Biomedical subjects

G Darai

Publications and source records attributed to G Darai.

At least 73 records · Page 4Linked to original sources

PubMed1993

Identification of the gene encoding the major capsid protein of fish lymphocystis disease virus.

The gene encoding the major capsid protein (MCP) of fish lymphocystis disease virus (flounder isolate; FLCDV-f) has been identified by PCR using oligonucleotide primers corresponding to different regions of the MCP of Tipula iridescent virus (TIV), iridescent virus 22 (IV22) and Chilo iridescent virus (CIV). DNA fragments of 0.4 kbp, 0.5 kbp and 0.27 kbp in size were amplified using oligonucleotide primers corresponding to amino acids (aa) 146 to 153 (primer 1) and 274 to 268 (primer 6), or aa 146 to 153 (primer 1) and 313 to 304 (primer 8), or aa 304 to 312 (primer 7) and 385 to 381 (primer 9) of the MCP of TIV, respectively. The PCR products were used as hybridization probes for screening the gene library of FLCDV-f. The MCP gene of FLCDV-f(1377 bp; 459 aa; 51.4K) was identified within the DNA sequence of the EcoRI FLCDV-f DNA fragment C (11.2 kbp; 0.611 to 0.718 map units). A high degree of aa sequence identity/similarity was detected between the MCP of FLCDV-f and TIV (50.3%/33.8%), IV22 (49.1%/34.2%). CIV (53%/29.5%) and African swine fever virus (16%/38.1%).

Amino Acid Sequence↗

PubMed1993

Identification of the gene encoding the major capsid protein of insect iridescent virus type 6 by polymerase chain reaction.

The gene encoding the major capsid protein of Chilo iridescent virus (CIV) has been identified by PCR using oligonucleotide primers corresponding to different regions of the major capsid proteins of Tipula iridescent virus (TIV) and iridescent virus 22 (IV22). A DNA fragment of 0.5 kbp was amplified using two oligonucleotide primers corresponding to the amino acid positions 146 to 153 and 304 to 313 of the major capsid protein of TIV, respectively. The radioactively labelled DNA fragment derived from PCR was hybridized to a CIV gene library. This analysis revealed that only the EcoRI CIV DNA fragment X [2.85 kbp; 0.589 to 0.603 viral map units (m.u.)] hybridized to the amplified DNA fragment. An RNA transcript of about 1.5 kb was identified when the PCR product was used as a hybridization probe. The same RNA transcript was detected when the EcoRI fragments X and Q (5.9 kbp; 0.603 to 0.631 viral m.u.) were used as probes. This indicates that the expected gene is located within map coordinates 0.589 to 0.631 and harbours part of the DNA sequences of fragments Q and X. The analysis of the DNA sequences of this particular region of the CIV genome revealed the presence of one open reding frame of 1401 bp. The DNA sequences of this region encode a protein of 467 amino acid residues with an M(r) of 51.4K. A high degree (64.7%) of amino acid sequence identity was detected between the major capsid protein of TIV and/or IV22 and the amino acid composition of the identified CIV protein.

Amino Acid Sequence↗

PubMed1993

A novel mu-capture enzyme-linked immunosorbent assay based on recombinant proteins for sensitive and specific diagnosis of hemorrhagic fever with renal syndrome.

Hantavirus nucleocapsid protein has recently been identified as a major antigen inducing an early and long-lasting humoral immune response in patients with hemorrhagic fever with renal syndrome. A mu-capture enzyme-linked immunosorbent assay utilizing recombinant nucleocapsid proteins of Hantavirus strains Hantaan 76-118 (Hantaan serotype) and CG 18-20 (Puumala serotype) as diagnostic antigens and specific monoclonal antibodies as the detection system has been developed. Histidine-tailed recombinant proteins were expressed in Escherichia coli and purified in a single step by affinity chromatography on a nickel-chelate resin. The assay was evaluated with a panel of sera from patients with hemorrhagic fever with renal syndrome originating from various geographic regions. The overall sensitivity of the mu-capture enzyme-linked immunosorbent assay (both recombinant antigens) was 100%, and its specificity was also found to be 100%. Immunoglobulin M antibodies were detected as early as on day 3, and maximum titers were obtained between days 8 and 25 after onset of the disease. The assay was regularly found to be positive within 3 to 4 months but in some cases up to 2 years after the acute phase of the disease.

