Human T cell leukemia/lymphoma virus-infected antigen-specific T cell clones: indiscriminant helper function and lymphokine production

The ability of human T cell leukemia/lymphoma virus (HTLV)-I to alter the function of infected T lymphocytes was examined directly by investigating the properties of an antigen-specific T cell clone before and after transformation with HTLV-I. Following infection, the T4 antigen-specific clone manifested a tenfold increase in its surface interleukin 2 (IL 2) receptor (Tac) density and acquired the viral determinants p19, p24, and 4D12 not present in the uninfected clone. Prior to infection, the T cell clone responded to antigen stimulation in the presence of histocompatible antigen-presenting cells with proliferation and secretion of multiple lymphokines, including IL 2, B cell growth factor (BCGF), B cell differentiation factor (BCDF), and interferon-gamma (IFN-gamma). Following infection, the T cell clone both proliferated and produced constitutively three of these lymphokines (BCGF, BCDF, and IFN-gamma) in the absence of accessory cells or antigen. Co-cultivation with any accessory cells regardless of histocompatibility resulted in increased proliferation and lymphokine production. IL 2 production by the HTLV-I-transformed cell, however, could not be detected. Similarly, the uninfected clone was able to provide B cell help for Ig production only when stimulated with both histocompatible cells and antigen. In contrast, the infected cell provided T cell help to B cells in an unregulated manner, independent of antigen or histocompatibility. Thus, functions such as the induction of proliferation, B cell help, and lymphokine production, which are finely regulated in uninfected antigen-specific T cell clones, became indiscriminant after HTLV-I infection.     

Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus. VI. Migration and activity in vivo in acute and persistent infection

Cloned cytotoxic T lymphocytes (CTL) specific for lymphocytic choriomeningitis virus (LCMV) were adoptively transferred to syngeneic mice acutely or persistently (carrier mice) infected with LCMV. Although infectious virus was cleared from the spleens during acute LCMV infection begun 24 hr earlier and the spleens remained clear of virus for the 4 days of testing, there was no concomitant reduction of viral titers in lymph nodes. In contrast, adoptive transfer of cloned CTL into animals with persistent rather than acute LCMV infection resulted in deaths of syngeneic but not allogeneic recipients. LCMV-immune spleen cells taken 30 to 50 days after a primary immunization and activated by in vitro stimulation before transfer also caused death of syngeneic carrier mice. However, LCMV-immune spleen cell per se provoked no clinical manifestations when transferred but cleared infectious virus and viral nucleic acid sequences from syngeneic carrier mice. The migration of 51Cr-labeled, LCMV-specific, H-2-restricted cloned CTL was assessed in vivo. The circulation of these CTL clearly differed from that of spleen cells freshly isolated from uninfected mice and from non-LCMV-specific CTL clone. Further, the circulatory pattern of LCMV-specific, H-2-restricted, cloned CTL in carrier mice was markedly different than in uninfected animals; only 7% of the injected cells remained in the lungs of uninfected mice 8 hr after injection, whereas 30% had accumulated in the liver. However, 55% of the cells injected into carrier mice still remained in their lungs 8 to 16 hr later. Hence, LCMV-specific, H-2-restricted, cloned CTL have unique trafficking patterns in the presence of LCMV antigens and immune activities in vivo.     

Human T-cell leukemia virus type I infection of CD4+ or CD8+ cytotoxic T-cell clones results in immortalization with retention of antigen specificity.

