Precise localization of genes on large animal virus genomes: use of lambda gt11 and monoclonal antibodies to map the gene for a cytomegalovirus protein family.

Abstract

We describe an efficient procedure, which uses monoclonal antibodies directed against specific viral proteins, for the precise mapping of genes on large DNA virus genomes. We have used the technique to locate the gene encoding a family of antigenicaily related DNA-binding proteins on the 240-kilobase-pair human cytomegalovirus (CMV) genome. A random library of CMV DNA fragments was generated using the prokaryotic vector Xgtll, which expresses open reading frames as ,-galactosidase fusion proteins in infected Escherichia coli. The library was screened with a mixture of monoclonal antibodies directed against the gene products of interest. The coding sequence for infected cell protein 36 (ICP36) was localized to a 2800-base-pair EcoRI fragment (map coordinates 0.228-0.240) on the CMV(Towne) and CMV(AD169) genomes by using DNA from immunoreactive Xgtll as probe. A 5000-nucleotide transcript from this region was detected during the early and late phases of the CMV growth cycle. This transcript directed the synthesis of the predominant member of the ICP36 family when hybrid-selected and translated in vitro. Immunoprecipitation of the in vitro translation product with the same monoclonal antibodies used in the initial mapping confirmed the location of the ICP36 gene. These studies establish the utility of the Agtll expression system for rapid and precise mapping of CMV genes (or other large animal virus genes) that encode proteins for which serological reagents exist.Human cytomegalovirus (CMV), a herpesvirus carried by most of the population, causes disease in infants and immunocompromised adults. CMV has a large DNA genome of -240 kilobase pairs (kbp) on which few viral genes have been precisely localized (1-4). Even less is known concerning the functional organization of the viral genome. Studies on the temporal expression of viral proteins and mRNA transcripts (4-11) have suggested that, as for herpes simplex virus (12), CMV gene expression is coordinately regulated and sequentially ordered; however, viral regulatory functions have not been identified. Major obstacles to locating viral genes have been (i) the large size of the CMV genome, (ii) the slow growth of the virus in cell culture, (iii) the continued synthesis of host proteins during infection, (iv) the lack of viral mutants, and (v) a host range limited to human fibroblast cells. We have modified the recently described procedures for cloning in the prokaryotic expression vector Xgtll (13,14), thereby establishing a system using serological reagents for rapid and precise mapping of viral gene products.We constructed a Xgtll library of randomly generated 400-to 500-base-pair (bp) CMV DNA fragments and probed this library with monoclonal antibodies to isolate clones expressing portions of viral proteins as f3-galactosidase fusion proteins. DNAs from immunoreactive phage were used as hybridization probes for precise localization of the coding sequences on the CMV genome.We used two monoclonal antibodies isolated by Pereira et al. (15) to map a major DN...

Role of apical and basolateral membranes in replication of human cytomegalovirus in polarized retinal pigment epithelial cells

“…CH160-5 to HCMV immediate early proteins (IE1 and IE2, UL123 and UL122) was used in the rapid infectivity assay (described below). The pool of MAbs used to detect expression of HCMV proteins in foci of infected RPE cells was as follows: CH16-1 (ICP36, UL44), CH167-1 (ICP8, UL57), CH19-1 (pp28, UL99) and CH253-1 (gB, UL55) (Mocarski et al, 1985(Mocarski et al, , 1988Pereira et al, 1982;Qadri et al, 1992).…”

Abundant 5 kb RNA of Human Cytomegalovirus without a Major Translational Reading Frame

“…Only a few proteins of HCMV-infected cells can at present be assigned to defined regions on the genome (Cranage et al, 1986;Heilbronn et al, 1987;Kouzarides et al, 1987;Mach et al, 1986;Mocarski et al, 1985;Riiger et al, 1987); from such studies it appears that E and late genes of HCMV are scattered over the entire genome. In contrast, the IE genes are clustered in a restricted area (Jahn et al, 1984a;Stinski et al, 1983).…”

Identification and Preliminary Use of Recombinant Lambda gt11 Fusion Proteins in Human Cytomegalovirus Diagnosis

“…Individual HCMV proteins expressed via recombinant DNA techniques are a promising approach to solve this problem. In this work a 2gtl 1 HCMV DNA library (Mocarski et al, 1985) was screened with a pool of human sera with high Ab titres to HCMV. Positive plaques were identified, purified to homogeneity and studied.…”

“…Positive plaques were identified, purified to homogeneity and studied. Two recombinant Escherichia coli proteins, one containing part of a major HCMV structural phosphoprotein (p 150) and the other part of a major non-structural protein (p52), were used to detect Ab in human sera and proved useful in distinguishing between acute and inactive HCMV infection.Human embryo fibroblasts were grown as described by Landini et al (1985) and the Towne strain of HCMV purified as previously described (Landini & Ripalti, 1982) was used in all the experiments.A 2gtl 1 library of randomly generated 400 to 500 bp HCMV DNA fragments inserted into the EcoRI site of the lacZ gene (Mocarski et al, 1985) was used. In this library the translational 0000-8558 O 1989 SGM …”

“…A 2gtl 1 library of randomly generated 400 to 500 bp HCMV DNA fragments inserted into the EcoRI site of the lacZ gene (Mocarski et al, 1985) was used. In this library the translational 0000-8558 O 1989 SGM products of inserted open reading frames are expected to be part of a fl-galactosidase fusion protein.…”