Amino Acid Sequence↗

PubMed1993

Characterization of the genome of molluscum contagiosum virus type 1 between the genome coordinates 0.045 and 0.075 by DNA nucleotide sequence analysis of a 5.6-kb HindIII/MluI DNA fragment.

The complete DNA nucleotide sequence of a HindIII/MluI genomic DNA fragment (0.045-0.075 viral map units) from molluscum contagiosum virus type 1 (MCV-1) was determined. The HindIII/MluI DNA fragment comprises 5,646 bp with a base composition of 64.4% G + C and 35.6% A + T. The DNA sequence contains many perfect direct repeats. A cluster of three repetitive DNA elements R1, R2 and R3, with a complex structural arrangement was detected between nucleotide positions 1802 and 2107. The unit length (box) of the repetitive DNA sequences was found to be 6 bp (15 boxes) and 9 bp (24 boxes) for R1 and R2, respectively. The repetitive DNA element R3 is organized in fifteen boxes (15 bp) in which a unit length of R1 is combined with a unit length of R2. The arrangement of the repetition R3 within the DNA sequences of this particular region of the MCV-1 genome was found to be (5 x R3) + (2 x R2) + (1 x R3) + (6 x R2) + (1 x R3) + (1 x R2) + (8 x R3). Twenty-three open reading frames (ORFs) of 60-1,175 amino acid (AA) residues were detected. The largest ORF (number 17) comprises 1,175 AA with a predicted molecular weight of 126 kD. This ORF harbors a promoter signal which is located 21 nucleotides upstream from the start codon and is very similar to the early promoter signals known for vaccinia virus. This putative protein contains glutamine-enriched regions between AA residues 427 and 682 which show homologies to the corresponding glutamine-enriched regions of a variety of cellular genes like human transcriptional initiation factor (TFIID: TATA box factor).

Amino Acid Sequence↗

PubMed1993

[Recent information about the etiopathogenesis of paretic-paralytic forms of herpesvirus infection in horses].

From spring 1990 to summer 1991 we investigated 21 horses with clinical symptoms of EHV-infection by means of serological and virological methods including DNA-hybridization to identify the causative agents. The results indicated that, as already reported by us, EHV4 may also cause the paralytic form of the infection. The possibility of double infection with EHV4 and EHV1 cannot be excluded. In 3 out of 21 affected horses we could investigate brain tissue and/or spinal fluid by Dotblot hybridization with EHV1 and EHV4-DNA. The investigated samples of all three horses showed hybridization with EHV4-DNA, without or with less pronounced reaction with EHV1-DNA. The results were confirmed by serological investigation. Brain tissue and cerebrospinal fluid from two horses with paretic or paralytic disorders (1979 and 1980) was also investigated by DNA hybridization. In the liquor of one horse--a 5-month-old foal with neonatal ataxia--we detected EHV1-DNA. The other horse showed a strong reaction with EHV1 and a weaker reaction with EHV4 in its brain material and no hybridisation in the cerebrospinal fluid. The results are discussed.

Animals↗

PubMed1992

Determination of the coding capacity of the BamHI DNA fragment B of apathogenic Herpes simplex virus type 1 strain HFEM by DNA nucleotide sequence analysis.