The human T-cell leukemia virus type I (HTLV-I) is capable of chronically infecting various types of T cells and nonlymphoid cells. The effects of chronic infection on the specific functional activities and growth requirements of mature cytotoxic T lymphocytes (CTL) have remained poorly defined. We have, therefore, investigated the results of HTLV-I infection of both CD4+ and CD8+ human CTL clones. HTLV-I infection resulted in the establishment of functional CTL lines which propagated indefinitely in culture many months longer than the uninfected parental clone. The infected cells became independent of the need for antigen (target cell) stimulation as a requirement for proliferation and growth. Like their uninfected counterparts, however, these HTLV-I-infected clones remained strictly dependent on conditioned medium from mitogen-stimulated T lymphocytes for their growth. This growth factor requirement was not fulfilled by recombinant interleukin-2 alone. Furthermore, the infected lines remained functionally identical to their uninfected parental CTL clones in their ability to specifically recognize and lyse the appropriate target cells. Our findings indicate that the major effects of HTLV-I infection on mature CTL consist of (i) the capacity for proliferation in the absence of antigen stimulation and (ii) a prolonged or immortal survival in vitro, but they also indicate that the fine specificity and cytolytic capacity of these cells remain unaffected.     

Composition and molecular organization of lipids and proteins in the envelope of mycoplasmavirus MVL2.

MVL2 virus was purified from culture supernatants of infected Acholeplasma laidlawii cells by differential centrifugation, followed by velocity centrifugation in sucrose gradients. The purified virus contained 0.08 to 0.1 mumol of lipid phosphorous per ml of viral protein. Thin-layer chromatography of viral lipids revealed the presence of phospho-, glyco-, and phosphoglycolipids identical with those found in the host cell membrane, but the relative amount of phosphatidylglycerol was much lower than that in the virus. The fatty acid composition of lipids incorporated into the virus included lipids synthesized before and after infection. The freedom of motion of spin-labeled fatty acids in MVL2 depended markedly on temperature and on the position of the nitroxide group on the hydrocarbon chain of the probe, suggesting that the local environment of the probe has the properties of a lipid bilayer. Nevertheless, the lipid hydrocarbon chains in MVL2 appear to be less mobile than those in membranes of the host cells. Polyacrylamide gel electrophoresis of purified MVL2 revealed four major and about five minor polypeptide bands. None of the polypeptide bands gave a positive periodic acid-Schiff reaction. Lactoperoxidase-mediated iodination, followed by proteolytic digestion of intact MVL2 particles, revealed that at least two major polypeptides are localized on the external surface of the viral envelope.     

DNA distribution and respiratory activity of Spodoptera frugiperda populations infected with wild-type and recombinant Autographa californica nuclear polyhedrosis virus.

Spodoptera frugiperda cells were infected with a wild-type Autographa californica nuclear polyhedrosis virus and with a recombinant Autographa californica nuclear polyhedrosis virus. The recombinant virus was derived from the wild-type virus and produced beta-galactosidase instead of polyhedrin. The changes in cell size, cell growth, viability, DNA distribution, and respiratory activity were followed through the time course of the infection. The DNA content as measured by flow cytometry of infected cells increased to approximately 1.8 times the value of uninfected cells and the distributions of single-cell DNA content of the infected cells were strongly deformed. Early in the infection the respiratory activity passed through a maximum. The mitochondrial activity based on Rhodamine 123 labelling of cells infected with the recombinant virus, as determined by flow cytometry, also passed through a maximum at 24 h post infection while the mitochondrial activity of cells infected with the wild-type virus continued to increase. Evolution of single-cell mitochondrial activity was different in uninfected populations and in populations infected with wild-type and with recombinant virus. In all experiments performed, the recombinant virus influenced cell behavior and the measured parameters earlier than the wild-type virus. The influence of the multiplicity of infection was stronger for the wild-type virus than for the recombinant virus.     

Characteristics of serially propagated monkey kidney cell cultures with persistent rubella infection

Maassab, H. F. (University of Michigan, Ann Arbor), and J. A. Veronelli. Characteristics of serially propagated monkey kidney cell cultures with persistent rubella infection. J. Bacteriol. 91:436-441. 1966.-A persistent infection of LLC-MK(2) cells with rubella virus has been established and maintained for over 3 years. This "carrier culture," designated as LLC-MK(2)-RAL, possesses distinct morphological and biological characteristics when compared with the original uninfected LLC-MK(2) line. The mechanism of viral persistance has not been entirely elucidated, but available data suggest a regulated infection with transmission of the virus directly from cell to cell or through cell division. Interferon was isolated from RAL (rubella-associated line) culture, which explains partly the wide spectrum of resistance to superinfecting viruses. Amantadine, although inhibiting cultures of LLC-MK(2) cells infected with rubella virus, failed to cure the "carrier culture."     

