Fast screening procedures for random transposon libraries of cloned herpesvirus genomes: mutational analysis of human cytomegalovirus envelope glycoprotein genes.

JOURNAL OF VIROLOGY,
0022-538X/00/$04.0010
Sept. 2000, p. 7720–7729 Vol. 74, No. 17
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Fast Screening Procedures for Random Transposon Libraries of
Cloned Herpesvirus Genomes: Mutational Analysis of Human
Cytomegalovirus Envelope Glycoprotein Genes
URS HOBOM, WOLFRAM BRUNE, MARTIN MESSERLE, GABRIELE HAHN,
AND ULRICH H. KOSZINOWSKI*
Lehrstuhl fu¨r Virologie, Max von Pettenkofer-Institut, Ludwig-Maximilians-Universita¨t Mu¨nchen, 80336 Munich, Germany
Received 28 December 1999/Accepted 23 May 2000
We have cloned the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromo-
some (BAC) in Escherichia coli. Here, we have subjected the HCMV BAC to random transposon (Tn) mu-
tagenesis using a Tn1721-derived insertion sequence and have provided the conditions for excision of the BAC
cassette. We report on a fast and efficient screening procedure for a Tn insertion library. Bacterial clones
containing randomly mutated full-length HCMV genomes were transferred into 96-well microtiter plates. A
PCR screening method based on two Tn primers and one primer specific for the desired genomic position of
the Tn insertion was established. Within three consecutive rounds of PCR a Tn insertion of interest can be
assigned to a specific bacterial clone. We applied this method to retrieve mutants of HCMV envelope glyco-
protein genes. To determine the infectivities of the mutant HCMV genomes, the DNA of the identified BACs
was transfected into permissive fibroblasts. In contrast to BACs with mutations in the genes coding for gB, gH,
gL, and gM, which did not yield infectious virus, BACs with disruptions of open reading frame UL4 (gp48) or
UL74 (gO) were viable, although gO-deficient viruses showed a severe growth deficit. Thus, gO (UL74), a
component of the glycoprotein complex III, is dispensable for viral growth. We conclude that our approach of
PCR screening for Tn insertions will greatly facilitate the functional analysis of herpesvirus genomes.
Human cytomegalovirus (HCMV) infection is widespread
and usually without symptoms in healthy adults, but it can
cause severe disease in the immunologically immature or im-
munodeficient host and is a leading cause of birth abnormali-
ties in industrialized countries (7). Its 230-kbp linear double-
stranded DNA genome is among the largest in the herpesvirus
family, encoding well over 150 proteins. The nucleic acid se-
quence predicts about 55 open reading frames (ORFs) coding
for transmembrane glycoproteins (10). Some of these glyco-
proteins modulate the communication of the infected cell with
the host’s immune system (20, 53). Only a limited number of
glycoproteins represent virion components (8, 23). The enve-
lope of HCMV consists of at least three distinct types of co-
valently linked glycoprotein complexes (18). The homology to
herpes simplex virus type 1 (HSV-1) virion glycoproteins indi-
cates a certain degree of conservation among herpesvirus gly-
coproteins (8). While the 12 HSV-1 glycoprotein ORFs have
already been subjected to extensive mutational analysis (43),
their HCMV counterparts so far have escaped such studies in
the context of viral infection (8).
F-factor-based bacterial artificial chromosomes (BACs) can
efficiently be used for the propagation of the genomes of large
recombinant DNA viruses in Escherichia coli (36). Cloning a
herpesvirus genome, exemplified by the murine CMV (MCMV)
genome, as an infectious BAC made herpesviruses generally
accessible to the methods of bacterial genetics (38). By now,
several other herpesvirus genomes have been cloned as BACs,
representing members of alpha-, beta-, and gammaherpesvi-
ruses (1, 6, 14, 21, 44, 47, 51; G. Hahn, M. Mach, M. Messerle,
and U. H. Koszinowski, Abstr. 24th Int. Herpesvirus Work-
shop, abstr. 13.013, 1999). Transposons (Tn) are well estab-
lished tools for random insertion mutagenesis of bacterial ge-
nomes (4). Using the MCMV BAC as an example, we have
introduced this method for the random mutagenesis of cloned
herpesvirus genomes (9). More recently, the general feasibility
of this approach has been independently confirmed by others
with a different Tn system (47).
Here, we report on the application of this technique for the
mutational analysis of the full-length infectious genome of
HCMV. We have developed a fast screening procedure for a
Tn insertion library of HCMV genomes. For retrieval of Tn
insertions in a gene of interest, only three consecutive rounds
of PCR analysis on hierarchically pooled DNA samples are
required. To demonstrate the efficacy of the method, we re-
trieved and analyzed mutants of known HCMV envelope gly-
coprotein genes. This method should significantly speed up the
access to genomes with mutations within specific ORFs and
thus facilitate the assignment of specific functions to individual
herpesvirus genes.
MATERIALS AND METHODS
Recombinant viruses and cells. Virus propagation and viral DNA extraction
were essentially performed as previously described (6). MRC-5 cells (human
fetal lung fibroblasts; BioWhittaker, Verviers, Belgium) were used for transfec-
tion and propagation of reconstituted viruses. The original HCMV BAC, re-
ferred to as pHB-5, represents an infectious derivative of AD169 (American
Type Culture Collection) lacking the genes US2 to US6 (nucleotides [nt] 193360
to 196045) (6). Nucleotide numbering and delineation of ORFs are given ac-
cording to the sequence published by Chee et al. (10) (GenBank accession no.
X17403), irrespective of the additional 929 bp contained in the EcoRI c9 frag-
ment of the cloned virus (13, 39).
