In this investigation, we studied the ability of human cytomegalovirus to infect peripheral blood mononuclear cells. With monoclonal antibody technology, we demonstrated that cytomegalovirus could infect human lymphocytes of T-and B-cell (PBM). Infectious CMV has occasionally been found in buffy-coat preparations (4-6) obtained from patients with clinical CMV infection; only rarely has the virus been isolated from healthy donors (7). Neither the exact cell involved in the leukocyte fraction nor the state of the virus in such cells has been established. When transformed lymphoblastoid and erythroleukemia cell lines were studied for the ability to support CMV replication, some researchers found evidence of short-term virus replication (8, 9), but rarely was infectious virus produced (10, 11). There have been occasional reports of expression of CMV antigens on B-lymphoblastoid cell lines (12). Attempts to infect normal PBM from healthy donors with laboratory-adapted strains of CMV have failed (11, 13).In the cascade regulation of the CMV genome (14), immediate-early protein synthesis precedes early polypeptide synthesis, both of which set the regulatory state for late polypeptide synthesis and production of mature virions. The early class polypeptides appear to have predominantly regulatory functions; the late polypeptides have mainly structural functions. We reasoned that if lymphocytes were abortively infected, techniques such as cocultivation assays and probes for late CMV gene products would be negative. Hence, we looked for CMV expression in peripheral blood lymphocytes (PBL) with monoclonal antibodies specifically produced to detect polypeptides relevant to major epochs in the cascade regulation of the viral genome. Prompted by observations of biological differences between strains of virus recently derived from infected patients and the fibroblastadapted strains of virus (15), we also studied both forms of this virus. MATERIALS AND METHODSLymphocyte Infection. PBM were removed from the blood of healthy human donors by density gradient centrifugation on Ficoll-Paque and infected at a multiplicity of 0.01-1.0 with CMV recently isolated from patients with various CMV syndromes (Table 1), or with stocks of plaque-purified laboratory-strain AD-169. The recent isolates were propagated in human foreskin fibroblasts (Flow Laboratories) for <12 passages. Because low-passage isolates of human virus are associated predominantly with the cell matrix (15), infected or mock-infected fibroblasts were sonicated, and this material was cultured with PBM for up to 6 days. Thereafter, the PBM were washed 3 times and prepared for immunofluorescence studies by air-drying and fixing the cells with acetone on glass slides. In some experiments, T-lymphocytes were positively selected from the PBM cultures by erythrocyterosetting techniques (17) or by sorting on a fluorescence-activated cell sorter (16).Immunofluorescence Techniques. Monoclonal antibodies were raised against CMV-infected fibroblasts with standard techniques...
One of the most challenging problems in medicine concerns latent virus infections, in particular the specific identification of tissues that harbor these viruses, the ways viruses persist, and the mechanism(s) by which such agents are activated . Cytomegalovirus (CMV),' like other herpes viruses, can persist within an organism in a latent form despite the presence of a vigorous antiviral immune response (reviewed in references 1 and 2) . Several epidemiologic surveys show that 40-80% of humans over the age of 40 have serologic evidence of prior CMV infection (1-3) and less than 1% of seropositive healthy individuals shed virus into their urine (4) . CMV infection would be regarded as an ordinary acute infection associated with numerous subclinical attacks, complete recovery, and eradication of the virus except that reappearance of this virus is common . For example, pregnancy is often associated with active CMV infection (5), and this virus has been recovered from up to 28% of pregnant women (6, 7) . Acute CMV infection occurs in nearly 90% of patients 1-2 mo after surgery for kidney transplantation (8) . CMV has been implicated as the cause of a mononucleosis-like illness occurring in patients after open heart surgery, and available evidence suggests that this infection is apparently transmitted by blood transfusions (9-13) . Both terminal leukemia and Hodgkin's disease in patients undergoing immunotherapy may also be associated with CMV infection (14) . The frequent occurrence of CMV infection in patients undergoing pregnancy, renal transplantation, and blood transfusions suggests that this virus may be activated by means of immunologic reaction to foreign antigens .Host-specific CMVs have been demonstrated in several species including guinea pig, rat, hamster, mouse, and man. The primary CMV infection in mice (MCMV) is very similar to that in man (15)(16) . Using the murine model, we studied whether MCMV was carried in lymphocytes taken from adult mice * This is publication no . 898
Murine cytomegalovirus (MCMV) does not productively infect OTT6050AF1 BrdU, F9, or PCC4 undifferentiated murine teratocarcinoma cell lines, as shown by immunofluorescence assays for viral antigens and by plaque assays for infectious virus. However, these cells were infected by a variety of other viruses. MCMV does productively infect PYS2 and OTT F12 differentiated murine teratocarcinoma cell lines. The replication of MCMV in the pluripotent PCC4 cell line was examined in detail. Undifferentiated PCC4 cells could be differentiated when propagated in the presence of dimethylacetamide, as judged by changes in the expression of H-2 antigens on the cell surface. Several viruses, including lymphocytic choriomeningitis virus, herpes simplex virus type 1, and vesicular stomatitis virus, replicated to a similar extent in differentiated and undifferentiated PCC4 cells. MCMV did productively infect differentiated PCC4 cells. In contrast, MCMV did not produce infectious virus, viral antigens, or substantial viral RNA in undifferentiated PCC4 cells. The molecular block of MCMV replication occurred at the level of MCMV RNA transcription. Undifferentiated PCC4 cells have receptors for MCMV and bind similar amounts of radiolabeled virus as differentiated PCC4 cells. After MCMV binds to its receptors on undifferentiated cells, MCMV penetrates the plasma membrane and is transported to the cells' nuclei. MCMV DNA was present in the cytoplasm, and small amounts of MCMV RNA (less than 17 percent of that found in MCMV-infected differentiated PCC4 cells) were found in the nucleus. However, MCMV RNA was not detected in the cytoplasm of undifferentiated cells. A latent infection was established by infecting undifferentiated PCC4 cells with MCMV, inactivating residual infectivity with antibodies to MCMV, and propagating cells under conditions that maintained the undifferentiated state. These MCMV-infected undifferentiated cells did not produce infectious virus, viral antigens, or viral RNA but did contain viral DNA detectable by DNA-DNA hybridization kinetics. Latency was terminated and infectious virus was made when such undifferentiated cells were induced to differentiate.
The expression of viral antigens on the surfaces of lymphocytic choriomeningitis virus (LCMV)-infected L-929 cells peaked 2-4 days postinfection and thereafter precipitously declined. Little or no viral antigen was expressed on the plasma membrane surfaces of persistently infected cells, but LCMV antigens were clearly present in the cytoplasms of most of those cells. Cells early after acute infection (days 2-4) were lysed by both virus-specific antibody and complement (C) and immune T lymphocytes. To the contrary, antibody and C did not kill persistently infected cells, but T lymphocytes did kill such cells although at a lower efficiency than acutely infected cells. The expression of viral antigens on the surfaces of infected cells was regulated by the virus- cell interaction in the absence of immune reagents and was closely associated with defective interfering (DI) LCMV interference. DI LCMV, per se, blocked the synthesis and cell surface expression of LCMV antigens, and DI LCMV generation immediately preceded a precipitous reduction in cell surface antigenicity during the acute infection. Persistently infected cells produced DI LCMV but no detectable S LCMV. Peritoneal cells isolated from mice persistently infected with LCMV resembled cultured persistently infected cells in their reduced expression of cell surface antigens and their resistance to LCMV superinfection. It is proposed that DI virus-mediated interference with viral protein synthesis may allow cells to escape immune surveillance during persistent infections.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.