Herpes simplex virus type 1 (HSV-1) strain HFEM acquired an apathogenic phenotype due to a deletion within the DNA sequences of the BamHI DNA fragment B of the viral genome. In order to investigate the coding strategy of this particular region of the genome of HSV-1 strain HFEM the DNA nucleotide sequence of the BamHI DNA fragment B was determined. This analysis revealed that the BamHI DNA fragment B of HSV-1 strain HFEM comprises 6593 bp, corresponding to the nucleotide positions (np) 113322 to 117088 and np 120643 to 123465 of the genome of HSV-1 strain 17. According to these data the deletion of the genome of HSV-1 strain HFEM occurred between the np 117089 and 120642. The promoter region of the UL56 gene of HSV-1 strain HFEM is a part of the deleted DNA sequences. Therefore, this gene of HSV-1 strain HFEM is affected and cannot be expressed. The first 35 amino acid (AA) residues of the deduced amino acid sequence of the UL56 open reading frame (ORF) were found to be identical to the amino acid sequence of the UL56 genes of HSV-1 strains 17 and F. However, due to a deletion at np 3494 of the BamHI DNA fragment B of HSV-1 strain HFEM the amino acid composition of the predicted UL56 gene of HSV-1 strain HFEM is different from HSV-1 strain 17 between amino acid positions 36 and 233. In addition the deduced amino acid sequence of the IRL (inverted repeat of the long segment) copy of the IE110 gene of HSV-1 strain HFEM was found to be about 342 amino acids shorter than the amino acid sequence of IE110 gene of HSV-1 strain 17 (775 AA). This was based on a point mutation which was detected within the DNA sequences of Exon 3 of this copy of IE110 gene of HSV-1 strain HFEM.

Amino Acid Sequence↗

PubMed1992

Characterization of the third origin of DNA replication of the genome of insect iridescent virus type 6.

The structure of the third origin of DNA replication (CIV-ori-M) of the genome (209 kbp) of Chilo iridescent virus (CIV) was determined by DNA nucleotide sequence analysis. The CIV-ori-M is located within the DNA sequences of the EcoRI CIV DNA fragment M (7 kbp; 0.310-0.345 viral map units) between the genome coordinates 0.310 (EcoRI site) and 0.317 (NcoI site). The DNA nucleotide sequence of the EcoRI/NcoI CIV DNA fragment (1601 bp) was determined for identifying the DNA sequence of the corresponding origin of DNA replication. The analysis of the DNA sequences of this region revealed the presence of a 12-mer inverted repeat at nucleotide positions 485-496 and 503-513 (485-AGATATTTGACT-496-TATGT-503-AGTCAAATATCT-513) that are able to form a hairpin-loop structure. A double-stranded DNA fragment was synthesized that corresponds to the nucleotide positions 485-513 that were cloned into the phages M13mp18 and M13mp19, and were screened for their ability to be amplified in CF-124 cell cultures infected with CIV. The successful amplification of the DNA sequence of the CIV-ori-M is strong evidence that this particular region of the CIV genome indeed serves as the origin of DNA replication.

Base Sequence↗

PubMed1992

Identification and mapping of origins of DNA replication within the DNA sequences of the genome of insect iridescent virus type 6.