Plasmid construction. To reinsert the deleted US2 to US6 genes and to make
the BAC cassette, a fusion construct of pMBO131 (40) with a gpt selection
marker (17) excisable from the genome, the following plasmids were used for
homologous recombination in bacteria. For construction of pUH-16, one loxP
site, excised as a PstI/SacI fragment (59-ctc gag ctc cac cgc ggt ggc ggc cac gga tgc
atc cgt ggc cgc GCA TAA CTT CGT ATA GCA TAC ATT ATA CGA AGT
TAT cta gca gat ctg cag-39) from pllNsi (M. Messerle, unpublished), a derivative
* Corresponding author. Mailing address: Max von Pettenkofer-
Institut, Pettenkofer Str. 9a, 80336 Munich, Germany. Phone: 49 89
5160 5290. Fax: 49 89 5160 5292. E-mail: koszinowski@m3401.mpk
.med.uni-muenchen.de.
7720

of pP4 (12), was cloned into pSL301 (Invitrogen, San Diego, Calif.). The loxP site
was flanked with homologous sequences from HCMV (a BssHII/NheI fragment
spanning nt 191395 to 193360 of AD169) and the BAC cassette (an XbaI/SpeI
fragment excised from pEB1097 [6]) (see Fig. 1C). From this construct BamHI
and MscI fragments were excised and placed between the BamHI and SmaI sites
of shuttle plasmid pST76K-SR (kindly supplied by G. Po´sfai, Szeged, Hungary).
pST76K-SR carries the recA and sacB genes and allows mutagenesis of BAC
plasmids in the recombination-deficient E. coli strain DH10B to be performed
essentially as described in reference 55. For construction of pUH-15, one loxP
site was excised from pllNsi by digestion with SacI and EcoRI and inserted into
an oligonucleotide linker (59-cat gGA TCC GCG GCC GCT TTC TCG AGC
TCA TGC ATT TTG AAT TCG GCG CGC CTT TTC TAG AGG ATC CAa
gct-39), replacing the multiple cloning site (NcoI-HindIII) of pSL301. Next, a PstI
fragment excised from pMin-1 (26), which codes for the RP4 origin of transfer,
was inserted into a unique PstI site imported with the loxP fragment. Subse-
quently, US2 to US6 (nt 193003 to 198197) were added as a XhoI/EagI fragment
of pCM1052 (16). After addition of the BAC homology domain (AscI to XbaI of
pEB1097) the whole insert was excised with BamHI and transferred into shuttle
vector pST76K-SR. Allelic exchange (see below) of pHB-5 with pUH-16 and
pUH-15 yielded HB-5loxP and AD169-BAC, respectively (Fig. 1C).
Tn mutagenesis. Insertion mutagenesis was performed as previously described
(9). Briefly, the temperature-sensitive Tn donor plasmid pTsTM8 was electro-
porated into E. coli strain DH10B harboring AD169-BAC and plated at 30°C on
Luria-Bertani (LB) agar plates containing chloramphenicol (13.6 mg/ml) and
ampicillin (100 mg/ml). Bacterial clones containing both the HCMV BAC plas-
mid and the Tn donor plasmid were grown as liquid cultures at 30°C in the
presence of both antibiotics. Small aliquots (approximately 2 ml per plate) were
spread on LB agar plates at 43°C and selected with chloramphenicol and kana-
mycin (50 mg/ml) for transposition events. Bacterial colonies (about 200 per
plate) were replated for another round of purification at 43°C in the presence of
chloramphenicol and kanamycin and were then grown as liquid cultures in
96-well microtiter plates. Aliquots of the liquid culture of individual bacterial
clones were pooled by rows, and DNA was prepared by following the alkaline
lysis protocol (45) to generate DNA pools. DNA pools of the microtiter plate
were generated by combining samples of the eight DNA row pools (see Fig. 3A).
PCR screening for Tn insertions within specific genes. For the detection of Tn
insertions at positions of interest, three rounds of PCR were performed (PCR
conditions: 7 min at 94°C, followed by 40 cycles of 16 at 95°C, 11 s at 55°C, and
1 to 2 min at 72°C, using AmpliTaq Gold and the GeneAmp 9700 PCR cycler
[Perkin-Elmer, Darmstadt, Germany]). The Tn can insert in both orientations
into the BAC (see Fig. 3A). Therefore, two Tn-specific primers (M13-for and
M13-rev) were included in the reaction. The position-specific search primers
used in this study are listed in Table 1. In the first round of PCR, the DNA pools,
each representing all mutants contained in one microtiter plate, were examined.
In the second round, the DNA pools representing the mutants stored in specific
rows of those individual plates that tested positive in the first round were
screened. From the individual wells of the rows identified as positive, crude DNA
extracts were made by boiling aliquots of the frozen bacteria briefly in 0.1%
Triton X-100 (45). These DNA samples were subjected to a third round of PCR
screening in order to determine the exact location of the desired mutant within
the Tn library. With a second distal primer oriented in the opposite direction
(listed in Table 1) and located about 2 kbp from the search primer, a confirma-
tory PCR was performed to exclude illegitimate deletion events at the Tn inte-
gration site. The exact location of the Tn insertion was determined by restriction
enzyme analysis and direct sequencing of BAC DNA with primers M13-for
(59-GCC GCT GTA AAA CGA CGG CCA GT-39) and M13-rev (59-GGC CGC
AGG AAA CAG CTA TGA CC-39) as described previously (9).
