Overexpression of cellular dihydrofolate reductase abolishes the anticytomegaloviral activity of methotrexate

Summary.  Cytomegalovirus (CMV) stimulates numerous cellular pathways upon infection. One of these pathways involves activation of dihydrofolate reductase (DHFR), an essential enzyme in the biosynthesis of purines and thymidylate. Here we report that methotrexate (MTX), an inhibitor of DHFR, suppresses murine CMV replication at the level of DNA synthesis in quiescent NIH 3T3 cells. However, MTX has no antiviral activity in NIH 3T3 sublines resistant to MTX due to DHFR overexpression. These results directly link MTX antiviral activity to DHFR and demonstrate that DHFR plays an essential role for CMV replication in quiescent cells. received December 21, 1998 Accepted March 10, 1999     

Conditions for Cytomegalovirus Stimulation of Lymphocytes

Lymphocytes from healthy donors or from patients with chronic lymphocytic leukaemia were subjected to live or inactivated cytomegalovirus (CMV) or the mitogen phytohaemagglutinin. No early or late CMV antigens could be demonstrated in the lymphocytes, indicating that neither abortive nor replicative CMV infection takes place. Only cells from CMV antibody-positive leukaemic and non-leukaemic donors were stimulated by CMV to DNA synthesis, with a maximum on day 5. Cells from all individuals responded to phytohaemagglutinin stimulation, the peak of activity occurring on day 3. The stimulation with CMV occurred in T cells and was independent of early CMV antigen production, viral DNA synthesis, or viral replication. CMV is thus not an in vitro lymphocyte mitogen like Epstein-Barr virus but is a very potent antigen for memory T cells.     

Selective inhibition of human cytomegalovirus DNA synthesis by (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine [(S)-HPMPC] and 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG)

The novel acyclic nucleoside phosphonate, (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine [(S)-HPMPC], is a potent and selective inhibitor of human cytomegalovirus (CMV) replication in cell culture. (S)-HPMPC inhibits CMV DNA synthesis in a concentration-dependent manner within the concentration range of 0.04-4 micrograms/ml. At 4 micrograms/ml, viral DNA synthesis is completely suppressed. (S)-HPMPC proved more inhibitory to CMV replication and CMV DNA synthesis than 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG, ganciclovir), the current drug of choice for the treatment of CMV infections. Both compounds affected cell proliferation and cellular DNA synthesis only at a concentration that was 100- to 500-fold higher than the antivirally effective concentrations. In accord with the postulated target (viral DNA synthesis) for its antiviral action, (S)-HPMPC did not prevent immediate early antigen expression in CMV-infected cells. A limited exposure time (as short as 6 hr postinfection) of the CMV-infected cells to (S)-HPMPC sufficed to afford a pronounced and prolonged inhibition of viral DNA synthesis and virus replication. This gives (S)-HPMPC a definite advantage over DHPG, which only afforded a weak and transient inhibition of CMV DNA synthesis and virus replication after it had been exposed to the cells for a short exposure time. The long-lasting antiviral action of (S)-HPMPC is a unique property that opens new therapeutic modalities for the treatment of virus infections.     

Enhanced poliovirus replication in cytomegalovirus-infected human fibroblasts.

Replication of poliovirus in human cytomegalovirus (CMV)-infected cells is enhanced 5-to 10-fold over replication in uninfected cells. Enhanced poliovirus replication in dually infected cells was not due to a difference in adsorption on infected cells and was supported by evidence of increased synthesis of polio-specific RNA. A functional CMV genome appeared to be required for the enhancement of polio replication since enhanced replication was not seen in cells infected with uv-irradiated CMV or in cultures treated with the inhibitors of CMV replication. Enhanced polio replication in CMV-infected cells may be due to the enhanced cellular metabolism in these cells.     

Effect of butyrate on adenovirus infection in semipermissive cells

Adenovirus 2 stimulated cellular DNA synthesis in quiescent cultures of semipermissive tsAF8 cells and 3T3 cells. Such stimulation was inhibited by Na-butyrate, which also inhibited viral DNA replication in tsAF8 cells. In addition, butyrate inhibited the expression of early regions E1A and E2 of adenovirus 2 in both tsAF8 and 3T3 cells, while it had little effect on permissive HeLa cells.     