Immunity to virus-free syngeneic tumor cell transplantation in the BALB/c mouse after immunization with homologous tumor cells infected with type C virus.

Syngeneic tumor cell lines free of endogenous type C virus or viral antigen antigen expression were derived from spontaneously occurring tumors of the BALB/cCr mouse. Two cell lines free of endogenous type C virus were examined and found to be highly tumorigenic in tumor growth kinetic studies. In vitro inoculation of these cell lines with Rauscher-murine leukemia virus (R-MuLV) resulted in their chronic infection in which 95 to 100% of the cells were scored as virus positive. These infected lines showed a highly significant increase in their immunogenicity as compared to their uninfected controls. Animals in which these virus-positive tumors regressed were then shown to be highly resistant to challenge with the uninfected tumor cell lines as well as to live R-MuLV. This observed resistance to uninfected tumor cell lines could not be induced by immunization of the mouse with uninfected tumor cells and R-MuLV simultaneously at the same injection site, nor could it be induced with lethally irradiated virus-infected tumor cells, subtumorigenic doses of uninfected cells, or inactivated R-MuLV or Gross leukemia virus (G-MuLV). Cell-mediated cytotoxicity studies revealed that spleen cells obtained from animals whose virus-infected tumors regressed were cytotoxic to homologous infected and uninfected tumor cells as well as to other uninfected tumor cell lines syngeneic to the BALB/c mouse. Correlation of in vitro cytotoxicity with in vivo immunity was provided by the Winn assay, by inoculation into susceptible mice of immune and nonimmune spleen cells premixed with uninfected tumor cells. The immune cells were highly effective in preventing this tumor cell transplantation. It was concluded that type-C virus infection of these syngeneic tumor cells resulted in their acquiring strong transplantation antigens that were in part due to the virion, but were at least in part due to alterations of antigens or haptens that are present in a less immunogenic form on the uninfected tumor cell.     

A human cell line selected for resistance to adenovirus infection has reduced levels of the virus receptor.

To investigate determinants of host cell susceptibility to infection, cells partially resistant to infection were selected from the rare cells which remained adherent after infection of a culture of A549 cells with Ad2RAE, a mutant of adenovirus type 2 whose vertex capsomers lack an Arg-Gly-Asp (RGD) sequence which mediates binding of wild-type virus to integrins. Integrins promote the internalization of attached virions, whereas adsorption itself results from binding of the viral fibers to an unidentified cellular receptor. Following three rounds of selection, a persistently infected culture was established in which virus replication was detected in approximately 5% of the cells. Uninfected cells were readily cloned from the culture, indicating that at any particular time the majority of cells in the culture were uninfected. The resistance of one clone of uninfected cells to infection was correlated with a 10-fold reduction in the concentration of fiber receptors on these cells compared with the parental A549 cell line, indicating that efficiency of virus adsorption depends on the receptor concentration. Surprisingly, the rate at which host cells internalized RGD-negative virus also was strongly dependent on the fiber receptor concentration. While internalization of wild-type virus is promoted by the binding of integrins to the penton base RGD sequence, these results suggest that virus also can enter cells by an alternate pathway which requires binding of virions to multiple fiber receptors.     

Stimulation of lymphokines in Jurkat cells persistently infected with vaccinia virus.