Allelic exchange. For the generation of revertant BACs, 4 to 5 kbp of viral
DNA was excised from BAC pHB-5 using appropriate restriction enzymes and
cloned into plasmid pST76A-SR, a derivative of pST76A (41) carrying the recA
and sacB genes from pST76K-SR (M. Wagner and C. Me´nard, unpublished
data). Plasmids pUH-25 to pUH-34, which were used for the construction of
revertants, are listed in Table 2. The principles of recA-mediated allelic replace-
ment in E. coli have been described elsewhere (38, 40, 55). Resolution of cointe-
grates was significantly improved by counterselecting against sacB (6, 22). Briefly,
temperature-sensitive shuttle plasmids (pUH-15, -16, and -25 to -34) were co-
transformed with the HCMV BAC plasmid into electrocompetent E. coli
DH10B, and transformants were selected at 30°C on agar plates containing
chloramphenicol, ampicillin, and/or kanamycin. Cultivation at nonpermissive
43°C in the presence of chloramphenicol and ampicillin or kanamycin led to the
selection of clones containing cointegrates. By recA-mediated recombination,
cointegrates are resolved with an ;50% chance to either the initial BAC or the
BAC variant carrying the desired mutation (38). Resolved cointegrates were
selected for in the presence of 5% sucrose at 30°C by making use of the sacB
gene carried on the shuttle plasmid (5, 6). To confirm allelic replacement and
loss of the Tn insertion, potentially positive colonies were replica plated on
chloramphenicol and kanamycin plates and screened for kanamycin-sensitive
clones.
Reconstitution of BAC cassette-free mutant viruses. One microgram of BAC
DNA purified on Nucleobond columns (Macherey Nagel, Du¨ren, Germany) was
cotransfected with 0.3 mg of pcDNApp71tag, a plasmid expressing pp71, for
enhancement of infectivity (3, 6, 35) (kindly provided by B. Plachter, Mainz,
Germany) and 0.5 mg of plasmid pBRep-Cre using 12 ml of Superfect according
to the manufacturer’s instructions (Qiagen, Hilden, Germany). pBRep-Cre con-
tains an XhoI fragment of pMC-Cre (19) fused to pBRep (W. Brune, unpub-
lished) expressing recombinase Cre for excision of the BAC cassette. In parallel,
transfections were performed with complementing plasmids carrying the respec-
tive HCMV DNA fragment that spans the Tn insertion site (pUH-25 to pUH-34;
listed above). Five days posttransfection cells were split 1:3. A marked cytopathic
effect usually became apparent 10 to 12 days posttransfection. When no plaques
became visible, cells were split 1:3 at day 15 after transfection. Cells were
monitored for 1 month after transfection. When after repeated transfection
experiments no plaques could be obtained unless the complementing plasmid
was included, the continuity of the sequence interrupted by Tn insertion was
classified as essential for virus growth.
RESULTS
Completion of the HCMV BAC. We wanted to construct a
Tn insertion library with a full-length HCMV AD169 genome.
Published infectious BAC clone pHB-5 was constructed by
deleting genes US2 to US6 to accommodate the BAC cassette
and to restrict the overlength of the pHB-5 BAC genome to
about 5 kbp (6). Tn insertion adds an additional 2 kbp of
overlength, and the reintroduction of the missing US2-to-US6
sequence would result in a propensity for random deletions
imposed by the packaging restraints of the virion during virus
reconstitution (49) (data not shown). Therefore, in order to
minimize genome instability, the BAC cassette was made ex-
cisable by attached loxP sites, similar to the completion of the
MCMV BAC and the pseudorabies virus (PrV) BAC (48, 54).
Homologous recombination of pHB-5 with pUH-16 and
pUH-15 in E. coli yielded HB-5loxP and AD169-BAC, respec-
tively (Fig. 1). Recombinase Cre-mediated excision of the BAC
TABLE 1. Search primers used for the identification of Tn
insertions into envelope glycoprotein genes
Primer Sequence
UL55-for....................59-AAA ACA TAG CGG ACC GTG AG-39
UL55-afo ...................59-GCA AGG CAT CAA GCA AAA ATC-39
UL55-are ...................59-CAC CGA TTC CAT GCT GGA C-39
UL55-rev ...................59-AGT GGC GAC GTG CCA ACA GC-39
UL75-for....................59-AGA CCC ATA ACA GTA CCT CG-39
UL75-rev ...................59-ATC TCC GCA GAG CGT TCC CC-39
UL100-are .................59-TTT GTG TGT GTT TGC GCC-39
UL100-rev .................59-CCT CTA GAA GGC CGT ACC AG-39
UL115-are .................59-GGA CAC AGA TAG CTC CAG-39
UL115-rev .................59-AGG ACG ACG ACG AGT ACG AC-39
UL4-for......................59-GCG ATG AGT CCA TAA AGC ACC-39
UL4-rev .....................59-CCA ACC ACA CAA AAG ACA ACA-39
UL74-for....................59-CTA CGA CAT TGC TGC TTC-39
UL74-rev ...................59-TCG TTG TAA TAA AGT ACA CGC C-39
TABLE 2. Plasmids used for the generation of revertant genomes
Plasmid Enzyme 1a Nucleotideposition Enzyme 2
a Nucleotide
position
pUH-25 EcoRV (SmaI) 105127 PstI (NsiI) 110694
pUH-26 SpeI (XbaI) 80099 XhoI (XhoI) 84790
pUH-27 SphI (SphI) 159669 NheI (NheI) 168330
pUH-28 SphI (SphI) 107919 SalI (SalI) 112129
pUH-29 KpnI (KpnI) 144435 KpnI (KpnI) 147618
pUH-30 XhoI (XhoI) 79366 SstI (SstI) 83906
pUH-31 NsiI (NsiI) 81000 Eco47III (SmaI) 85583
pUH-32 SnaBI (HpaI) 11427 SstI (SstI) 15544
pUH-33 SnaBI (HpaI) 105042 SnaBI (HpaI) 109189
pUH-34 XbaI (XbaI) 106258 PmlI (HpaI) 110389
a Enzymes used for cleaving shuttle vector pST76A-SR are in parentheses.
VOL. 74, 2000 FAST SCREENING PROCEDURE FOR HCMV GLYCOPROTEIN MUTANTS 7721

cassette yielded AD169-RV, a viral genome reduced to only a
small amount of overlength with respect to the wild-type (wt)
genome (Fig. 1E). To avoid the disruption of upstream regu-
latory sequences of US1 by the retained loxP site, a small
duplication surrounding the loxP site was introduced. The in-
termediate construct HB-5loxP (Fig. 1C), containing a single
loxP site, and AD169-BAC were digested with BglII and NsiI
and compared with plasmid pHB-5 (Fig. 2A, lanes 1 to 6).