Induction of cellular DNA synthesis and increased mitotic activity in syrian hamster embryo cells abortively infected with human cytomegalovirus.

The effect of human cytomegalovirus (CMV) on cell DNA synthesis and mitotic activity in hamster embryo fibroblasts was examined. The results indicated that CMV infected cells had increased rates of cell DNA replication and mitotic activity. Detection of the effect of CMV on these two parameters necessitated arrest of cells prior to infection with low serum concentrations. This lowered the background levels of DNA synthesis and cell division so that the effect of virus infection could be detected. The data indicate that cells arrested prior to infection demonstrate increased susceptibility to virus infection. It was also observed that the effect of CMV on both DNA replication and mitotic activity could be enhanced by irradiation with ultraviolet light of the virus prior to infection.     

An Amino-Terminal Fragment of SV40 T Antigen Induces Cellular DNA Synthesis in Quiescent Rat Cells

SV40 large T antigen can stimulate cellular DNA synthesis in quiescent cells. Here we report that an SV40 mutant, which expresses only the amino-terminal 147 amino acids of this protein and which does not make small t antigen, stimulates cellular DNA synthesis in quiescent rat cells at levels similar to those achieved with serum stimulation. Thus, the amino-terminal 147 amino acids of large T antigen define a domain of this protein that is sufficient for efficient stimulation of cellular DNA synthesis in the absence of small t antigen.     

Induction of Immunoglobulin Secretion and DNA Synthesis in Human Lymphocytes in Vitro by Cytomegalovirus Preparations

Immunoglobulin secretion as evidenced by plaque-forming cells (PFC) in an indirect haemolysis-in-gel assay, and DNA synthesis were induced in human blood lymphocytes by the following preparations of cytomegalovirus (CMV): Nucleocapside Nc-CMV antigen, membrane M-CMV antigen, crude C-CMV preparations and CMV-incubated adherent cells. Peak stimulations occurred around day 6 in culture. Nc-CMV and M-CMV only stimulated PFC and DNA synthesis in lymphocytes from CMV seropositive individuals. C-CMV also stimulated lymphocytes from CMV seronegative individuals but gave better responses in lymphocytes from CMV seropositive individuals. CMV-incubated adherent cells occasionally stimulated lymphocytes from CMV seronegative individuals but always in CMV seropositive blood donors. Nc-, M-, and C-CMV gave high numbers of PFC in B cells enriched by sheep erythrocyte sedimentation and in co-cultures of enriched T and B cells. Almost no PFC were induced in enriched T cells. DNA synthesis induced by all the three antigenic CMV preparations increased after removal of adherent cells. Strong DNA synthesis was induced in enriched T cells compared to almost none in enriched B cells. It is concluded that some pure CMV preparations act as antigens and more crude preparations induce polyclonal activation. Different CMV preparations may be used to diagnose CMV, to study immune reactivity against CMV and as a model for CMV infections in vitro.     

The HIV-1 Nef protein has a dual role in T cell receptor signaling in infected CD4+ T lymphocytes

The phenotypic changes that are induced by immune activation in CD4⁺ T lymphocytes provide an optimal environment for efficient HIV-1 replication in these cells. The pathogenic Nef protein of HIV-1 modulates the T cell receptor (TCR) signaling, but whether this has a positive or negative effect on cellular activation is a matter of debate. Here we have investigated the response to TCR stimulation of primary CD4⁺ T lymphocytes infected with wt or Nef-deficient HIV-1. Results show that, in freshly isolated quiescent T cells, Nef superinduces NFAT and IL-2 production bypassing early TCR effector molecules. Conversely, the early phosphorylation of PLC-γ1, the induction of NFAT, and the expression of IL-2 are impaired by Nef in sub-optimally activated/resting T cells. Our data indicate that Nef has a dual role in the modulation of TCR signaling aimed at favoring HIV-1 replication and spread in both quiescent and metabolically active CD4⁺ T lymphocytes.     

