Detlev H. Krüger

Type III restriction endonucleases translocate DNA in a reaction driven by recognition site-specific ATP hydrolysis.

Type III restriction/modification systems recognize short non‐palindromic sequences, only one strand of which can be methylated. Replication of type III‐modified DNA produces completely unmethylated recognition sites which, according to classical mechanisms of restriction, should be signals for restriction. We have shown previously that suicidal restriction by the type III enzyme EcoP15I is prevented if all the unmodified sites are in the same orientation: restriction by EcoP15I requires a pair of unmethylated, inversely oriented recognition sites. We have now addressed the molecular mechanism of site orientation‐specific DNA restriction. EcoP15I is demonstrated to possess an intrinsic ATPase activity, the potential driving force of DNA translocation. The ATPase activity is uniquely recognition site‐specific, but EcoP15I‐modified sites also support the reaction. EcoP15I DNA restriction patterns are shown to be predetermined by the enzyme‐to‐site ratio, in that site‐saturating enzyme levels elicit cleavage exclusively between the closest pair of head‐to‐head oriented sites. DNA restriction is blocked by Lac repressor bound in the intervening sequence between the two EcoP15I sites. These results rule out DNA looping and strongly suggest that cleavage is triggered by the close proximity of two convergently tracking EcoP15I‐DNA complexes.

Frequent and rapid emergence of mutated pre-C sequences in HBV from e-antigen positive carriers who seroconvert to anti-HBe during interferon treatment

Hepatitis B virus (HBV) variants which cannot express e-antigen (HBeAg) are characteristic for many viremic anti-HBe positive chronic carriers who often have particularly severe and fluctuating hepatitis. Whether such variants are selected for and are less amenable to interferon treatment is under dispute. Therefore, by DNA amplification and direct sequencing we have investigated the emergence of HBV pre-C sequence variants in nine e-antigen positive chronic carriers, all of whom seroconverted to anti-HBe or lost HBeAg during interferon treatment, and in three of whom no viral DNA was detectable after interferon treatment. In most, but not all of the patients we found newly emerging pre-C sequences in a subpopulation of the viral genomes that included silent point mutations, amino acid changes, start and stop codon and frameshift mutations. The emergence of these mutations was paralleled by a drastic decrease of viremia during treatment. The observed mutations appeared most frequently during interferon treatment. Some of the mutations appeared or disappeared late after interferon treatment concomitant with anti-HBe antibody development. The appearance or lack of mutations in the pre-C region of a subpopulation of HBV of these patients was independent of successful virus elimination. These data indicate that interferon treatment is frequently associated with the simultaneous fall in titer of viral DNA by several orders of magnitude and the emergence of novel pre-C sequences, some of them preventing HBeAg expression. However, the presumably immune-mediated selection for pre-C mutant viruses and decrease in viremia under interferon treatment appears not to be prognostic for successful or unsuccessful virus elimination.

EcoRII: a restriction enzyme evolving recombination functions

The restriction endonuclease EcoRII requires the cooperative interaction with two copies of the sequence 5′CCWGG for DNA cleavage. We found by limited proteolysis that EcoRII has a two‐domain structure that enables this particular mode of protein–DNA interaction. The C‐terminal domain is a new restriction endonuclease, EcoRII‐C. In contrast to the wild‐type enzyme, EcoRII‐C cleaves DNA specifically at single 5′CCWGG sites. Moreover, substrates containing two or more cooperative 5′CCWGG sites are cleaved much more efficiently by EcoRII‐C than by EcoRII. The N‐terminal domain binds DNA specifically and attenuates the activity of EcoRII by making the enzyme dependent on a second 5′CCWGG site. Therefore, we suggest that a precursor EcoRII endonuclease acquired an additional DNA‐binding domain to enable the interaction with two 5′CCWGG sites. The current EcoRII molecule could be an evolutionary intermediate between a site‐specific endonuclease and a protein that functions specifically with two DNA sites such as recombinases and transposases. The combination of these functions may enable EcoRII to accomplish its own propagation similarly to transposons.

Active protection by bacteriophages T3 and T7 againstE. coli B-and K-specific restriction of their DNA

SummaryThe bacteriophages T3 and T7 are not modified and restricted byE. coli strains with different host specificity (E. coli B, K, O) in vivo. The phages code for a gene product with the ability toovercomeclassicalrestriction (ocr):ocr− mutants are subject to modification and restriction via DNA methylation vs cleavage. The T3 genome possesses recognition sites for the restriction endonuclease R.EcoB which, unless the DNA is B-specifically modified, trigger 5–7 DNA cleavages. Theocr gene function of T3 and T7 is located within the gene 0.3 region of these phages and is not identical with thesam (SAMase) function of T3. The mechanisms ofocr protection remains unclear, while it is certain that this protection by the gene 0.3 protein is exerted in the infected cell and not through “over-all” modification in the preceding growth cycle of the phage.

GENETIC CHARACTERIZATION OF A NEW HANTAVIRUS DETECTED IN MICROTUS ARVALIS FROM SLOVAKIA

A new hantavirus, called Malacky, has been identified in lung tissue specimens of a vole,Microtus arvalis, by the reverse transcriptase polymerase chain reaction (RT-PCR). The voles were trapped in a geographical area in Slovakia where hemorrhagic fever with renal syndrome (HFRS) is endemic in the human population. Sequence analysis of a major part of the S segment showed this virus to represent a new subtype within Tula, a new hantavirus genetic group defined very recently.