Upon introduction of a loxP site into pHB-5, a 25.5-kbp BglII
fragment (Fig. 2A, lane 1) was cleaved into two subfragments
of 3.6 and 22.1 kbp (Fig. 2A, lane 2). Subsequent introduction
of genes US2 to US6 resulted in additional bands at 13.7 and
2.2 kbp (Fig. 2A, lane 3). The constructions outlined in Fig. 1
converted a 7.1-kbp NsiI fragment of pHB-5 (Fig. 2A, lane 4)
into two new fragments of 6.2 and 1.1 kbp (Fig. 2A, lane 5).
Further manipulation of HB-5loxP as depicted in Fig. 1C
yielded additional NsiI fragments of 2.2, 6.7, and 13.0 kbp (Fig.
2A, lane 6).
Transfection of the completed AD169-BAC into human em-
bryonic fibroblast cells followed by recombinase Cre-mediated
excision of the BAC cassette should yield recombinant virus
AD169-RV (Fig. 1E). Comparison of the DNAs of the recom-
binant viruses AD169-RV and RVHB5 (6) with that of the
parental strain, AD169 (Fig. 2A, lanes 7 to 12) displayed the
alterations of the restriction patterns as predicted in Fig. 1C. A
18.9-kbp XbaI fragment present in prototype AD169 (Fig. 2A,
lane 9) was cleaved into two subfragments of 7.5 and 11.8 kbp
(Fig. 2A, lane 7). In RVHB5 these fragments were absent, and
6.3- and 5.7-kbp fragments of the BAC cassette were retained
in the viral DNA (Fig. 2A, lane 8). The single 13.3-kbp HpaI
fragment of wt AD169 (AD 169-wt) (Fig. 2A, lane 12) yielded,
due to the insertion of an HpaI site, the 2.7- and 11.1-kbp HpaI
fragments of AD169-RV (Fig. 2A, lane 10), whereas the 2.6-
kbp HpaI fragment carrying US6 was absent from recombinant
virus RVHB5 (lane 11). Recombinant virus AD169-RV
showed growth properties indistinguishable from those of
AD169-wt (Fig. 2B). Therefore, the completed AD169-BAC
provided a useful substrate for Tn mutagenesis. Single Tn
insertions into BAC vector-deficient AD169-RV resulted in an
overlength of the genome well below that of the 235 kbp of
RVHB5 or conventional lacZ insertion mutants, a genome size
that is still packaged and that yields intact viral progeny (6, 29).
FIG. 1. Completion of the full-length HCMV BAC and excision of the BAC cassette. (A) In HCMV BAC plasmid pHB-5, the BAC cassette replaces genes US2
through US6 (nt 193360 to 196045). (B) Details of the region of BAC insertion. X, XbaI; N, NsiI; H, HpaI; B, BglII. (C) For construction of HB-5loxP, a loxP site flanked
with 2.0-kbp fragments homologous to HCMV and BAC sequences was cloned into pST76K-SR. Site-directed introduction into the HCMV genome by shuttle
mutagenesis yielded an additional BglII site and two additional NsiI sites (I). Subsequently, the genes US2 to US6 were introduced by shuttle mutagenesis together with
a loxP site and an RP4 origin of transfer (oriT) (II). (D) Cotransfection of AD169-BAC with a plasmid expressing recombinase Cre into permissive cells leads to the
removal of the BAC cassette from the HCMV genome. (E) The virus progeny AD169-RV is distinguishable from AD169-wt by XbaI and HpaI restriction sites located
next to the loxP site.
7722 HOBOM ET AL. J. VIROL.

As shown exemplarily with mutant B-B1 containing a sequence
disruption at the extreme end of ORF UL78 (nt 114124),
insertion mutagenesis with a Tn-derived mobile DNA se-
quence has no apparent effect on viral growth (Fig. 2B).
Generation and screening of a library of genomic HCMV Tn
mutants. The Tn previously described (9, 26) has a high pref-
erence for insertion into plasmids or BACs. In more than 90%
of the transposition events the BAC plasmid represented the
target, while insertions into the bacterial genome occurred
with a frequency of less than 10%. Therefore, a positive selec-
tion of Tn-inserted BACs via bacterial conjugation was not
required. Bacterial clones with Tn insertions were generated
and stored as glycerol stocks in 96-well microtiter plates.
These, together with a series of DNA pools of the mutant
genomes, constitute a library of Tn mutants (Fig. 3A). Assum-
ing a completely random distribution of Tn insertions over the
entire genomic BAC, such a library would have to comprise
5,294 individual clones to have a 99% chance of finding an
insertion every 200 nt. About 1,050 mutants would suffice for a
Tn insertion somewhere within every 1.0-kb fragment. In a first
approach, our library included 2,000 random mutant bacterial
clones.
To test the hypothesis of random insertions, half of this
library of genomic HCMV mutants was screened for insertion
FIG. 2. Recombinase Cre-mediated excision of the BAC vector sequences
results in a nearly wt AD169-RV. (A) Lanes 1 to 6, restriction enzyme digests of
HCMV BAC plasmids pHB-5, HB-5loxP, and AD169-BAC isolated from bac-
teria; lanes 7 to 12, digestion of viral DNA extracted from cells infected with
viruses reconstituted from BAC plasmids AD169-BAC (AD169-RV) and pHB-5
(RVHB5), or the parental strain AD169-wt. Relevant restriction sites are de-
picted in Fig. 1. Arrowheads, restriction endonuclease fragments; dots, addi-
tional fragments resulting from manipulations described in the legend for Fig. 1.
