The use of hypertonicity to selectively inhibit host translation in murine cytomegalovirus-infected cells.

A low molecular weight fifth RNA component was found in a 1976 peanut stunt virus (PSV) isolate from Virginia. This PSV-associated RNA 5 (PARNA 5) occurred both in tobacco and cowpea infections with the 1976 PSV, but not with a very similar 1974 PSV isolate from Virginia. PARNA 5, isolated and purified from the 1976 PSV infections in tobacco, by itself caused no infection in tobacco. Neither PARNA 5 nor double-stranded (ds) PARNA 5 could be isolated from such plants. Production of PARNA 5 in tobacco was also supported by the 1974 PSV when PARNA 5 was present in the inoculum, except when it had first been irradiated with ultraviolet light. Host plants infected with PSV + PARNA 5 yielded the dsRNAs of all four PSV-RNAs as well as large quantities of dsPARNA 5. PARNA 5 was found to have a somewhat slower electrophoretic mobility than cucumber mosaic virus-associated RNA 5 (CARNA 5), suggesting that PARNA 5 was a slightly larger molecule than CARNA 5. In competition hybridization experiments, neither CARNA 5 nor 1974 PSV-RNA competed to any extent with PARNA 5 for duplex formation with (?) strands of dsPARNA 5. This confirmed that CARNA 5 and PARNA 5 are different molecules and demonstrated that PARNA 5 has a satellite-like relationship with PSV rather than being a defective RNA.     

The effect of heat-inactivated murine cytomegalovirus on host DNA synthesis of different cells.

Heat-inactivated murine cytomegalovirus (MCMV) stimulates cellular DNA synthesis in WME, NMG, 3T3, WgIA, chick and NIK-8 cells, but active or u.v.-irradiated MCMV does not. The stimulation of DNA synthesis in NIL-8 and chick cells was studied in detail. We found that both the nuclear and the mitochondrial DNA synthesis were stimulated in these cells. There was no virus DNA synthesis during the period we studied (48 h). The stimulation of nuclear DNA synthesis was about threefold in NIL-8 and 2.5-fold in chick cells as measured by the rate of incorporation of 3H-thymidine (3H-dThd) in the CsCl fractions which banded at the density of cell DNA. The stimulation was about 9.5-fold in NIL-8 and 1.7-fold in chick cells as detected by autoradiography. There was a 3-fold and a 2.2-fold increase in the degree of incorporation of 3H-dThd into mitochondrial DNA of NIL-8 and chick cells, respectively. The amount of mitochondrial DNA obtained in infected cells of both kinds was about twice that in control cells. The synthesis of mitochondrial DNA was also stimulated by a factor of 2 in the thymidine kinaseless 3T3 cells which incorporate exogenous thymidine exclusively into mitochondrial DNA. There were no MCMV specific antigens detectable by immunofluorescence 5 h after infection, but diffuse nuclear fluorescence could be demonstrated 24 h after infection. Our results indicate that the heat-inactivated virus penetrates the cells, stimulates host DNA synthesis and induces synthesis of early MCMV antigens.     

The enumeration of viral genomes in murine cytomegalovirus-infected cells.

Murine cytomegalovirus (MCMV) DNA synthesis in infected cells was measured by the technique of DNA-DNA reassociation kinetics, using ¹²⁵I-labeled MCMV DNA as the probe. This method circumvented the problems inherent in thymidine incorporation studies due to the low level of thymidine kinase activity in infected cells. MCMV-DNA synthesis was detected as early as 8 hr p.i. in mouse embryo cells.     

Cell-mediated cytotoxicity of murine cytomegalovirus-infected target cells allows for release of residual infectious virus

Summary. Although cell-mediated cytotoxicity effectively kills target virus-infected cells, no careful consideration has been given to the fate of infectious progeny virus contained within target cells following the cytolytic event. To address this issue with respect to murine cytomegalovirus (MCMV) pathogenesis, we developed a rapid semiquantitative assay for infectious MCMV based on expression of -galactosidase using a LacZ-expressing recombinant MCMV. Simultaneous use of this assay in combination with a modified cell-mediated cytotoxicity assay revealed that cytotoxicity of MCMV-infected target cells mediated by MCMV-immune splenic cells does not lead to inactivation of intracellular infectious virus.     

DNA synthesis in mouse embryo fibroblast cells infected with murine cytomegalovirus.

