transiently deflect the cells from lymph nodes and inflammatory exudates induced in the peritoneal cavity. T cells are affected regardless of age, state of activation, or position in the mitotic cycle. The effect is reversible and is mediated not only by infectious (I)-NDV, but also by UV-NDV which cannot achieve a complete replication cycle in eggs. But I-NDV has another lasting effect on activated T cells. It is revealed in the failure of virus-treated thoracic duct lymphocytes to transfer cellular resistance to Listeria monocytogenes, delayed-type hypersensitivity to soluble antigens of the parasite, and the permanent exclusion of labeled S-phase lymphocytes from inflammatory foci. Activated T cells are inhibited by virus multiplicites which have little if any effect upon the proliferative potential of antigen-sensitive T cells or localization of labeled small lymphocytes in lymph nodes. The underlying mechanism has not been determined; however, there are reasons for thinking that NDV has a lethal effect upon activated T cells, because the latter are permissive for virus replication.

Formation in the dark, of virus-induced deoxyribonuclease activity in Anacystis nidulans, an obligate photoautotroph

In Anacystis nidulans, upon infection with cyanophage AS-1, after a lag period of 1 h the level of deoxyribonuclease (DNase) activity increaded rapidly up to 15- to 20-fold in 4 to 5 h in the light. In contrast, the ribonuclease and phosphomonoesterase activities increased significantly only 4 to 5 h after infection, i.e. as late as 1 h prior to lysis. In complete darkness, the nuclease levels remained unaltered. However, when the infected cells were exposed to light for 1 or 2 h after infection, the DNase level increased essentially to the same extent in the dark as in continuous light, although the complete replication cycle of the virus was impaired in the dark and cells lysed only in the continuously illuminated cultures. Inhibition of photosystem II with 3-(3,4-dichlorophenyl)-1-dimethylurea during the early illumination period strongly decreased the subsequent, infection-dependent increase in DNase activity in the dark. The virus-induced increase in DNase activity was also inhibited by chloramphenicol. The data suggest that, in spite of the obligate photoautotrophic nature of A. nidulans, dark metabolism is able to support fully the formation of some specific proteins if the triggering of their synthesis takes place in light.

The Use of Protoplasts in Plant Virology

Understanding virus infection in higher plants depends heavily upon knowledge of infection in the individual cells that constitute the whole plant body. Unfortunately, however, experimental materials suitable for studying infection at the cellular level have not been available. Materials such as plants, organs, or tissues are inadequate for this purpose, because the number of cells initially infected by inoculation is extremely small and because the stage of infection in individual cells becomes random as infection spreads. The concept of one-step virus growth was introduced into virology when Ellis & Delbriick (24) devised a procedure to simultaneously inoculate large numbers of Escherichia coli cells with a bacteriophage and to eliminate the chance of secondary infection. With the cells inoculated in this way, it is possible readily to follow one complete replication cycle of a virus. Such one-step growth experiments are one of the methodological foundations of modern bacterial and animal virology. In order to realize one-step growth of plant viruses, two major requirements must be fulfilled in an experimental material: (a) a substantial proportion of cells present must be infected simultaneously and, (b) the material must consist of separated single cells to minimize the possibility of infection being spread. Both of the require­ ments are not fulfilled by plant cells grown either as callus or in suspension cultures (41). The leaf systems developed by NiIsson-Tillgren et al (59) and by Dawson & Schlegel (21) satisfy the first but not the second requirement, whereas the separated cells from infected leaves (I, 8, 38, 75, 94, 100) meet the second but not the first. Since plant viruses are unable to penetrate rigid plant cell walls, one way to achieve a high frequency of cell infection is to remove the walls, that is, to use cell proto­ plasts.

Growth of adeno-associated satellite virus under conditions of arginine deprivation

Adeno-associated satellite viruses need participation of a helper adenovirus for their complete replication cycle in susceptible cells. Omission of exogenous arginine from the growth environment renders the adenovirus helper incapable of complete maturation. However, even in the absence of adenovirus particles, viable satellite virus can be isolated from the system. These particles, although originally infectious, lose infectivity rapidly and are less stable morphologically than satellite viruses produced under standard nutritional conditions. Particles are found in the nucleus and in the cytoplasm indicating that transport of viral components is not inhibited. In addition, type 4 satellite virus particles harvested from arginine-deficient growth conditions lack the hemagglutinin normally associated with the mature type 4 virion.

Posttranscriptional block to adenovirus replication in nonpermissive monkey cells

Events occurring during the abortive infection of monkey cells by human adenoviruses have been compared to those occurring during the complete replicative cycle which results from adenovirus and SV40 coinfection of these cells. Previous studies had indicated a block to replication at a posttranscriptional level. This paper describes the examination of two posttranscriptional events: attachment of polyadenylic acid sequences to adenovirus mRNA, and association of this mRNA with polyribosomes. Posttranscriptional addition of poly(A) sequences to mRNA appears to take place normally in the absence of SV40 coinfection. “Late” adenovirus mRNA, though synthesized in the nonpermissive infection, appears to be deficient in its association with polyribosomes. This finding is consistent with the previously described defect in late adenovirus protein synthesis in the unenhanced infection of monkey cells.

Genetic evidence for SV40 gene function in enhancement of replication of human adenovirus in simian cells.

HUMAN adenoviruses undergo a complete cycle of replication in human cells, but cause an abortive infection in simian cells. In the simian cells infected with the human adenovirus, early adenovirus-specific T antigen is induced1,2, viral DNA is replicated to the same level as in productive cycles of replication3,4, and no change in the synthesis of viral mRNA can be detected5,6. However, only limited amounts of viral capsid proteins are made in relatively few cells1,2,6–8. If the simian cells are coinfected with simian virus 40 (SV40), the nonproductive cycle of replication of human adenoviruses is changed to a productive cycle9,10. This helper effect of infection by SV40 is thought to be under the control of SV40 gene function, but there has been no direct evidence for this. Using temperature-sensitive (ts) mutants of SV40, I have now found such evidence.

Properties of a measles virus neuropathic for rhesus monkeys.

A highly hamster brain adapted strain of measles virus, capable of producing encephalitis in monkeys, inoculated into Vero cell cultures replicated in a suppressed growth cycle. No virus was released into the medium and the infected cells lacked specific virus determinants for hemadsorption. A complete replicative cycle was achieved after six subcultures of Vero cells and was accompanied by a partial drop in neurovirulence. However, hamster neuropathogenicity persisted throughout 26 Vero passages and was markedly higher than in non-brain passaged strains. High neurovirulence was associated with pronounced giant cell cytopathic effect and thermolability. On continuous passage these three properties dissociated at different rates indicating that they are independent properties of measles virus. The importance of their coincidence for the initiation of an encephalitic process in the brain of primates is stressed.