(B) Single-step growth curve of recombinant viruses. AD169-RV is compared to
parental HCMV strain AD169-wt and B-B1-RV, an AD169-BAC-based mutant
virus with the Tn sequences stably integrated at the 39-terminal end of UL78, nt
114124. MRC-5 cells were infected at an MOI of 0.1. Virus titers from cells and
supernatant were determined in duplicate by a standard plaque assay at the
indicated time points.
FIG. 3. Schematic outline for the PCR-based screening procedure for Tn
insertions. (A) Three rounds of PCR were performed on the hierarchically
pooled aliquots of the Tn library to locate a candidate mutant to a specific well
in one of the 96-well plates. (B) A primer binding specifically to a genomic
position of interest and the Tn-specific primers M13-for and M13-rev were
included in the reaction. If an insertion event occurred close to the position of
the specific primer binding site, a PCR product was generated irrespective of the
orientation of the Tn. kan, ORF encoding the kanamycin resistance marker;
upside down lettering indicates orientation opposite to that of the two other
ORFs shown. The size of the PCR amplificate indicates whether the insertion
lies within or outside the area of interest. In a confirmatory PCR with a distal
primer of opposite orientation, mutants were checked for the absence of dele-
tions at the Tn insertion site.
VOL. 74, 2000 FAST SCREENING PROCEDURE FOR HCMV GLYCOPROTEIN MUTANTS 7723

events located in the ORFs coding for known virion glycopro-
teins. DNA pools of each plate were tested by a PCR using
three oligonucleotide primers as outlined in Fig. 3B. Since the
AD169 genome has been completely sequenced (10), search
primers can be designed to bind selectively to any position of
interest, whereas the Tn-specific primers M13-for and M13-rev
hybridize to sites near the inverted repeat structures of the
transposed element (26). A PCR product should be generated
wherever a Tn has inserted near the search primer position,
irrespective of the Tn orientation (Fig. 3B). The observed
fragment sizes of the PCR products obtained from the first
round of search should permit a choice among suitable candi-
dates. Figure 4A and B show two representative experiments
for a first round of PCR search scanning DNA plate pools. In
Fig. 4A 13 plates were tested with a primer specific for gB
(UL55-rev; listed in Table 1). The detection of numerous PCR
products indicated Tn insertion events at various positions
close to the search primer position, suggesting that the library
comprises a large number of insertions into gene UL55 (gB)
(Fig. 4A). However, when this library of about 1,000 mutants
was probed with a gM-specific primer (UL100-for), candidate
mutants were detected much less frequently (Fig. 4B). This
difference suggested that insertions are not equally distributed
over the entire viral genome.
When the plate pool tested positive for the gene of interest,
two more rounds of PCR analysis were necessary to identify
the bacterial clone carrying the respective BAC viral mutant
(Fig. 3A). In plate 13 (Fig. 4B, lane 13) the size of the amplified
PCR product predicted a Tn insertion in the gene encoding the
C-terminal part of gM. In a second round of PCR screening,
the mutant was traced by examining the DNA pools represent-
ing all mutants stored in rows A to H of plate 13 (Fig. 4C).
After the mutant was localized to row D, individual wells of
that row were subjected to a third round of PCR analysis in
order to identify the well containing the clone of interest (Fig.
4D, lanes 1 to 12). According to this protocol only the mutant
clones that carried an insertion close to the position of interest
were analyzed, while all other clones were left uncharacterized.
This method of mutant isolation is fast and involves only three
steps of PCR screening of hierarchically pooled samples of
DNA and bacterial clones.
Characterization of the Tn mutants. A first estimate of the
distance between the Tn insertion site and the binding site of
the search primer can be achieved by determining the size of
the observed PCR product. In rare instances, Tn mutagenesis
can result in adventitious deletions at the Tn insertion site (9),
which probably occur via an illegitimate resolution of nearby
double Tn insertions or by intramolecular transposition (4). To
exclude such deletion mutants, a confirmatory PCR with an
oligonucleotide primer (e.g., UL100-rev) located about 2 kbp
downstream from the search primer and distal from the Tn
integration site was performed (Fig. 4D, lane 14). When the
resulting PCR fragment matched the expected size, the mutant
genome was further analyzed by restriction enzyme digestions
with several enzymes. Figure 5 shows examples of how Tn
FIG. 4. Localization of candidate mutants to individual wells of the Tn li-
brary. (A) First-round PCR search for mutants within the gene coding for gB
using the UL55-rev primer. Each lane on the gel shows PCR products generated
on secondary DNA pools representing all Tn mutants in one plate. A size marker
(1-kbp ladder) is shown between lanes 4 and 5. (B) The PCR search was
performed on the same DNA samples as in panel A but a primer specific for the
gM gene (UL100-for) was used. (C) Pooled DNA samples representing the
mutant clones stored in lanes A to H of plate 13 were subjected to PCR analysis
with primers UL100-for, M13-for, and M13-rev. (D) All 12 individual wells
(lanes 1 to 12) of row D identified in panel C were subjected to a third round of
PCR screening. The desired mutant viral genome was detected in position D-11
on plate 13 of the Tn library. Pooled DNA from row D was included as a positive
control (lane 13). Lane 14, PCR performed with the same DNA sample as in lane
13 using the distal primer UL100-rev with M13-for and M13-rev. The detection
of the expected 1.2-kbp band was indicative of the absence of irregular deletion
events.
FIG. 5. Tn insertion mutants show random insertions within chosen genes.
Lane 1, HindIII digest of the AD169-BAC DNA isolated from bacteria; UL100
(gM) is located on the 6.6-kbp HindIII R fragment (black arrow, white arrow-
head); lanes 2 to 4, BAC mutants with insertions into UL100 digested with
HindIII; the exact nucleotide positions of the Tn insertion are indicated above
the lanes; lane 5, NotI digest of parental AD169-BAC; genes UL74 (gO) and
UL75 (gH) are located on a 9.0-kbp NotI fragment (black arrow, white arrow-
head); lanes 6 to 13, NotI digest of AD169-BAC-derived genomic HCMV mu-
tants carrying Tn insertions in the genes coding for gH and gO. DNA fragments
were separated on a 0.6% agarose gel, and molecular size markers are given in
kilobase pairs on either side. Grey arrows, Tn-specific 1.8-kbp fragments; dots,
subfragments resulting from cleavage of restriction endonuclease fragments.