Effect of 2'5'-oligoadenylic acid on a mouse cell line partially resistant to interferon

We have investigated the sensitivity of a mouse cell line, NIH 3T3 (clone 1), that was found to be resistant to the anticellular and certain antiviral activities of interferon, to 2′,5′-oligoadenylate (2′5′A). The 2′5′A was introduced into the cells by coprecipitation with calcium phosphate and by increasing cell permeability with lysolecithin. In both cases neither cellular protein nor DNA synthesis was inhibited by 2′5′A in the NIH 3T3 cells. In contrast, the synthesis of proteins and DNA in L cells, an IFN sensitive line, was inhibited by low concentrations of 2′5′A. Furthermore, treatment of the infected cell cultures with 2′5′A resulted in the inhibition of vesicular stomatitis virus (VSV) replication in L cells but not in NIH 3T3 cells. The production of MuLV in NIH 3T3 cells was also not affected by 2′5′A. These observations, together with our previous finding that NIH 3T3 (clone 1) cells are deficient in RNase F activity suggest that activation of the RNase F by 2′5′A is responsible for the inhibition of both protein and DNA synthesis in sensitive cells.     

Cell cycle dependence of synthesis of unintegrated viral DNA in mouse cells newly infected with murine leukemia virus

We have studied Moloney murine leukemia virus (MuLV) replication in newly infected NIH/3T3 cells brought to a stationary phase by serum depletion. Progeny viruses were markedly decreased under these conditions. Studies of the early phase of the virus cycle by the Southern blot hybridization procedure revealed that levels of unintegrated linear double-stranded and supercoiled viral DNAs were decreased in quiescent NIH/3T3 cells as compared to levels detected in serum-replenished cells. When serum was added to quiescent cells up to 48 hr postinfection, we could detect an increase of viral DNA, suggesting the presence of a stable intermediate encoding viral information. In order to characterize this intermediate, stationary cells were labeled with BrdU at the time of serum addition so that substituted viral DNA molecules made under serum stimulation could be separated on CsCl gradients from those made under serum depletion. The analysis of this experiment revealed that upon serum addition, the majority of viral DNA was fully substituted (HH), indicating that it must have been synthesized from an RNA template. Also, an important part of viral DNA made after serum addition had an intermediate density (HL), suggesting that incomplete molecules made in quiescent cells were completed after serum addition. Our results clearly show that host factors are required for synthesis of viral DNA in NIH/3T3 cells newly infected with MuLV.     

Effect of co-incubation with cytomegalovirus on growth of interleukin 2-dependent lymphocytes

We have tested the ability of human cytomegalovirus (CMV) to interfere with the interleukin-2 (IL-2)-dependent proliferation of T lymphocytes in long-term tissue culture. The results indicate that CMV was able to establish an apparently abortive infection in approximately 40% of such cells, although productive viral replication could not be detected, and was able to impede cellular proliferation almost completely. The addition of high concentrations of exogenous IL-2 to cultures of CMV co-incubated cells was not readily able to overcome the anti-proliferation inhibitory effect induced by this virus. Exposure to CMV led to an approximate 50% decrease in the number of cells which expressed Tac Ag, or IL-2 receptor, at the cell surface.     

Analysis of cellular DNA synthesis during polyoma virus infection of mice: acute infection fails to induce cellular DNA synthesis.

It is widely believed that infection with various DNA viruses stimulates quiescent host cells to divide in preparation for virus replication. To examine this issue, the effects of acute polyoma virus infection on cellular DNA synthesis are observed in newborn mice. Using [3H]thymidine incorporation and fluorography of whole mouse sagittal sections, we observed clear, high-resolution images of organ-specific patterns of cellular DNA synthesis in newborn animals. No alteration in these patterns was observed during acute polyoma virus infection. Other methods, including measurements of [3H]thymidine-labeled DNA-specific activities in various tissues and in situ autoradiography, also failed to detect virus-induced alterations in cellular DNA synthesis. These results indicate that newborn animals have high endogenous levels of DNA synthesis and imply that acute polyoma virus infection may not be associated with further induced levels of cellular DNA synthesis.