The plasmid construct pSVO

The E2a gene of human adenovirus type 2 (Ad2) encodes the 72-kilodalton DNA-binding protein. We previously described perfect inverse correlations between the methylation of all 5' C-C-G-G 3' sequences in the Ad2 E2a gene in virus-transformed hamster cells containing viral DNA sequences in an integrated state and the extent to which this gene is expressed. We subsequently showed that in vitro methylation of all 14 5' C-C-G-G 3' sequences in the cloned E2a gene by prokaryotic Hpa II DNA methyltransferase leads to transcriptional inactivation after microinjection into Xenopus laevis oocytes. The unmethylated cloned E2a gene is expressed in these cells. We report here the construction of partly methylated clones of the E2a gene. In the promoter (5')-methylated construct, three 5' C-C-G-G 3' sequences at the 5' end of the subclone were methylated. One of these sites is located 215 base pairs (bp) upstream (bp 26,169 of Ad2 DNA), and two sites are located 5 and 23 bp downstream from the cap site (bp 25,931 and 25,949 of Ad2 DNA) of the E2a gene. This construct was transcriptionally inactive upon microinjection into nuclei of X. laevis oocytes. In the gene (3')-methylated construct, 11 5' C-C-G-G 3' sequences in the main part of the transcribed gene region were methylated in vitro. This construct was transcribed in X. laevis oocytes, and at least some of the Ad2-specific RNA synthesized was initiated at the same sites as in Ad2-infected human KB cells. Both mock-methylated constructs were transcribed into Ad2-specific RNA in X. laevis oocytes. These results demonstrate that DNA methylations at or close to the promoter and 5' end of the E2a gene cause transcriptional inactivation. Perhaps only one methyl group would be adequate for inactivation; in vivo methylation of more than one cytosine may be a form of safeguard or redundancy.

The severity of glomerular injury in the heterologous phase of NTN is dependent on proinflammatory cytokines including TNF alpha and IL-1 beta, and can be enhanced by LPS. We have previously shown that passive immunization against IL-1 beta and TNF partially abrogated the LPS effect in this model. In the present work, we have assessed the effects on glomerular injury of blocking and binding of IL-1 to its receptor by rh IL-1 receptor antagonist (IL-1ra) and by neutralizing IL-1 and TNF with rm soluble IL-1 receptor type1 (sIL-1Rt1) and rh sTNF receptor (sTNFr p55), respectively. Pretreatment with either IL-1ra, sIL-1Rt1, or sTNFr partially abrogated the effects of LPS and reduced albumin excretion from 45 +/- 8, 66 +/- 9, and 101 +/- 17 mg/24 hr at 13 +/- 4 (P < 0.02), 14 +/- 4 (P < 0.001), and 21 +/- 7 mg/24 hr (P < 0.001), respectively. Similarly, these inhibitors reduced the prevalence of glomerular capillary thrombi and the intensity of glomerular neutrophil infiltration. Glomerular thrombosis was reduced from 18 +/- 3%, 28 +/- 5%, and 25 +/- 7% to 3 +/- 2% (P < 0.002), 6 +/- 2% (P < 0.001), and 3 +/- 2 (P < 0.001), respectively, and glomerular neutrophil infiltration was reduced from 46 +/- 3, 54 +/- 2, 59 +/- 8 to 19 +/- 2 (P < 0.001), 25 +/- 2 (P < 0.001), and 28 +/- 2 neutrophils/50 glomeruli in section, respectively. Coadministration of both soluble receptors of IL-1 and TNF caused a further decrease in glomerular injury. The protective effect was also noticed at four hours after induction of nephritis, and even when these inhibitors were administered after the LPS injection and at the same time of induction of nephritis. All three treatments reduced circulating TNF concentration (down to 20%, 34%, and 0%, respectively) but without detectable glomerular TNF gene expression. Glomerular IL-1 beta mRNA levels were also reduced by 41%, 53%, and 67%, respectively, when assessed by densitometric analysis of Northern blots. In contrast, the glomerular expression of IL-1ra was not affected by its exogenous administration but was mildly reduced by sIL-1Rt1 and sTNFr, which demonstrates the potential role for host derived IL-1ra as an endogenous negative feedback mediator in the glomerulus. These results confirm the direct involvement of IL-1 and TNF in LPS-enhanced hNTN and demonstrate the potency of these inhibitors in modulating injury even when administered after LPS and in time of induction of nephritis. They were more specific and effective than passive immunization with polyclonal antibodies, and this demonstrates their potential usefulness in the management of nephritis.

Virus adsorption and uptake of human rhinovirus 14 (HRV14) were studied with HeLa cells and baby hamster kidney (BHK) cells which were transfected with the HRV14 receptor intercellular adhesion molecule-1 (ICAM-1). Transmission electron microscopy of HeLa cells revealed that HRV14 was internalized via clathrin-coated pits and -coated vesicles. A minority of virus particles also used uncoated vesicles for entry. The internalization showed the characteristics of receptor-mediated endocytosis. Presence of the carboxylic ionophore monensin inhibited viral uncoating, indicating a pH-dependent entry mechanism. The expression of ICAM-1 on the surface of the ICAM-1 transfected baby hamster kidney cells (BHK-ICAM cells) allowed extensive virus adsorption and internalization through membrane channels. Virus particles were lined up in these channels like pearls on a string, but did not induce a productive infection. Although ICAM-1 was expressed to the same degree on BHK-ICAM and HeLa cells, HRV14 induced neither viral protein and RNA syntheses nor infectious virus progeny in BHK-ICAM cells. ICAM-1 on the transfected BHK cells was a functional active receptor as it rendered these cells permissive to coxsackievirus A21. These results suggest that HRV14 uptake into BHK-ICAM cells is blocked directly in or shortly after its final step of internalization, the uncoating. Our findings underline that the receptor ICAM-1 determines virus uptake into cells, however, is not sufficient to confer susceptibility of BHK cells to HRV14 infection.

No abstract