Generation of Covalently Closed Circular DNA of Hepatitis B Viruses via Intracellular Recycling Is Regulated in a Virus Specific Manner

Persistence of hepatitis B virus (HBV) infection requires covalently closed circular (ccc)DNA formation and amplification, which can occur via intracellular recycling of the viral polymerase-linked relaxed circular (rc) DNA genomes present in virions. Here we reveal a fundamental difference between HBV and the related duck hepatitis B virus (DHBV) in the recycling mechanism. Direct comparison of HBV and DHBV cccDNA amplification in cross-species transfection experiments showed that, in the same human cell background, DHBV but not HBV rcDNA converts efficiently into cccDNA. By characterizing the distinct forms of HBV and DHBV rcDNA accumulating in the cells we find that nuclear import, complete versus partial release from the capsid and complete versus partial removal of the covalently bound polymerase contribute to limiting HBV cccDNA formation; particularly, we identify genome region-selectively opened nuclear capsids as a putative novel HBV uncoating intermediate. However, the presence in the nucleus of around 40% of completely uncoated rcDNA that lacks most if not all of the covalently bound protein strongly suggests a major block further downstream that operates in the HBV but not DHBV recycling pathway. In summary, our results uncover an unexpected contribution of the virus to cccDNA formation that might help to better understand the persistence of HBV infection. Moreover, efficient DHBV cccDNA formation in human hepatoma cells should greatly facilitate experimental identification, and possibly inhibition, of the human cell factors involved in the process

OLIGOPEPTIDES ARE THE MAIN SOURCE OF NITROGEN FOR LACTOCOCCUS-LACTIS DURING GROWTH IN MILK

The consumption of amino acids and peptides was monitored during growth in milk of proteinase-positive (Prt(+)) and -negative (Prt(-)) strains of Lactococcus lactis, The Prt(-) strains showed monophasic exponential growth, while the Prt(+) strains grew in two phases. The first growth phases of the Prt(+) and Prt- strains were the same, and no hydrolysis of casein was observed, Also, the levels of consumption of amino acids and peptides in the Prt(+) and Prt(-) strains were similar. At the end of this growth phase, not all free amino acids and peptides were used, indicating that the remaining free amino acids and peptides were unable to sustain growth. The consumption of free amino acids was very low (about 5 mg/liter), suggesting that these nitrogen sources play only a minor role in growth. Oligopeptide transport-deficient strains (Opp(-)) of L. lactis were unable to utilize oligopeptides and grew poorly in milk, However, a di- and tripeptide transport-deficient strain (DtpT(-)) grew exactly like the wild type (Opp(+) DtpT(+)) did, These observations indicate that oligopeptides represent the main nitrogen source for growth in milk during the first growth phase. In the second phase of growth of Prt(+) strains, milk proteins are hydrolyzed to peptides by the proteinase, Several of the oligopeptides formed are taken up and hydrolyzed internally by peptidases to amino acids, several of which are subsequently released into the medium (see also E. R. S. Kunji, A. Hagting, C. J. De Vries, V.Juillard, A. J. Haandrikman, B. Poolman, and W. N. Konings, J. Biol. Chem. 270:1569-1574, 1995). It is concluded that growth of L. Iactis in milk depends on oligopeptides as nitrogen source for 98% of growth and that the oligopeptide transport system plays a crucial role in the utilization of these peptides

LOCALIZATION OF PEPTIDASES IN LACTOCOCCI

The localization of two aminopeptidases, an X-prolyl-dipeptidyl aminopeptidase, an endopeptidase, and a tripeptidase in Lactococcus lactis was studied. Polyclonal antibodies raised against each purified peptidase are specific and do not cross-react with other peptidases. Experiments were performed by immunoblotting after cell fractionation and by electron microscopy of immunogold-labeled peptidases. All peptidases were found to be intracellular. However, immunogold studies showed a peripheral labeling of the X-prolyl-dipeptidyl aminopeptidase, the tripeptidase, and the endopeptidase. This peripheral location was further supported by the detection of these three enzymes in cell membrane fractions in which none of the two aminopeptidases was present