The overall rate of DNA synthesis in mouse embryo fibroblast (MEF) cells infected with murine cytomegalovirus (MCMV) decreased immediately after infection, reaching its minimum at 10–12 hr postinfection (PI), and then increased gradually. CsCl equilibrium centrifugation analysis of synthesis of both host and viral DNA in MEF cells infected with MCMV revealed the following: (1) Host cell DNA synthesis was inhibited by more than 95% by 10–12 hr PI. (2) Viral DNA synthesis began at 10–12 hr PI, and by 22–24 hr PI the rate was slightly higher than the rate of host cell DNA synthesis observed at 0–2 hr PI. (3) Viral DNA synthesis occurred without the concurrent synthesis of any significant amount of host cell DNA, indicating that there was preferential viral DNA synthesis. The inhibition of host DNA synthesis occurred when cells were infected with either uv- or heat-inactivated MCMV. This occurred in the absence of both cytopathic effects (CPE) and the synthesis of viral-induced proteins. In these instances, the suppression of host DNA synthesis was observed only during the first 12 hr PI. At 12–14 hr PI, host DNA synthesis began to increase, and finally reached or surpassed the level seen in mock-infected cells. Since the suppression of the host DNA synthesis occurred in the absence of viral-induced protein synthesis, the possibility that the suppression is caused by one or more of the structural proteins of the infecting viral particles is discussed.     

A cycloheximide-enhanced protein in cytomegalovirus-infected cells

Following the treatment of cytomegalovirus (CMV, strain Colburn)-infected cells with the drug cycloheximide early after infection, dramatically enhanced amounts of an otherwise minor protein are synthesized. Size and charge estimates, based on one- and two-dimensional separations in denaturing polyacrylamide gels, indicate that this protein has a molecular weight of 94K and an acidic net charge. Results of experiments to investigate its synthesis indicate that (i) the 94K protein is expressed by 2–4 hr after infection, (ii) its appearance requires de novo RNA synthesis, (iii) both the protein and its mRNA appear to be metabolically stable, and (iv) the increased amount of this protein following cycloheximide inhibition appears to be the result of selective mRNA amplification. On the basis of its partitioning during detergent fractionation procedures, it is suggested that the 94K protein may function in the cytoplasm.     

Coordinate control of host DNA synthesis and measles virus production in persistently infected BHK-21 cells

Summary Measles virus, upon infection, caused cytopathic effects in BHK-21 cells while it readily established persistent infection in a mutant strain of BHK-21 cells which possessed a temperature sensitive defect in DNA synthesis (BN2). This finding demonstrated that host function can be involved in allowing the establishment of persistent infection. When BN2 and infected BN2 cells were incubated at the non-permissive temperature (39.5° C), DNA synthesis was inhibited in a similar manner in both cells types, suggesting that viral functions do not interfere with the expression of the host mutation. Thets mutation which blocked the DNA synthesis also caused inhibition of viral yield, viral antigen expression and viral RNA synthesis at 39.5° C. The results suggest that continued DNA synthesis is necessary for the expression of measles functions.With 3 Figures     

The synthesis of coliphage T1 DNA: Degradation of the host chromosome

T1 derives most of the thymine for synthesis of its DNA from the host DNA. Nevertheless, degradation of host DNA to acid-soluble products cannot be observed even when synthesis of phage DNA is inhibited. The methods of inhibition were infection of a nonpermissive host with amber mutants with a DO (no DNA synthesis) phenotype, infection of a nonpermissive host with an amber mutant with a DA (DNA synthesis arrest) phenotype, shift to a nonpermissive temperature after infection with a ts mutant with a DO phenotype, and addition of nalidixic acid. We conclude that degradation of the host chromosome is linked to ongoing synthesis of phage DNA. During T1's development cycle, the bacterial nuclear region maintains a normal appearance by electron microscopy. Filling and filled phage heads are observed chiefly near the boundary of the nuclear region.     

pUL25 immunolocalization in human cytomegalovirus-infected and gene-transfected cells

Summary.  By means of confocal and electron microscope immunocytochemistry we have studied the localization of a recently described structural protein (pUL25) of human cytomegalovirus, in both infected cells and in cells transiently transfected with UL25. pUL25 localization in infected cells was observed in typical cytoplasmic structures characterized by a very electrondense texture previously reported to accumulate other tegument proteins. At the virion level pUL25 seems to localize at the interface between the tegument and the capsid of both intracytoplasmic and extracellular virions. In UL-25-transfected cells, pUL25 has been found in characteristic para-crystalline cytoplasmic aggregates, suggesting its intrinsic ability to aggregate in a regular subunit pattern. Received August 6, 1999/Accepted November 3, 1999     

Evidence for early nuclear antigens in cytomegalovirus-infected cells.

Human cytomegalovirus (CMV) induces nuclear antigens resembling the Epstein-Barr nuclear antigen (EBNA) as early as 3 h after infection. These early antigens can be detected only with the anti-complement immunofluorescence staining (ACIF) technique. Synthesis of these new antigens is not influenced by cytosine arabinoside (ara-C).     

The role of defective cytomegalovirus particles in the induction of host cell DNA synthesis.