7724 HOBOM ET AL. J. VIROL.

insertions result in new restriction enzyme cleavage products.
The gene encoding the integral membrane protein gM is lo-
cated in the 6.6-kbp HindIII R fragment of HCMV (32) (Fig.
5, lane 1). In the Tn mutants the 6.6-kbp HindIII R fragment
is cleaved into two subfragments of corresponding sizes (Fig. 5,
lanes 2 to 4) and an additional Tn-specific 1.8-kbp fragment
appears due to a HindIII site located within each of the in-
verted repeats of the Tn (26). In lanes 6 to 13 Tn insertions
into gene UL75 (gH) and the immediately adjacent gene UL74
(gO) are shown. These genes are located in a 9.0-kbp NotI
fragment of AD169-BAC (Fig. 5, lane 5). This fragment is
cleaved into two subfragments of corresponding sizes (Fig. 5,
lanes 6 to 13). A Tn-specific 1.8-kbp fragment results from two
NotI sites located near the Tn ends and is present in all mu-
tated HCMV BACs but not in the parental clone, AD169-BAC
(Fig. 5, lane 5). Finally, the exact nucleotide position of the Tn
insertion was determined by direct sequencing of the BAC
using the M13 primers.
Our library was large enough to isolate at least two mutants
of each glycoprotein gene we were interested in. The individual
BAC clones characterized are summarized in Fig. 6. For the gB
gene the insertions were located at nt 83389, 83171, 83108*,
83012, 82743, 82305*, 82166, and 81329*; disruptions of the gH
gene were found at nt 110107, 109543*, 108347, 108090*,
108018, and 107933; gL gene insertions were found at nt
164159*, 163840, and 163712; insertions into the gM gene were
detected at nt 146185*, 146082, 145422, and 145420; gp48 gene
insertions were found at nt 13470*, 13614, and 13798; those
disrupting the gO gene mapped to nt 107507* and 106317. For
mutants corresponding to nucleotides denoted with an aster-
isk, a revertant genome was also created by allelic replacement
in E. coli.
Identification of essential glycoprotein genes by reconstitu-
tion of mutant viruses. We tested whether insertions in genes
coding for envelope glycoproteins still allow virus replication in
cultured fibroblasts. For this purpose, each of the mutant ge-
nomes depicted in Fig. 6 was transfected into MRC-5 cells.
Mutant genomes that did not give rise to plaques were com-
plemented by cotransfection with plasmids carrying 4 to 5 kb of
viral DNA overlapping the Tn insertion site (pUH-25 to pUH-
34). In all cases, complementation in vitro reproducibly res-
cued plaque formation, proving that the Tn insertion was the
reason for the failure to replicate. In addition, a number of
revertant genomes were constructed by allelic exchange in E.
coli (13470-Rev, 81329-Rev, 82305-Rev, 83108-Rev, 107507-
Rev, 108090-Rev, 109543-Rev, 146185-Rev, and 164159-Rev;
numbers indicate the nucleotide position of the Tn insertion).
Both types of revertants (obtained either by recombination in
eukaryotic cells or by allelic exchange in bacteria) showed wt
growth properties along with wt restriction patterns (data not
shown). This suggested that mutant genomes with Tn inser-
tions within the genes encoding gB, gH, gM, and gL are not
viable. As reported previously (42, 52) the UL4 gene product,
gp48, is a nonessential component of the viral envelope. Our
data confirm these findings. Mutated BACs with a Tn inte-
grated into gene UL4 gave rise to a mutant viral progeny
without significantly impaired replication kinetics, irrespective
of the multiplicity of infection (MOI) applied (data not
shown). The results of the transfection experiments are sum-
marized in Fig. 6.
FIG. 6. Schematic representation of Tn insertions into the HCMV virion glycoprotein genes. Mutants were characterized by appropriate restriction enzyme digests
and direct sequencing on BACs prior to transfection. ., insertion of a Tn sequence resulting in the disruption of that gene. For gB many more candidates are present
in the library, but these have not been confirmed by sequencing (grey symbols). Transfection results for insertion mutants are summarized at the right. When upon
repeated transfection no viral progeny could be obtained (2), plaque formation could reproducibly be rescued by cotransfection of subgenomic fragments of HCMV
DNA (4 to 5 kbp) spanning the insertion site. p, mutant with revertant genome constructed by allelic exchange in bacteria and found to be infectious.
VOL. 74, 2000 FAST SCREENING PROCEDURE FOR HCMV GLYCOPROTEIN MUTANTS 7725

Remarkably, the mutant BACs with disruptions of gene
UL74 (gO) also yielded viable virus, suggesting that the
HCMV gO is not essential for the infectious cycle of HCMV.
Still, all genomes with an insertion into UL74 led to mutant
viruses with an attenuated growth phenotype in cell culture
(Fig. 7C), which remained conserved over several rounds of
replication. The gO-defective viruses exhibited a severely re-
duced plaque size, which was observed irrespective of whether
the Tn insertion was at the beginning (18G3: insertion at nt
107507), corresponding to an alteration in the coding sequence
at amino acid position 8 leading to a premature stop 11 amino
acids later, or at the end (10H5: insertion at nt 106317), cor-
responding to a truncation of the protein at amino acid 404, of
the UL74 gene (nt 107525 to 106128) (Fig. 7A). These data do
not yet allow the conclusion that a mutated protein with a stop
at this position is associated with a loss of gO function. An
inserted Tn sequence can also destabilize the mRNA transcript
and thus result in a functional gene knockout (56). If a trun-
cated gene product should be expressed, a set of insertion
mutants would help to identify functionally important do-
mains. Figure 7B shows the DNA extracted from the mutant
viruses with a Tn stably integrated into the gene encoding gO
compared to that from a virus reconstituted from the parental
AD169-BAC, which is indistinguishable from revertant virus
Rev18G3-RV, obtained by allelic replacement. Repair of the
mutated UL74 gene with an intact sequence restored wt
growth properties (Fig. 7C).