Coding strategy of the S and M genomic segments of a hantavirus representing a new subtype of the Puumala serotype

SummaryThe hantavirus strain Vranica was previously reported to have been isolated from a bank vole in Bosnia-Hercegovina and associated with the occurrence of hemorrhagic fever with renal syndrome (HRFS) in humans. The complete cDNA nucleotide sequences of the small (S) and medium (M) genomic RNA segments of this virus were determined. Major open reading frames were found in the S and M segment between nucleotide positions 43 and 1341 coding for a polypeptide of 433 amino acid residues and between nucleotide positions 41 and 3 484 coding for 1 148 amino acid residues, respectively. The analysis and the alignment of the nucleotide and the derived amino acid sequences with known sequences of other hantavirus strains demonstrate that Vranica resembles Swedish strains and represents a new virus subtype of the Puumala serotype distinct from the subtypes represented by virus strains CG18–20 and Sotkamo.

Fine Mapping and Functional Characterization of Two Immuno-Dominant Regions from the preS2 Sequence of Hepatitis B Virus

A set of monoclonal antibodies (mAbs) directed against the preS2 region of hepatitis B virus (HBV) surface antigen (HBsAg) was generated by immunization of mice with native HBsAg isolated from the blood of HBV carriers. According to (1) mutual competition binding of mAb to natural HBsAg, (2) recognition of full-length preS2 displayed on hepatitis B core particles, (3) recognition of synthetic partial preS2 peptides, and (4) Western blotting using a fusion protein library of truncated preS2 fragments of different legths, mAbs were assigned to two groups which coincided with groups I and III described by Mimms et al. [Virology 1990; 176:604-619]. All mAbs recognized linear epitopes and were glycosylation independent. Six out of eight fine-mapped mAbs recognized common epitopes located in the amino-terminal part of the preS sequence between amino acids 131 and 144 (group I), and inhibited binding of HBsAg to polymerized human serum albumin. Only two mAbs recognized a carboxy-terminal HBV-genotype-specific epitope covering amino acid residues 162 to 168 (group III). These mAbs bound to the highly variable proteolysis-sensitive hinge of preS2. Although four out of six mAbs targeted to immunodominant region I require the full-length sequence 131-L[Q/L]DPRVRGLY[F/L]PAG-144, two mAbs recognize the shorter and slightly carboxy-terminal-shifted sequences 133-DPRVRGLY[F/L]-141 or 135-PVRGLY[F/L]PAG-144. Together with previously identified preS2 epitopes 133-DPRVRGL-139, 137-RGLYFPA-143, and 132-QDPR-135, these data indicate diversity of the immune response against epitopes within the same immunodominant region. This diversity may be generated by a labile secondary structure. Sequence analysis suggests the transition from an alpha-helix to a loop structure at this site.

CAPSID PROTEIN-ENCODING GENES OF HAMSTER POLYOMAVIRUS AND PROPERTIES OF THE VIRAL CAPSID

On the basis of its genome organization the hamster polyomavirus (HaPV) is closely related to the murine polyomavirus Py. But HaPV infection, in contrast to Py infection, gives rise to two different tumor types; depending on the hamster strain used for infection, HaPV induces either epitheliomas or lymphomas. Although the HaPV virions were shown to be similar to those of Py and SV40, more precise information about the structure and protein composition of the HaPV capsid was still missing. Here we describe the primary structure of the capsid protein-encoding HaPV genes and the structure and protein composition of the HaPV capsid. Virions isolated from epitheliomas in HaPV-infected hamsters were shown by electron microscopy to be spherical particles with the typical icosahedral structure of polyomaviruses. However, in contrast to the capsids of SV40 and Py, a T = 7 laevo symmetry of HaPV capsids was observed. Separation of HaPV virions in SDS polyacrylamide gels and Western blotting with VP1-specific antisera identified VP1 as the major capsid protein species corresponding in its molecular weight to the predicted value of 41.8 kDa. Because of the presence of two potential translational initiation sites in the VP1 gene, the N-terminal amino acid sequence of virion VP1 was determined and found to start at the second initiation site. The amino acid homologies of HaPV capsid proteins shared with Py varied between 65.5% (VP1), 45.4% (VP3) and 44.6% (VP2), whereas the homologies to the relevant proteins of other polyomaviruses were found to range between 49.6–57.9% for VP1 and 28.9–41% for VP2/VP3.

Mutations in the hemagglutinin gene associated with influenza virus resistance to norakin

SummaryThe nucleotide sequences of the hemagglutinin genes of four norakinresistant mutants of Influenza A/FPV/Weybridge were determined and compared to the wild-type hemagglutinin. All mutants show one or two amino acid substitutions which are discussed to destabilise the pH 7 conformation of hemagglutinin.

Host-dependent modification of bacteriophage T7 and SAMase-negative T3 derivatives affecting their adsorption ability

SummaryWhen passaging phage T7 and SAMase-negative T3 mutants betweenE. coli strains with identical (EcoB) or without (EcoO) DNA host specificity, phenotypically a host-controlled modification and restriction is observed. This phenomenon is not due to “classical” modification and restriction of the bacteriophage DNA but depends on the reversibly altered adsorption capacity of the phages on the different host strains.