Two distinct endogenous type C viruses isolated from the asian rodent Mus cervicolor: conservation of virogene sequences in related rodent species.

The cocultivation of a lung cell line from the Southeast Asian mouse Mus cervicolor with cells from heterologous species has resulted in the isolation of two new distinct type C viruses. Both viruses are endogenous to M. cervicolor and are present in multiple copies in the cellular DNA of these mice. One of the viruses, designated M. cervicolor type CI, replicates readily in the SIRC rabbit cell line and is antigenically related to the infectious primate type C viruses isolated from a woolly monkey (simian sarcoma-associated virus) and gibbon apes (gibbon ape leukemia virus). This virus is also closely related by both immunological and nucleic acid hybridization criteria to a type C virus previously isolated from a second Asian murine species, Mus caroli. The isolation of the M. cervicolor type C I virus thus provides further evidence that the infectious primate type C viruses originated by trans-species infection of primates by an endogenous virus of mice. The second virus, designated M. cervicolor type C II, replicates well in various cell lines derived from the laboratory mouse Mus musculus. While antigenically related to type C viruses derived from M. musculus, the M. cervicolor type C II virus isolate can be readily distinguished from standard murine leukemia viruses. Both new type C viruses from M. cervicolor are unrelated to the previously described retrovirus (M432) isolated from the same Mus species. The DNA of M. cervicolor therefore contains multiple copies of at least three distinct classes of endogenous viral genes. An examination of the cellular DNA of other rodent species for nucleic acid sequences related to the genomes of both M. cervicolor type C I and II reveals that both viruses have been highly conserved evolutionarily, and that other species of rodents, such as laboratory mice and rats, contain endogenous virogenes related to those in the DNA of M. cervicolor.     

Isolation and characterization of a human T cell leukemia virus type II from a hemophilia-A patient with pancytopenia.

Human T cell leukemia virus (HTLV) type I has been isolated from the cultured T cells of several patients with adult T cell leukemia (ATL) and has been etiologically linked to ATL. However, HTLV-type II has been isolated only once, from the T cells of a patient with a T cell variant of hairy-cell leukemia. We report here the isolation of HTLV-II-related virus from the cultured T cells of a hemophilia-A patient with pancytopenia. The T cell line (CM) grows in the absence of T cell growth factor. Cord blood T cells were rapidly transformed when co-cultivated with irradiated CM cells. Heterologous competition radioimmunoassays using purified HTLV-I p24 showed the expression of HTLV-IIMO-related protein in these cells. Electron microscopy of the CM cells showed the presence of intracellular and extracellular type C viral particles. Comparison of the proviral genome in the CM cell line and the prototype HTLV-IIMO-containing cell line (MO) by molecular hybridization with probes specific for HTLV-IIMO indicated that restriction cleavage sites were identical. The fresh peripheral blood leukocytes of the patient contained two complete copies of the proviral genome, despite the lack of HTLV-II p24 expression. The virus from the cell line CM is designated as HTLV-IICM to distinguish it from the original HTLV-IIMO isolate.     

Differentiated mouse epithelial cell line with hepatocyte characteristics

An epithelial cell line, 3105, with an unusual growth pattern has been derived from the liver of an (NZB x NZW)F1 mouse. When confluent, it forms a monolayer of closely packed cells interspersed with holes that do not fill in during cultivation. By electron microscopy, the line has tight and intermediate junctions as well as desmosomes typical of epithelial cells. It produces several enzymes normally present in liver including hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, glucose-6-phosphatase, and alkaline phosphatase; has cytochromes P-450 and b5; and spontaneously release xenotropic but not ecotropic endogenous mouse type C viruses. Inoculation of the cell line into athymic nude mice gives rise to benign cysts in 2-3 months. This mouse epithelial line with hepatocyte characteristics should be helpful to investigators as a cell model of normal liver cell differentiation.     

Infectious primate type C virus proviral DNA in productively infected cells.

Cell lines competent to infection by DNA from cultures chronically infected by type C viruses of the simian sarcoma virus and baboon endogenous virus groups were identified. Significant differences were observed in the relative susceptibility of some cell lines to infection by a given proviral DNA. Practical applications of transfection techniques for the separation of viruses from dually infected cultures and to free virus stocks from mycoplasmal contamination are described.     

Biological properties of a type C virus isolated from a human X mouse hybrid cell line.