The response of the human CD4+ T-cell line Jurkat to infection with vaccinia virus was investigated. Virus titers peaked approximately 3 to 4 days after infection, while cell growth paralleled that of uninfected cells, indicating that growth rates were not appreciably affected by viral infection. Results from plaque assays and fluorescence-activated cell sorter (FACS) analyses of virus antigens demonstrated that a persistent infection in which the percentage of infected cells and the virus titers fluctuated from passage to passage was established. Further characterization of the persistent infection revealed that the virus influences cellular functions. Induction of interleukin-2 (IL-2) and IL-2 receptor alpha (IL-2R alpha) in Jvac cells was shown by enzyme-linked immunosorbent assay and FACS analysis, respectively. Hybridization of cellular RNA with cloned probes confirmed the increased IL-2 expression and demonstrated that Jvac cells also expressed more IL-6 but not gamma interferon (IFN-gamma) or IL-1 beta. Dual-antibody staining and FACS analysis for vaccinia virus antigens and IL-2R alpha indicated that IL-2R alpha expression was restricted to the infected cells. Jvac cells were also resistant to superinfection, an additional proof that persistent infection elicited phenotypic changes in the cell population.     

Mathematical model of influenza A virus production in large-scale microcarrier culture

A mathematical model that describes the replication of influenza A virus in animal cells in large-scale microcarrier culture is presented. The virus is produced in a two-step process, which begins with the growth of adherent Madin-Darby canine kidney (MDCK) cells. After several washing steps serum-free virus maintenance medium is added, and the cells are infected with equine influenza virus (A/Equi 2 (H3N8), Newmarket 1/93). A time-delayed model is considered that has three state variables: the number of uninfected cells, infected cells, and free virus particles. It is assumed that uninfected cells adsorb the virus added at the time of infection. The infection rate is proportional to the number of uninfected cells and free virions. Depending on multiplicity of infection (MOI), not necessarily all cells are infected by this first step leading to the production of free virions. Newly produced viruses can infect the remaining uninfected cells in a chain reaction. To follow the time course of virus replication, infected cells were stained with fluorescent antibodies. Quantitation of influenza viruses by a hemagglutination assay (HA) enabled the estimation of the total number of new virions produced, which is relevant for the production of inactivated influenza vaccines. It takes about 4-6 h before visibly infected cells can be identified on the microcarriers followed by a strong increase in HA titers after 15-16 h in the medium. Maximum virus yield Vmax was about 1x10(10) virions/mL (2.4 log HA units/100 microL), which corresponds to a burst size ratio of about 18,755 virus particles produced per cell. The model tracks the time course of uninfected and infected cells as well as virus production. It suggests that small variations (<10%) in initial values and specific rates do not have a significant influence on Vmax. The main parameters relevant for the optimization of virus antigen yields are specific virus replication rate and specific cell death rate due to infection. Simulation studies indicate that a mathematical model that neglects the delay between virus infection and the release of new virions gives similar results with respect to overall virus dynamics compared with a time delayed model.     

Tamiflu-resistant but HA-mediated cell-to-cell transmission through apical membranes of cell-associated influenza viruses

The infection of viruses to a neighboring cell is considered to be beneficial in terms of evasion from host anti-virus defense systems. There are two pathways for viral infection to "right next door": one is the virus transmission through cell-cell fusion by forming syncytium without production of progeny virions, and the other is mediated by virions without virus diffusion, generally designated cell-to-cell transmission. Influenza viruses are believed to be transmitted as cell-free virus from infected cells to uninfected cells. Here, we demonstrated that influenza virus can utilize cell-to-cell transmission pathway through apical membranes, by handover of virions on the surface of an infected cell to adjacent host cells. Live cell imaging techniques showed that a recombinant influenza virus, in which the neuraminidase gene was replaced with the green fluorescence protein gene, spreads from an infected cell to adjacent cells forming infected cell clusters. This type of virus spreading requires HA activation by protease treatment. The cell-to-cell transmission was also blocked by amantadine, which inhibits the acidification of endosomes required for uncoating of influenza virus particles in endosomes, indicating that functional hemagglutinin and endosome acidification by M2 ion channel were essential for the cell-to-cell influenza virus transmission. Furthermore, in the cell-to-cell transmission of influenza virus, progeny virions could remain associated with the surface of infected cell even after budding, for the progeny virions to be passed on to adjacent uninfected cells. The evidence that cell-to-cell transmission occurs in influenza virus lead to the caution that local infection proceeds even when treated with neuraminidase inhibitors.     

