We describe a technique for transferring electrophoretically separated bands of RNA from an agarose gel to paper strip The RNA is coupled covalently to diazoenzylomethyl groups on the paper. After transTer and appropriate treatment of the paper to destroy remaining diazo goups, specific RNA bands can be detected by hybridization with mP~labeled DNA probes followed by autoradiography. This procedure allows detection of specific RNA ban s wit high sensitivity and low background. was allowed to crystallize. It was collected on a sintered glass filter, washed with pyridine, then washed thoroughly with petroleum ether and dried under reduced pressure. The NBPC (267 g) was stored at -20°in a desiccator.Preparation of Aminobenzyloxymethyl-Paper. The method for making aminobenzyloxymethyl (ABM)-paper and its subsequent conversion to the diazobenzyloxymethyl form (DBM-paper) by diazotization is outlined in Fig. 1. The ABM-paper was prepared by a modification of methods described previously (5-7) for preparing aminobenzyloxymethyl-cellulose powder. A sheet of Whatman 540 paper (14 X 25 cm) in a flat enameled pan was soaked with 10 ml of an aqueous solution of 0.8 g of NBPC and 0.25 g of sodium acetate. Air bubbles under the paper were squeezed out and the paper was dried at 600 and then heated to 130-135°for 35 min. The paper was washed twice for 20 min with water, dried at 600, washed twice for 20 mm with benzene, and dried in the air. The nitrobenzyloxymethyl paper was reduced to ABM-paper by treating it with 150 ml of 20% sodium dithionite (wt/vol) for 30 min at 600 with shaking. The ABM-paper was washed for 20 min with water, 20 min with 30% acetic acid, and then with water until there was no further odor of H2S. The ABM-paper was dried in the air and stored at 40 in a desiccator. It is stable for several weeks under these conditions. Diazotization of ABM-Paper. Just before reaction with single-stranded nucleic acids, ABM-paper was converted to the diazobenzyloxymethyl (DBM) form by treatment with a solution containing 40 ml of water, 80 ml of 1.8 M HCO, and 3.2 ml of a freshly prepared solution of NaNO2 (10 mg/ml) for 30 min at 40. The solution was checked for free HNO2 with starchiodide paper, which turns black. After 30 min, the DBM-paper was washed five times for 5 min each with 100 ml of cold water and then twice for 10 min with ice-cold sodium borate buffer, 50 mM, pH 8. Upon washing, the paper turns bright yellow. It should be kept cold until transfers begin, no more than 15 min later. DBM-paper had the capacity to couple [16][17][18][19][20][21][22][23][24]

Viral infection causes stress to the endoplasmic reticulum. The response to endoplasmic reticulum stress, known as the unfolded protein response (UPR), is designed to eliminate misfolded proteins and allow the cell to recover by attenuating translation and upregulating the expression of chaperones, degradation factors, and factors that regulate the cell's metabolic and redox environment. Some consequences of the UPR (e.g., expression of chaperones and regulation of the metabolism and redox environment) may be advantageous to the viral infection; however, translational attenuation would not. Thus, viruses may induce mechanisms which modulate the UPR, maintaining beneficial aspects and suppressing deleterious aspects. We demonstrate that human cytomegalovirus (HCMV) infection induces the UPR but specifically regulates the three branches of UPR signaling, PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE-1), to favor viral replication. HCMV infection activated the eIF2␣ kinase PERK; however, the amount of phosphorylated eIF2␣ was limited and translation attenuation did not occur. Interestingly, translation of select mRNAs, which is dependent on eIF2␣ phosphorylation, did occur, including the transcription factor ATF4, which activates genes which may benefit the infection. The endoplasmic reticulum stress-induced activation of the transcription factor ATF6 was suppressed in HCMV-infected cells; however, specific chaperone genes, normally activated by ATF6, were activated by a virus-induced, ATF6-independent mechanism. Lastly, HCMV infection activated the IRE-1 pathway, as indicated by splicing of Xbp-1 mRNA. However, transcriptional activation of the XBP-1 target gene EDEM (ER degradation-enhancing ␣-mannosidase-like protein, a protein degradation factor) was inhibited. These results suggest that, although HCMV infection induces the unfolded protein response, it modifies the outcome to benefit viral replication.Human cytomegalovirus (HCMV) is a betaherpesvirus which can cause significant medical problems in individuals with immature or compromised immune systems. The genome of HCMV is 230 kb of double-stranded DNA with the potential to encode over 200 proteins. Like that of other herpesviruses, HCMV viral gene expression occurs in an ordered temporal pattern having immediate-early, early, delayed-early, and late kinetics, with increasing viral protein synthesis over time.

Human fibroblasts infected with human cytomegalovirus (HCMV) were more viable than uninfected cells during glucose starvation, suggesting that an alternate carbon source was used. We have determined that infected cells require glutamine for ATP production, whereas uninfected cells do not. This suggested that during infection, glutamine is used to fill the tricarboxylic acid (TCA) cycle (anaplerosis). In agreement with this, levels of glutamine uptake and ammonia production increased in infected cells, as did the activities of glutaminase and glutamate dehydrogenase, the enzymes needed to convert glutamine to ␣-ketoglutarate to enter the TCA cycle. Infected cells starved for glutamine beginning 24 h postinfection failed to produce infectious virions. Both ATP and viral production could be rescued in glutamine-starved cells by the TCA intermediates ␣-ketoglutarate, oxaloacetate, and pyruvate, confirming that in infected cells, a program allowing glutamine to be used anaplerotically is induced. Thus, HCMV infection activates the mechanisms needed to switch the anaplerotic substrate from glucose to glutamine to accommodate the biosynthetic and energetic needs of the viral infection and to allow glucose to be used biosynthetically.

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