DISCUSSION
We report on the generation of a library of Tn insertion
mutants of the reconstituted full-length AD169 genome cloned
as an infectious BAC in E. coli. Pooled DNA samples of this
library were used to screen for single Tn insertions in various
genes of interest. As several individual candidates can be han-
dled in parallel, a whole set of mutants can be identified for any
position of interest in less than a week’s time. For the six genes
encoding glycoproteins reported here, a library of approxi-
mately 2,000 clones proved large enough to contain at least two
independent Tn insertions per gene studied. Transfection of
mutant genomes gave rise to infectious viral progeny only for
insertions into UL4 and UL74. Insertions within the genes
coding for gB, gH, gL, and, interestingly, gM were not com-
patible with virus replication in fibroblasts.
Our studies show that any genomic region can be rapidly
screened for insertion events by a sequential three-step PCR
analysis of the Tn insertion library. BACs usually are present
only as a single copy per bacterial cell (46). Exposure of the
BAC-containing bacteria to the Tn donor plasmid for a re-
stricted period of time for mutagenic conditions led to rare
transposition events resulting in mainly single Tn insertions
(9). The known preference of this minimal Tn1721-derived Tn
for supercoiled plasmids versus bacterial genomes (4) was also
FIG. 7. Mutant viruses defective in gene UL74 (gO) exhibit a reduction in
growth kinetics. (A) Schematic representation of the Tn insertions. kan, ORF
encoding the kanamycin resistance marker; upside down lettering indicates op-
posite orientation. Boldface numbers, exact nucleotide positions of the interrup-
tion of the ORF; lightface numbers, expected fragment sizes in kilobase pairs.
(B) Viral DNA isolated from cells infected with AD169-RV (lanes 1, 5, and 9)
compared to that from the UL74-deficient mutant viruses reconstituted from
clones 10H5 (lanes 2, 6, and 10) and 18G3 (lanes 3, 7, and 11), together with that
from a revertant genome generated by allelic exchange in bacteria and recon-
stituted to yield Rev18G3-RV (lanes 4, 8, and 12). Viral DNA was digested with
NdeI (lanes 1 to 4). The UL74 gene is located on a 3.8-kbp NdeI fragment (lane
1), which, due to the Tn insertion in variants 10H5 and 18G3, shifts to a 5.6-kbp
fragment (lanes 2 and 3, dots). In lanes 5 to 8 (BglII digests) and lanes 9 to 12
(EcoRI digests) the 18G3 revertant has a pattern indistinguishable from that of
AD169-RV, whereas both gO-deficient viruses show the expected altered frag-
ment sizes depicted in panel A (dots). (C) Single-step growth curve of gO
mutants compared to revertant virus Rev18G3-RV. Confluent monolayers of
MRC-5 cells were infected at an MOI of 0.01. Virus was harvested from both
media and infected cells at the indicated time points and titered by plaque assay.
Values shown are the results of duplicate assays from duplicate infections.
7726 HOBOM ET AL. J. VIROL.

valid for BACs. We found a Tn insertion into the MCMV (9)
and HCMV BAC plasmids in about 90% of the clones ana-
lyzed. Thus, for the generation of a library with single Tn
insertions, the bacteria with insertions into the bacterial chro-
mosome did not require practical consideration. However,
during the setup of the experimental system the choice of the
antibiotic resistance gene was not without impact on Tn mu-
tagenesis. A single copy of a tetracycline resistance gene ap-
parently confers only a weak resistance against the antibiotic.
Bacteria with two or more Tn insertions in AD169-BAC were
predominantly selected when TnMax13, encoding a tetracy-
cline resistance gene, was used together with a tetracycline
concentration of 10 mg/ml (data not shown). A switch to the
aphA3 gene conferring resistance to kanamycin reproducibly
resulted in single Tn insertions. However, by reducing the
tetracycline concentration single Tn insertions should also be
obtained.
Single insertions of the Tn selectively into the viral BAC are
important for establishing useful libraries for fast screening.
Some Tn, such as the Tn5 or Tn10 derivatives, have a consid-
erable propensity to integrate into the bacterial genome thus
requiring discrimination between insertion events into the viral
BAC and into the E. coli genome. Such a separation has been
achieved either by DNA extraction and retransformation of
BACs (47) or, alternatively, via bacterial conjugation using an
oriT element inserted into the BAC cassette. However, for
efficient transfer into E. coli the library had to be amplified,
which increases the risk of a repeated selection of identical
clones that probably did not arise from independent insertion
events (47).
The Tn mutagenesis which we have carried out previously
for the MCMV BAC and here for AD169-BAC is rapid and
direct, but is it also random? Whereas for Tn7 a strict sequence
specificity has been reported (4), Tn1721, a member of the Tn3
family, and Tn5 display an almost random distribution of in-
tegration sites (4). Tn insertions were observed at random
locations throughout the AD169-BAC genome. With about
250 Tn mutants of MCMV and HCMV analyzed so far, we
never found a repetition of an identical Tn insertion. Instead,
we have detected independent clonal insertion events 20 nt or
less from each other within the same gene. Thus, random
insertion mutagenesis could perhaps be used to generate a
series of gene truncation mutants to map functional domains
of a target protein. However, for unknown reasons the inser-
tions are not entirely equally distributed over the genome, as
indicated by an apparent accumulation of insertions in the
UL55 region. Nevertheless, our library of about 2,000 individ-
ual mutant clones has so far proved large enough to identify at
least two genomic HCMV mutants per gene studied.