The biological properties of the HMV-1 virus, spontaneously released from a human X C57BL/6 mouse hybrid cell line, were similar to those of RadLV, the prototype B-tropic virus of C57BL/6 mice. Both viruses replicated on B-type mouse cells and in the wild mouse cell line SC-1. The plaque-forming abilities of the two viruses were relatively low, but gradually increased after passage in new host cells. Both viruses were neutralized by AKR antisera but not by FMR antisera. HMV-1 virus could rescue the defective sarcoma genome from S+H- mouse cells. The pseudotype sarcoma virus so produced was deficient in "helper virus" activity. Newborn mice inoculated with HMV-1 virus remained tumor-free over a 1-yr observation period.     

Suppression of human lymphocyte mitogen response by disrupted primate retroviruses of type C (baboon endogenous virus) and type D (PMFV)

Disrupted primate retroviruses of type C (baboon endogenous virus, BaEV) and type D (human cell line-derived isolate PMFV) considerably suppressed Concanavalin A - induced blastogenic response of human lymphocytes. Rauscher mouse leukemia virus (RLV) displayed a suppressive activity on murine splenic lymphocytes when tested under analogous conditions. The immunosuppressive activities were shown not to result from cytotoxicity or from virus-mitogen binding.     

Replication of a macrophage-tropic strain of human immunodeficiency virus type 1 (HIV-1) in a hybrid cell line, CEMx174, suggests that cellular accessory molecules are required for HIV-1 entry.

To investigate the mechanism underlying one aspect of the cellular tropism of human immunodeficiency virus type 1 (HIV-1), we used a macrophage-tropic isolate, 89.6, and screened its ability to infect a number of continuous cell lines. HIV-1 (89.6) was able to replicate robustly in a T-cell/B-cell hybrid line, CEMx174, while it replicated modestly or not at all in either of its parents, one of which is the CD4-positive line CEM.3. Analysis by transfection of a molecular clone, a virus uptake assay, and polymerase chain reaction all provided strong evidence that the block to HIV-1(89.6) replication in the CEM.3 line lies at the level of cellular entry. These results were complemented by preparing a CD4-expressing derivative of the B-cell parent, 721.174, and demonstrating that it is permissive for productive HIV-1(89.6) replication. Given these experimental findings, we speculate that there exist cellular accessory factors which facilitate virus entry and infection in CD4-positive cells. Furthermore, these cellular accessory factors may be quite virus strain specific, since not all macrophage-tropic strains of HIV-1 were able to replicate in the CEMx174 hybrid cell line. This experimental model provides a system for the identification of one or more of these putative cellular accessory factors.     

Sequence arrangement and biological activity of cloned feline leukemia virus proviruses from a virus-productive human cell line.

We examined 14 different feline leukemia virus proviruses from the productively infected human cell line RD(FeLV)-2 after cloning in the modified lambda vector Charon 4A. Each isolate was characterized by restriction digestion and Southern blot analysis. The DNA of each isolate was tested for competence to express virus after uptake by sensitive animal cells (transfection). All but one isolate contained an apparently complete provirus, but only four were infectious. Seven isolates (four noninfectious, three infectious) were studied by heteroduplexing followed by electron microscopy or by S1 nuclease treatment and gel electrophoresis. No regions of nonhomology between proviruses were detected by either criterion, and in no case did we observe homology between flanking sequences. Random shearing or removal of flanking sequences by S1 nuclease had no effect on the status of infectivity of the clones. Thus, we were unable to find molecular differences between infectious and noninfectious proviruses. Our data are consistent with either of the following hypotheses: (i) that there is a short host sequence which is essential as a promoter for virus expression; or (ii) that lack of infectivity is due to small mutations within the proviral genome.     

Herpes simplex virus infection and propagation in a mouse L cell mutant lacking heparan sulfate proteoglycans.