Androgen-independent proliferation of LNCaP prostate cancer cells infected by xenotropic murine leukemia virus-related virus

Xenotropic murine leukemia virus-related virus (XMRV) is a novel gammaretrovirus that was originally isolated from human prostate cancer. It is now believed that XMRV is not the etiologic agent of prostate cancer. An analysis of murine leukemia virus (MLV) infection in various human cell lines revealed that prostate cancer cell lines are preferentially infected by XMRV, and this suggested that XMRV infection may confer some sort of growth advantage to prostate cancer cell lines. To examine this hypothesis, androgen-dependent LNCaP cells were infected with XMRV and tested for changes in certain cell growth properties. We found that XMRV-infected LNCaP cells can proliferate in the absence of the androgen dihydrotestosterone. Moreover, androgen receptor expression is significantly reduced in XMRV-infected LNCaP cells. Such alterations were not observed in uninfected and amphotropic MLV-infected LNCaP cells. This finding explains why prostate cancer cell lines are preferentially infected with XMRV.     

Alterations in chromatin of cells infected with RNA tumor viruses.

Chromatins were isolated from murine leukemia or sarcoma virus infected lymphocyte-like TB cells and compared by immunological and biochemical methods. Chromatin from virus infected cells did not fix complement as well as uninfected cell chromatin suggesting that conformational changes had occurred in chromatin from virus infected cells. This alteration was detected within 24 hours after infection. Infected cell chromatins, examined by electrophoretic methods after radiolabeling displayed alterations in nonhistone proteins, whereas the histones appeared unaltered. The non-histones were synthesized in greater amounts in infected compared to normal cells, particularly a 60,000 D protein, while the amount of histone did not vary. The above changes should not have been due to cell growth or cycle variations, for the cells had similar growth rates and were harvested from the same stage of cell confluency during exponential growth to ensure uniformity of culture conditions.     

Assessment of virus infection in cultured cells using metabolic monitoring

A rapid, in-process assessment of virus replication is disired to quickly investigate the effects of process parameters on virus infection, and to monitor consistency of process in routine manufacturing of viral vaccines. Live virus potency assays are generally based on plaque formation, cytopathic effect, or antigen production (TCID₅₀) and can take days to weeks to complete. Interestingly, when infected with viruses, cultured cells undergo changes in cellular metabolism that can be easily measured. These phenomena appear to be common as they has been observed in a variety of virus-host systems, e.g., in insect cells infected with baculovirus, Vero cells infected with Rotavirus, MRC-5 cells infected with Hepatitis A virus, and MRC-5 cells infected with the Varicella Zoster Virus (VZV). In this article, changes in glycolytic metabolism of MRC-5 cells as a result of CVZ infection are described. Both glucose consumption and lactate production in VZV infected MRC-5 cells are significantly elevated in comparison to uninfected cells. Based on this result, a rapid, in-process assay to follow VZV infection has been developed. The relative increase in lactate production in infected cells () increases as the infection progresses and then plateaus as the infection peaks. This plateau correlates with time of peak virus titer and could be used as a harvest triggering parameter in a virus production process.X_u = cell density of uninfected cellsX_i = cell density of infected cellsX_T = total cell densityL_i = cumulative lactate production in infected culturesL_u = cumulative lactate production in uninfected culturesq_Li = specific lactate production of infected cellsq_Lu = specific lactate production of uninfected cellsk₁, K₂ = constantsList of Symbols     

Effect of Vesicular Stomatitis Virus Infection on the Histocompatibility Antigen of L Cells