A targeted deletion of a complete ORF in the context of the
viral genome is considered a potent means to functionally
analyze an HCMV gene. This approach, however, may also
affect regulatory sequences controlling the function of other
genes, with the consequence that the phenotype of a mutant
cannot be attributed with certainty to the deleted ORF. This
problem is only partially addressed by the construction of a
revertant and is not excluded by studying the directly neigh-
boring genes. In principle, each individual Tn insertion mutant,
as the product of a random sequence disruption, suffers from
the same ambiguity. Therefore, a consistent phenotype of sev-
eral insertion mutants from different positions within one ORF
probably defines the property of the targeted gene with a
higher degree of confidence. A variability of phenotypes would
either indicate effects on other genes or suggest the presence of
functionally independent domains in the targeted gene. By
studying the properties of several independent Tn mutants
with insertions dispersed all over the area of interest, a rapid
and reliable assignment of functions may be achieved even for
overlapping ORFs and spliced genes. Therefore, we conclude
that, collectively, each set of gene mutants, although not nec-
essarily an individual mutant, describes the properties of the
targeted gene.
Products of six ORFs have been described as constituents of
the HCMV virion envelope, most of them being organized in
three distinct glycoprotein complexes numbered I to III (8, 18,
23). UL33 codes for a G protein-coupled receptor homolog
that has also been detected in the envelopes of purified virus
particles (37) but that is much less abundant than the glyco-
proteins mentioned above and thus may result from random
incorporation into budding particles. It had to be presumed
that, by analogy to HSV-1 (43) and all other herpesviruses
studied, deletions within UL55 (gB) and UL75 (gH) would not
be viable (8). For HCMV, the essential viral functions of at-
tachment and fusion have been assigned to gB and gH, respec-
tively (11). Proof for this assumption can only be derived from
defined genetic constructs and is now provided by this study.
This type of construction was not possible through virus mu-
tant construction by recombination in eukaryotic cells due to
the lack of complementing helper cells. Work on essential virus
genes requires two components, the defined viral gene mutants
and cell lines expressing the gene under study for complemen-
tation. Our defined sets of genomic mutants will be a useful
tool to evaluate transfected cell lines with regard to their
helper function. This should provide a basis for the subsequent
functional analysis of gene mutants.
The results obtained in this study provide at least two new
pieces of information. First, HCMV gM (UL100) was shown to
be essential for growth in fibroblasts. The function of the
integral membrane protein gM is largely unknown. This ex-
tremely hydrophobic protein is associated with at least one
antigenically distinct component in a multimeric complex (28).
Apart from a diffuse heparin binding potential (27) no molec-
ular functions have been proposed for glycoprotein complex II
(gCII). The requirement for HCMV gM contrasts with findings
for the alphaherpesviruses, where, despite its high conserva-
tion, the gM gene homolog UL10 in HSV-1 has been deleted
without severe consequences (2). Deletion of the gM gene
homolog UL10 in PrV also resulted in attenuated but viable
viral progeny (15). Data from HSV-1 and PrV suggest an
involvement in penetration of their respective gM homologs
(15).
Second, the HCMV gO function is not required for propa-
gation of the virus in fibroblasts. Along with two other protein
components, gH and gL, gO forms a trimeric, covalently linked
complex called gCIII (24, 33). Alphaherpesviruses do not con-
tain a gene homologous to the gO gene. Moreover, in Epstein-
Barr virus, which so far contains the only other example of a
heterotrimeric gH-related complex, the BZLF2 gene product,
a gO-related protein, can be omitted for infection in a cell-
type-specific manner (34). Likewise, for HCMV, which in vivo
infects multiple cell types, gO might act as a coreceptor binding
partner cooperating with the fusion-competent gH. It will be of
interest to see, whether gO is essential for the infection of
some target tissues by HCMV. The other two components of
the gCIII complex, gH and gL, are necessary for the formation
of infectious viruses. This is corroborated by similar findings
throughout the herpesvirus family (31, 43). HCMV gH (UL75)
has been proposed to be involved in attachment to a ubiqui-
tous cell surface receptor (8), whereas gL is essential for the
transfer of the essential component gH from the endoplasmic
reticulum to the cell surface (25, 30, 50). Careful analysis of the
molar ratios of the envelope components of HCMV suggested
VOL. 74, 2000 FAST SCREENING PROCEDURE FOR HCMV GLYCOPROTEIN MUTANTS 7727

that, in addition to a heterotrimeric disulfide-linked form of
gH (gCIII), additional noncomplexed gH molecules are pres-
ent on the surface of the virion (33).
The system described in this paper permits the rapid and
targeted identification of entire sets of insertion mutants in any
HCMV gene of interest. The number of mutants per gene
depends on the size of the library and can be adjusted to suit
one’s need. Assessment of the functional importance of virion
glycoproteins is just one application to demonstrate the poten-
tial use of a library of random Tn insertion mutants for a
detailed genetic analysis of HCMV or any other BAC-cloned
virus genome. By three consecutive steps of PCR screening on
hierarchically pooled samples of the random Tn library, several
independent mutant clones can be identified in less than a
week and insertion mutants can be characterized prior to
transfection. Sets of mutants with insertions at different loca-
tions in the gene help to faithfully attribute functional pheno-
types to a gene product. This method might be particularly
attractive for the analysis of viral genes that give rise to splice
variants and that are composed of functionally distinguishable
subdomains.
ACKNOWLEDGMENTS
We thank G. Po´sfai, M. Wagner, and C. Me´nard for providing
plasmids. We are indebted to A. Hegele and A. Colomar for expert
technical assistance.
This work was supported by grants from the Deutsche Forschungs-
gemeinschaft (SFB 455 and BR 1730/1-1) and from the Bundesminis-
terium fu¨r Bildung und Forschung.
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