We have isolated a variant line of mouse L cells, termed gro2C, which is partially resistant to infection by herpes simplex virus type 1 (HSV-1). Characterization of the genetic defect in gro2C cells revealed that this cell line harbors a specific defect in the heparan sulfate synthesis pathway. Specifically, anion-exchange high-performance liquid chromatography of metabolically radiolabeled glycosaminoglycans indicated that chondroitin sulfate moieties were synthesized normally in the mutant cells, whereas heparin-like chains were absent. Because of these properties, we have used these cells to investigate the role of heparan sulfate proteoglycans in the HSV-1 life cycle. In this report, we demonstrate that the partial block to HSV-1 infection in gro2C cells occurs in the virus entry pathway. Virus adsorption assays using radiolabeled HSV-1 (KOS) revealed that the gro2C cell surface is a relatively poor target for HSV-1 in that virus attachment was 85% lower in the mutant cells than in the parental L cell controls. A portion of the 15% residual virus adsorption was functional, however, insofar as gro2C cells were susceptible to HSV-1 infection in plaque assays and in single-step growth experiments. Moreover, although the number of HSV-1 plaques that formed in gro2C monolayers was reduced by 85%, the plaque morphology was normal, and the virus released from the mutant cells was infectious. Taken together, these results provide strong genetic evidence that heparan sulfate proteoglycans enhance the efficiency of HSV attachment to the cell surface but are otherwise not essential at any stage of the lytic cycle in culture. Moreover, in the absence of heparan sulfate, other cell surface molecules appear to confer susceptibility to HSV, leading to a productive viral infection.     

Multiplication of a granulosis virus isolated from the potato tuber moth in a new established cell line of Phthorimaea operculella

Summary A newly established cell line was obtained from the culture of embryonic cells of the potato tuber moth Phthorimaea operculella in low temperature conditions (19° C) using modified Grace’s medium supplemented with 10% fetal bovine serum. The population doubling time was about 80 h when cells were cultivated at 19°C and 38 h at 27° C. The cell line had a relatively homogeneous population consisting of various sized spherical cells. The cells were cultivated for more than 25 passages. Their polypeptidic profile was different from profiles of other P. operculella cell lines we previously described and from other lepidopteran cells. The new cell line was designated ORS-Pop-95. The complete replication of the potato tuber moth granulosis virus (PTM GV) was obtained in vitro by both viral infection and DNA transfection. PTM GV multiplied at a significant level during several passages of the cell line that was maintained at 19° C. As long as the cells were maintained at 19° C, virus multiplication could also be obtained at the same rate at 27° C. To compare PTM GV multiplied both in vivo and in vitro, we used morphological identification, serological, DNA probe diagnosis and endonuclease digest profile analysis and confirmed the identity of the virus.     

A retrovirus-producing transformed mouse cell line derived from a human breast adenocarcinoma transplanted in a nude mouse

Summary A transplantable tumor was established in NIH/Swiss/Nu mice from tissue derived from a human breast adenocarcinoma metastatic to the brain. Cultivation of dispersed cells from the third transplant generation of the tumor produced a rapidly growing, high-density culture of fibroblastlike cells. Chromosome and isozyme assays showed these cells to be of mouse origin. The cells behaved as an established line from initial culture. Cells of the tissue culture line, designated NM-1, produced rapidly growing fibrohistiocytomas in nude mice. Electron microscopy revealed that the cells produced large numbers of type C virus particles. Serological, biochemical, and infectivity assays indicated that the retrovirus produced by NM-1 cells is an ecotropic, infective, murine retrovirus antigenically related to, but distinguishable from, Gross and Moloney viruses. The virus did not transform mouse fibroblasts. The data support the conclusion that mouse stromal cells within the transplanted human tumor had undergone malignant transformation and induction to virus replication. The role of the virus in the malignant transformation remains to be clarified.     

A retrovirus-producing transformed mouse cell line derived from a human breast adenocarcinoma transplanted in a nude mouse

A transplantable tumor was established in NIH/Swiss/Nu mice from tissue derived from a human breast adenocarcinoma metastatic to the brain. Cultivation of dispersed cells from the third transplant generation of the tumor produced a rapidly growing, high-density culture of fibroblastlike cells. Chromosome and isozyme assays showed these cells to be of mouse origin. The cells behaved as an established line from initial culture. Cells of the tissue culture line, designated NM-1, produced rapidly growing fibrohistiocytomas in nude mice. Electron microscopy revealed that the cells produced large numbers of type C virus particles. Serological, biochemical, and infectivity assays indicated that the retrovirus produced by NM-1 cells is an ecotropic, infective, murine retrovirus antigenically related to, but distinguishable from, Gross and Moloney viruses. The virus did not transform mouse fibroblasts. The data support the conclusion that mouse stromal cells within the transplanted human tumor had undergone malignant transformation and induction to virus replication. The role of the virus in the malignant transformation remains to be clarified.     