The origins of DNA replication of the genome (209 kbp) of Chilo iridescent virus (CIV), which is circularly permuted and terminally redundant, were identified. The defined genomic library of CIV, which represents 100% of DNA sequences of the viral genome (e.g., all 32 EcoRI CIV DNA fragments), was used for transfection of Choristoneura fumiferana insect cell cultures (CF-124) that were previously infected with CIV. The plasmid rescue experiments were carried out to select those recombinant plasmids that were amplified during viral replication in CIV-infected cell cultures. It was found that six recombinant plasmids harboring the EcoRI DNA fragments C [13.5 kbp, 0.909-0.974 map units (m.u.)], H (9.8 kbp, 0.535-0.582 m.u.), M (7.25 kbp, 0.310-0.345 m.u.), O (6.5 kbp, 0.196-0.228 m.u.), Q (5.9 kbp, 0.603-0.631 m.u.), and Y (2.0 kbp, 0.381-0.391 m.u.) were able to be amplified under the conditions used. This indicates that the CIV genome possesses six DNA replication origins. Subclones of the EcoRI CIV DNA fragments C and H were screened under the same conditions. It was found that DNA sequences within the EcoRI DNA fragments C and H at the genome coordinates 0.924-0.930 and 0.535-0.548, respectively, contain origins of viral DNA replication. The DNA nucleotide sequences of the EcoRI CIV DNA fragment Y (1986 bp) were determined for identifying the DNA sequence of the corresponding origin of DNA replication. The computer-aided analysis revealed the presence of a 15-mer inverted repeat at nucleotide positions 661-675 and 677-691 (661-TAAATTTAATGAGAA-G-TTCTCATTAAATTTA-692). The analysis of the DNA sequence of the EcoRI DNA fragment H corresponding to the particular region at the genome coordinates 0.535-0.548 (1) showed that this region contains a 16-mer inverted repeat at the nucleotide positions 1315 and 1332 (1315-TAAATTTTAATGGTTA-A-TAACCATTAAAATTTA-1347), which is very similar to the inverted repetition found within the EcoRI DNA fragment Y. The successful recognition and amplification of the single-stranded synthetic DNA sequences of both strands of CIV-ori-Y (nucleotide position 661-691) using phage M13 system in CIV-infected cells is strong evidence that the CIV-ori-Y is bidirectionally active, and this DNA sequence is considered to be the origin of DNA replication within the EcoRI CIV DNA fragment Y.

Base Sequence↗

PubMed1992

Mutations in the UL53 gene of HSV-1 abolish virus neurovirulence to mice by the intracerebral route of infection.

The cell fusion protein, the product of the UL53 gene, is responsible for intracerebral (IC) pathogenicity of HSV-1. Recombinant HSV-1 R15 is apathogenic to mice by the IC route of inoculation, while intratypic recombinants, in which the UL53 gene in R15 was replaced by an analogous sequence from the pathogenic strain R19, regained IC pathogenicity. The nucleotide sequence of the UL53 gene of HSV-1 strains R15 (apathogenic) and R19 (pathogenic) was determined and compared to that of other pathogenic strains. Four mutations were found which are thought to be responsible for the apathogenic phenotype of HSV-1 strain R15. Northern blot hybridization of RNA extracted from BSC-1 cells infected with several HSV-1 strains indicated that all of the virus strains tested expressed equal amounts of UL53 mRNA in infected cell cultures. Demonstration of the expression of UL53 mRNA in brains of mice infected with HSV-1 strains was made possible by the combined use of a rapid method for mRNA extraction (Oligo dT-linked magnetic beads) and a highly sensitive technique for detection of the existence of the UL53-specific mRNA (cDNA synthesis followed by PCR). It was shown that both pathogenic (KOS and P42) and apathogenic (R15) HSV-1 strains expressed the UL53 gene in brains of IC infected mice.

Amino Acid Sequence↗

PubMed1992

Direct detection of Molluscum contagiosum virus in clinical specimens by in situ hybridization using biotinylated probe.

Molluscum contagiosum virus (MCV) is an unclassified poxvirus which has recently become recognized as causing a major sexually transmitted disease. At present no assay is available for specific detection of MCV because the virus cannot be serially propagated in cell culture. Since MCV produces an abortive, limited growth with some cytopathic effect in certain cell lines, we were able to develop an in situ hybridization assay for detection of MCV genome in clinical specimens. Human fetal diploid lung cell monolayers were infected with clinical specimens, and after proper incubation and fixation in paraformaldehyde, hybridization was performed under full stringency conditions with a molecularly cloned biotinylated probe. Only MCV infected cells showed homology to the MCV probe with a purple-brown cytoplasmic staining. Additionally, we have described an in situ hybridization assay for direct detection of MCV genome in formalin-fixed, paraffin-embedded biopsies. Characteristic intracytoplasmic Molluscum bodies (Henderson-Paterson bodies) were detected in stratum spinosum cells of the epidermis. Striking staining similarities have been observed between in situ hybridization and haematoxylin-eosin cytostaining. These procedures are the first successful identification of MCV genome in clinical samples by molecular hybridization, with sensitivity and specificity equal to or greater than electron microscopy.