Cytomegalovirus (CMV)-induced stimulation of cellular DNA synthesis was studied by autoradiography-immunofluorescence in susceptible human embryonic lung (HEL) cells in which DNA synthesis had been suppressed by serum starvation or treatment with 5-fluorouracil. Those cells in the culture which had been stimulated to synthesize detectable amounts of cellular DNA between 24 and 48 hr did not synthesize detectable amounts of viral antigens even as late as 96 hr postinfection, indicating that infection was abortive in these cells. To determine the role of defective CMV particles in the stimulation of cellular DNA synthesis, the ability of populations of virions obtained after undiluted serial passage to induce host cell DNA synthesis was compared to that of populations of recently plaque-purified virions. Stocks obtained after amplification at low multiplicity of three individual plaques were all poor inducers of cellular DNA synthesis. On the other hand, undiluted, serially passaged CMV stocks were very effective in stimulating cellular DNA synthesis. After low doses of uv irradiation the ability of standard virus stocks to induce host cell DNA synthesis also increased; stimulation of cellular DNA was apparent even at uv doses which eliminated by more than 99.5% the ability of the virus to induce the synthesis of viral antigens. We conclude that CMV particles that are incapable of giving rise to the synthesis of detectable amounts of either early or late viral antigens and that are presumably defective are present in standard CMV stocks and play an important role in the stimulation of cellular DNA synthesis in susceptible cells.     

Activated murine neutrophils induce unscheduled DNA synthesis in B lymphocytes

Activated neutrophils induce DNA damage in neighboring cells by secreting reactive oxygen compounds into the extracellular milieu. Repair of this damage is required to prevent mutagenesis and neoplastic transformation. Conditions were established to detect the activation of excision-repair pathways (unscheduled DNA synthesis) by measuring stimulated thymidine uptake in target B lymphocytes exposed to activated neutrophils. Murine neutrophils were cocultured in serum-free medium with splenic B cells or with murine plasmacytoma cells for 2 h. Unscheduled DNA synthesis in the B cells was detected at neutophil: target cell ratios of 1:1 to 4:1 when the neutrophils were activated with phorbol myristate acetate. Reagent H₂O₂ alone (≥6 μM) also induced UDS whereas HOCl (up to 4 mM) did not. No repair synthesis was observed within the neutrophils themselves. Control experiments indicated that the induction of UDS by neutrophils and H₂O₂ was not due to formation of a stable genotoxic compound from HU. On the contrary, scavenging of free H₂O₂ by HU probably lowered the levels of UDS that could be detected by these agents. Induction of unscheduled DNA synthesis by neutrophils and H₂O₂ occurred under conditions of less cytostasis than was found with other DNA-damaging agents such as 4-nitroquinoline 1-oxide or γ-irradiation. This may reflect a heightened responsiveness of the cells to repair of damage from physiological oxidants. The result demonstrate that DNA damage induced by reactive oxygen intermediates can be repaired by nucleotide excision-repair pathways.     

DNA and histone synthesis in mouse cells which exhibit temperature-sensitive DNA synthesis

It is demonstrated that temperature inactivation of histone synthesis is coupled to inhibition of DNA replication in ts AlS9 and ts Cl mouse L-cells, which are temperature-sensitive (ts) in an S-phase function. In contrast, uncoupling of histone and DNA synthesis occurs in BalB/C-3T3 ts 2 cells which are ts in a function of the pre-DNA-synthetic phase. Termination of histone synthesis in ts AlS9 and ts Cl cells is 16–18 h after onset of temperature inactivation of DNA replication and appears to be associated with general cessation of chromatin replication triggered by the earlier event. Synthesis of histone and other chromosomal proteins proceeds in ts 2 cells under conditions in which DNA synthesis undergoes temperature inactivation. It is suggested that the terminal phenotype of coupled temperature inactivation of DNA and histone synthesis may be diagnostic of cells ts in an S-phase function and may therefore be a useful secondary screen in designation of cell cycle mutants.     

Detection of cytomegalovirus-infected cells in autopsy material by in situ hybridization

A non-radioactive in situ hybridization (ISH) method was elaborated to detect cytomegalovirus (CMV)-infected cells in tissue specimens processed for diagnostic routine histopathology. A biotinylated CMV-DNA probe was hybridized following a) four different enzymatic predigestions, b) progressively increasing denaturation periods, and then detected by c) streptavidin-biotin, d) a monoclonal antibody against biotin using a three-stage alkaline phosphatase anti-alkaline phosphatase (APAAP)-technique, and e) combining c + d. Autopsy specimens obtained from an infant with acquired CMV-infection, six patients with AIDS, five patients clinically and serologically without CMV-infection, and preoperative needle core biopsies from six renal allografts served as material. ISH was specific and more sensitive when compared to immunohistochemical (IMH) detection of CMV-antigens by a monoclonal antibody. ISH was concluded to be a rapid, practical, and sensitive tool in daily diagnostic histopathology.     

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.