Sialidase activity in mouse neuroblastoma cell lines

Sialidase activity was determined for three different neuroblastoma clonal lines derived from the A/J strain mouse C1300 neuroblastoma line. For each cell line, the endogenous and exogenous activities were less than 1 nmol sialic acid released/mg protein/90 min and 50 min reaction time, respectively. The C1300 tumor had similarly low levels of sialidase activity. The sialidase activity associated with the neuroblastomas is less than that associated with synaptosomes. Each cell line had a distinctly different ganglioside pattern varying in complexity from G_M3 to G_D1a. Treatment of the cells withVibrio cholerae sialidase under isosmotic conditions showed that cell-surface sialyl residues were susceptible to sialidase activity, with some of the susceptible residues coming from the ganglioside constituents. Of the total number ofV. cholerae sialidase-releasable sialyl residues, 50–60% were released by the neuroblastoma sialidase acting on endogenous substrate.     

Restricted infectivity of ecotropic type C retroviruses in mouse teratocarcinoma cells: Studies on viral DNA intermediates

Replication of Gross strain N-tropic type C retrovirus was markedly restricted in a pluripotential undifferentiated embryonal cell line (PCC₄) of murine teratocarcinoma, whereas the same virus could cause productive infection in a myoblast-derived differentiated line (PCD₁) of the same tumor origin. To investigate the restriction mechanism, we compared the initial viral DNA formation in these two cell lines. Analyses by means of a modified Hirt extraction procedure and a modified Southern gel transfer method indicated that PCC₄ and PCD₁ cells supported the synthesis of viral DNA intermediates after inoculation of the Gross virus. In both cells, a linear DNA duplex (form III viral DNA) appeared at 4 hr, reached a maximal level at 8–9 hr, and declined rapidly thereafter, while two closed-circular supercoiled DNA duplexes (form I viral DNA) showed their appearance, increase and decline in the 8–24 hr period. During the period from 34 to 78 hr after virus inoculation, another burst of viral DNA synthesis occurred in PCD₁ cells, presumably due to secondary virus infection, while at this period both form III and form I viral DNAs became undetectable in PCC₄ cells. The Hirt supernatant DNAs prepared from PCD₁ and PCC₄ cells 10 hr after virus inoculation were equally infectious for NIH3T3 cells in a DNA transfection assay. Both PCD₁ and PCC₄ cells were very poor recipients for DNA transfection, although one positive result with PCD₁ cells might suggest a difference between the two cell types in this aspect. These results indicate that restriction of type C retrovirus in undifferentated embryonl carcinoma cells occurs at a step subsequent to formation and maturation of viral DNA intermediates.     

Comparison of the ability of lactate dehydrogenase-elevating virus and its virion RNA to infect murine leukemia virus-infected or -uninfected cell lines.

Lactate dehydrogenase-elevating virus (LDV) has a strict species specificity. Cells or cell lines other than a particular subset of mouse primary macrophages which can support LDV replication in vitro have not been identified. LDV induces neurological disorders in old C58 or AKR strains, in which the involvement of multiple copies of the endogenous N-tropic murine leukemia virus (MuLV) genome and the Fv-1 locus of the mouse has been implicated. Our previous studies have demonstrated that LDV could infect and replicate in cell lines of the mouse or other species in vitro when they were infected with MuLV. The significance of and the precise mechanism underlying this phenomenon, however, remain unclear. We demonstrated in this study the efficient infection and replication of the virus in vitro by inoculation of its RNA mixed with liposome. No significant difference either in the efficiency of RNA transfection or in the ability to support its replication was observed among the various species' cell lines examined. In addition, by RNA transfection the virus replicated with equal efficiency in MuLV-infected and -uninfected cells or in macrophages derived from mice irrespective of their age. In contrast, the pattern of the infection by virus particles was quite different; LDV replication was observed only in macrophages (particularly from newborn mice) and MuLV-infected cells. By using various LDV isolates, it was demonstrated that the capability of replication between neurovirulent, LDV type C, and the other avirulent strains was almost the same in mouse cell lines when their RNA was introduced into the cells. Higher infectivity of LDV-C to MuLV-infected cells may be due to its efficient incorporation of the particles into MuLV-infected cells.