Blotting, Southern↗

PubMed1991

Stability of molluscum contagiosum virus DNA among 184 patient isolates: evidence for variability of sequences in the terminal inverted repeats.

The stability of the Molluscum contagiosum virus Type 1 genome (188 kbp) was studied in 184 DNA isolates from 131 patients. Variability of up to 1.5 kbp at both ends of the genome symmetrically was observed using restriction analysis of the DNA isolates and by Southern Blot experiments using cloned and labeled HindIII terminal DNA fragments of MCV-1 prototype DNA. The variable sequences were mainly confined to the terminal fragments and parts of the MCV-1 terminal repeats. Labeled probes did not detect terminal sequences of MCV Type 2 under the applied stringency. A less marked instability of the central MCV-1 BamHI DNA fragment F was observed within the genome coordinates 0.431 to 0.454 mu. Reiteration of tandem repeats similar to those described for vaccinia virus might explain the variability of the terminal sequences and might be involved in viral replication.

Blotting, Southern↗

PubMed1991

Identification and characterization of a Hantavirus strain of unknown origin by nucleotide sequence analysis of the cDNA derived from the viral S RNA segment.

The genetic characterization of a serologically Hantaan-like virus but of unknown origin (termed DX) was carried out by molecular cloning and nucleotide sequencing of the corresponding cDNA of the viral S RNA segment. The S RNA was found to be 1765 nucleotides long with 3' and 5' termini being complementary for 24 bases. The virus messenger-sense RNA contains one major open reading frame (ORF) encoding 428 amino acids or a 50 kD polypeptide. A comparison of the DX S RNA segment to those of Sapporo rat, Hantaan, Puumala/Hällnäs B1, and Prospect Hill viruses reveals 95.4, 71.3, 55.3, and 60.9% homology at the nucleotide sequence level, and 94.7, 80.1, 58.4, and 59.8% at the deduced amino acid sequence level. Thus Hantavirus strain DX is very closely related to Sapporo rat virus. We also analyzed the S RNA segments of these Hantaviruses for the presence of a second ORF encoding a potential nonstructural NSs protein. All potential second ORFs detected in the different S RNA segments differ substantially in length and position among the viruses, despite the high conservation of the nucleotide sequences and the overall structure of the nucleocapsid proteins. This suggests that the nucleocapsid protein is the only polypeptide encoded by Hantavirus S RNA segments, setting them apart from the other members of the Bunyaviridae family.

Amino Acid Sequence↗

PubMed1991

In vitro transcription and translation of proteins encoded by the BamHI-B genomic fragment of herpes simplex virus-1.

The BamHI-B DNA fragment of herpes simplex virus type-1 (HSV-1) is associated with intraperitoneal pathogenicity. Among the recently mapped RNA transcripts from this fragment (15), one was reported to be associated with latency. To relate the RNA transcripts to virus pathogenicity, the in vitro-transcribed RNAs from BamHI-B fragments of three HSV-1 strains--F (pathogenic), R19, and HFEM (apathogenic), were studied by in vitro translation. When the BamHI-HpaI (0.738-0.755 map units) DNA fragment from HSV-1 strain F was transcribed rightward and translated, three proteins of 70, 63, and 51 kD were detected. The 63 kD protein resembles in size and orientation the protein encoded by the ICP-27 (IE-2) gene (0.740-0.749 mu). The 51 kD polypeptide is assumed to be a prematurely terminated form of this protein. No proteins were obtained from RNA transcribed in the opposite direction. The SalI-NcoI (0.746-0.761 mu) fragment of the three HSV-1 strains yielded two proteins of 25 and approximately 15 kD when transcribed rightward and a 35 kD polypeptide from RNA transcribed in the opposite direction. As a result of the genomic deletion in HFEM, it was possible to obtain the 35 kD protein from the SalI-SalI DNA fragment (0.746-0.761 mu) as well. In vitro transcription and translation of the PstI-SalI (0.778-0.790 mu) DNA fragment (the right-hand side of HpaI-P) did not result in protein synthesis. The possibility that the UL56 gene is connected with the intraperitoneal pathogenicity of HSV-1 is discussed.