When mouse L cells are infected for 22 hr with vesicular stomatitis virus (VSV), a ribonucleic acid-containing enveloped virus, greater than 70% of the major histocompatibility antigen (H-2), is no longer detectable by the method of inhibition of immune cytolysis. Infected cells prelabeled with (14)C-glucosamine also show a correspondingly greater loss of trichloroacetic acid-insoluble radioactivity than uninfected cells. The loss of H-2 antigenic activity is not due to the viral inhibition of host cell protein synthesis since cells cultured for 18 hr in the presence of cycloheximide have the same amount of H-2 activity as untreated controls. Also, cells infected with encephalomyocarditis virus, a picornavirus, show no loss of H-2 activity at a time when host cell protein synthesis is completely inhibited. VSV structural proteins associated in vitro with uninfected L-cell plasma membranes do not render H-2 sites inaccessible to the assay. Although antibodies may not combine with all the H-2 antigenic sites on the plasma membrane, anti-H-2 serum reacted with L cells before infection does not prevent a normal infection with VSV. H-2 activity can be detected in virus samples purified from the medium of infected L cells; this virus purified after being mixed with L-cell homogenates shows greater H-2 activity than virus purified after being mixed with HeLa cell homogenates. However, VSV made in HeLa cells shows no H-2 activity when mixed with L-cell homogenates.     

Structural and biological properties of mycoplasmavirus MVL3: an unusual virus-procaryote interaction.

The kinetics of adsorption and growth of mycoplasmavirus MVL3 in Acholeplasma laidlawii 1305/68 host cells have been studied with one-step growth, premature lysis, and single-burst experiments. The virus was found to kill infected host cells. Virus release starts 90 min after infection and continues for about 10 to 15 h. Hence, virus production is unlike the classical lytic bacteriophages and instead resembles nonlytic cytocidal animal viruses. Structural details of the virus are described, and the molecular weight of the viral linear DNA has beenfound to be 26 x 10(6).     

Altered cellular functions in a PC-12 cell clone chronically infected with retrovirus

We characterized retrovirus-induced changes in PC-12 cell function and neuronal differentiation. PC-12 cells were infected with a neurotropic retrovirus (temperature-sensitive Moloney murine leukemia virus, mutant BA-1). We isolated a cell clone from this infected culture that displayed altered response to nerve growth factor; increased choline acetyltransferase activity; and decreased basal and nerve growth factor-stimulated acetylcholinesterase activity. In addition, Kirsten murine sarcoma virus infection of and subsequent expression of the v-ras oncogene in PC-12 cells induced neurite extension, enhanced choline acetyltransferase activity, and limited the growth potential of the infected cells.     

Persistent measles virus infection enhances major histocompatibility complex class I expression and immunogenicity of murine neuroblastoma cells

The effect of persistent measles virus infection on the expression of major histocompatibility complex (MHC) class I antigens was studied. Mouse neuroblastoma cells C1300, clone NS20Y, were persistently infected with the Edmonston strain of measles virus. The persistently infected cell line, NS20Y/MS, expressed augmented levels of both H-2K^k and H-2D^d MHC class I glycoproteins. Activation of two interferon(IFN)-induced enzymes, known to be part of the IFN system: (2–5)oligoadenylate synthetase and double-stranded-RNA-activated protein kinase, was detected. Measles-virus-infected cells elicited cytotoxic T lymphocytes that recognized and lysed virus-infected and uninfected neuroblastoma cells in an H-2-restricted fashion. Furthermore, immunization of mice with persistently infected cells conferred resistance to tumor growth after challenge with the highly malignant NS20Y cells. The rationale for using measles virus for immunotherapy is that most patients develop lifelong immunity after recovery or vaccination from this infection. Patients developing cancer are likely to have memory cells. A secondary response induced by measles-virus-infected cells may therefore induce an efficient immune response against non-infected tumour cells.