Animals↗

PubMed1991

Primary structure of the large (L) RNA segment of nephropathia epidemica virus strain Hällnäs B1 coding for the viral RNA polymerase.

The L RNA segment of the nephropathia epidemica virus (NEV) strain Hällnäs B1 was characterized by molecular cloning of the corresponding cDNA and subsequent determination of the DNA nucleotide sequence. The L RNA segment is 6550 nucleotides long with complementarity of 20 bases at the 3' and 5' termini. The viral messenger sense RNA contains one major open reading frame (ORF) with a coding capacity of 2156 amino acid residues encoding a protein with a calculated molecular weight of 246 kDa and an IEP of pH 7.4. Comparison of the deduced amino acid sequences from NEV hantavirus and Bunyamwera virus (BWV) L segment messenger sense RNAs, revealed a high degree of diversity (overall amino acid identity, 17%). However, three clusters of 30-40% amino acid identity were detected. One of these domains, containing an Asp-Asp motif found in many RNA polymerases, also shares amino acid sequence homology with the PB1 polymerase component of influenza type A. These results indicate that the L RNA segment of the NEV codes for the viral RNA-dependent RNA polymerase. The data presented here complete our previous studies on the characterization of the NEV genome by cDNA sequencing of the viral M and S RNA segments.

Amino Acid Sequence↗

PubMed1991

The primary structure of the thymidine kinase gene of fish lymphocystis disease virus.

The DNA nucleotide sequence of the thymidine kinase (TK) gene of fish lymphocystis disease virus (FLDV) which has been localized between the coordinates 0.678 to 0.688 of the viral genome was determined. The analysis of the DNA nucleotide sequence located between the recognition sites of HindIII (0.669 map unit; nucleotide position 1) and AccI (nucleotide position 2032) revealed the presence of an open reading frame of 954 bp on the lower strand of this region between nucleotide positions 1868 (ATG) and 915 (TAA). It encodes for a protein of 318 amino acid residues. The evolutionary relationships of the TK gene of FLDV to the other known TK genes was investigated using the method of progressive sequence alignment. These analyses revealed a high degree of diversity between the protein sequence of FLDV TK gene and the amino acid composition of other TKs tested. However, significant conservations were detected at several regions of amino acid residues of the FLDV TK protein when compared to the amino acid sequence of TKs of African swine fever virus, fowlpox virus, shope fibroma virus, and vaccinia virus and to the amino acid sequences of the cellular cytoplasmic TK of chicken, mouse, and man.

Amino Acid Sequence↗

PubMed1991

Elimination of UL56 gene by insertion of LacZ cassette between nucleotide position 116030 to 121753 of the herpes simplex virus type 1 genome abrogates intraperitoneal pathogenicity in tree shrews and mice.

In order to investigate whether or not the UL56 gene is involved in those processes determining the viral pathogenicity and latency, a recombinant virus HSV-1-M-LacZ was constructed in which the DNA sequences between nucleotide position (np) 116030 and 121753 were replaced by the E. coli beta-galactosidase (LacZ) gene. This deletion spans from the carboxyterminus of UL55 (np 116030) to the second exon of IE110 (np 121753) eliminating UL56 and the variable region of the BamHI DNA fragment B which were implicated in intraperitoneal pathogenicity and latency. The host range and growth kinetics of the recombinant virus HSV-1 M-LacZ were comparable to the parental strain HSV-1 F. As expected it was found that HSV-1-M-LacZ lost its virulent phenotype and was not able to develop acute infection in animals. The state of the UL56 gene was investigated by determining the cDNA sequence of the UL56 gene transcript of HSV-1 F using PCR products obtained after amplification of the cDNA with oligonucleotide primers corresponding to the translational start and stop codons of this gene. This analysis revealed that the DNA sequence of the UL56 gene of HSV-1 F differed from those DNA sequences determined for the genomic DNA of HSV-1 strain 17. Between nucleotide position 116343 and 116344 two nucleotides -AG- are inserted which prolong the ORF of the UL56 gene to 233 amino acids with a predicted molecular weight of 30 kDa.

Adrenal Glands↗

PubMed1991

Antigenicity of hantavirus nucleocapsid proteins expressed in E. coli.

DNA clones representing the small genomic segment of Nephropathia epidemica virus strain Hällnäs B1 (NEV) and Hantaan virus strain 76-118 (HTV) encoding their nucleocapsid proteins were inserted into the E. coli vector pIN-III-ompA for secretion of proteins into the periplasmic space. The complete HTV and NEV nucleocapsid proteins and two truncated versions of the NEV nucleocapsid proteins were expressed as fusion proteins. Unexpectedly, all products accumulated as insoluble aggregates. Most of the ompA signal peptide remained uncleaved. However, nucleocapsid fusion proteins could be purified from the insoluble fraction by extraction with 8 M urea followed by separation on SDS-PAGE and electroelution. Rabbits were immunized with the eluted proteins and the resulting antibodies reacted specifically with authentic viral nucleocapsid proteins of HTV and NEV. The recombinant nucleocapsid proteins were found to react specifically with various hantavirus-immune sera, but not with human control sera, indicating their suitability as potential diagnostic antigens. This is the first report on the expression of a protein of a NEV serotype strain of hantaviruses by use of recombinant DNA techniques.

Antigens, Viral↗

PubMed1991

Identification and mapping of the UL56 gene transcript of herpes simplex virus type 1.

The herpes simplex virus type 1 (HSV-1) strain HFEM is apathogenic for tree shrews and mice by the intraperitoneal application route. This is due to a 4.1 kbp deletion [0.762 to 0.789 map units (mu)] within the BamHI DNA fragment B of the viral genome. With exception of 71 bp the DNA sequences of the deleted region are located within the repetitive DNA sequences of the inverted repeat of the L segment of the HSV-1 genome (IRL). A 1.5 kb RNA hybridizing to the DNA sequences of the HSV-1 genome at map position 0.760-0.762 (BssHII DNA fragment F, part of the BamHI DNA fragment B) was found to be missing in cells infected with HSV-1 HFEM and other apathogenic HSV-1 strains. A detailed analysis of the transcriptional profile of this region of the pathogenic prototype strain HSV-1 F and strand-specific hybridizations revealed that this 1.5 kb RNA species is transcribed at 2 to 4 h p.i. in leftward orientation. The corresponding open reading frame in the HSV-1 genome had been predicted as the UL56 gene. The absence of this 1.5 kb RNA in HSV-1 HFEM-infected cells is due to the fact that the promoter region of the UL56 gene is located within those DNA sequences which are deleted in the HSV-1 HFEM genome. A specific DNA fragment (650 bp) was amplified by reverse polymerase chain reaction using oligonucleotide primers corresponding to the predicted translational start and termination region of the UL56 gene. The corresponding cDNA had been derived from cellular RNA from HSV-1 F-infected cells using oligo(dT) priming. This indicates that the 1.5 kb RNA is the real transcript of the UL56 gene of HSV-1.

Amino Acid Sequence↗