INTRODUCTION

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Human cytomegalovirus (HCMV) is a widespread herpesvirus, which infects 50 to 100% of the human population. HCMV disease is a significant medical problem, although it is mostly restricted to patients with immature or compromised immune systems (5). HCMV gene expression during productive infection of cultured human fibroblast cells follows an ordered cascade of expression of immediate-early (IE or ) genes, followed by expression of delayed-early (DE or ) genes, followed after viral DNA replication by strong expression of late (L or ) genes. HCMV also modulates the expression of many cellular genes during the virus life cycle (47).

IE1 p72 (IE1_491aa) is the most abundant product of the strongly transcribed major IE locus of HCMV and is detected in the nuclei of infected cells both in culture and in infected individuals. RNA transcripts originating from the major IE enhancer-promoter (MIEP) immediately after infection span five major exons and are alternately spliced and polyadenylated to produce messages for either IE1 p72 (exons 1 to 4) or IE2 p86 (IE2_579aa) (exons 1 to 3 and 5) (74, 75). The large exon unique to the IE2 p86 message, originally termed exon 7 (75), is here termed exon 5 (47). Translation of IE1 p72 and IE2 p86 initiates in exon 2, and the proteins share 85 identical residues at their amino termini.

IE2 p86 is thought to be the major specific transcriptional regulator of the lytic cycle of HCMV. Consistent with a such a role, IE2 p86 is a sequence-specific DNA binding protein, which autoregulates by binding adjacent to the transcription start site of the MIEP (12, 32, 40, 41, 44, 53, 83) and also binds to specific sites in other HCMV promoters (3, 66, 67). IE2 p86 interacts with diverse components of the cellular transcription machinery, including TBP, TFIIB, CREB, CBP, and c-Jun (7, 23, 33, 39, 65, 67), and in transient-cotransfection assays IE2 activates transcription from a wide range of HCMV and cellular promoters (15, 28, 35, 45, 54, 70, 73). Transactivation by IE2 p86 may be mediated by upstream promoter elements (39, 65), but promiscuous activation of heterologous promoters is frequently TATA box mediated (23).

In contrast, IE1 p72 acts via discrete promoter elements to stimulate a relatively limited number of viral and cellular promoters. Notably, IE1 p72 transactivates its own promoter, the HCMV MIEP (13, 45, 73), acting via NF-B sites in the 18-bp repeat sequences of the enhancer (13, 62). IE1 p72 also activates the human immunodeficiency virus type 1 long terminal repeat and the cellular DNA polymerase , dihydrofolate reductase, and prointerleukin-1 gene promoters (26, 29, 80, 82). IE1 p72 physically interacts with the cellular transcription factors SP-1, E2F-1, and CTF-1 (26, 43, 46), but binding of IE1 to the basal transcription factors TFIIB and TFIID has not been detected (7, 23). As distinct from its promoter-specific transactivator functions, coexpression of IE1 p72 augments the promiscuous transactivating activities of IE2 p86 on a wide variety of viral and cellular promoters (9, 35, 43, 45, 73). However, the accessory viral transactivators TRS1/IRS1, UL36-UL38, and UL112-UL113 are in addition required for the maximal stimulation of a number of HCMV DE promoters (30, 71).

Several other distinct activities have been described for IE1 p72, but considered together, these do not form a clear picture of the probable functions of IE1 in the context of the virus life cycle. IE1 p72 colocalizes in stable cell lines and in infected cells with cellular chromatin, a property shared with the EBNA-1 latent antigen of Epstein-Barr virus (38). IE1 p72 also interacts with the pocket protein p107 (56), inhibits tumor necrosis factor alpha-driven apoptosis in HeLa cells (85), and causes the relocalization of PML protein from nuclear ND10 subdomains (1, 34, 37). Together with IE2 p86, IE1 p72 is one of six auxiliary factors required in trans, alongside core viral DNA replication proteins, for the replication in transfected cells of a plasmid containing the HCMV lytic origin of replication (51). Although this may partly reflect a requirement for IE1 p72 to fully activate core replication gene promoters (30), IE1 p72 was nevertheless required for efficient viral origin replication in permissive human fibroblasts, even when the core replication proteins were constitutively expressed (63).

IE1 p72 and IE2 p86 are widely thought to be the central regulators of the cascade of viral and cellular gene expression during lytic HCMV infection. There is, however, very little direct evidence for this assertion, other than that obtained by the use of antisense phosphorothioate oligonucleotides and antisense RNA expression to inhibit ie1 and ie2 expression and hence viral replication (2, 4, 6). For other herpesviruses, most notably herpes simplex virus type 1 (HSV-1), spontaneous and recombinant viral mutants with defects in gene-regulatory proteins have provided vital experimental information, facilitating a better understanding of the important functions of these proteins in the context of viral infection (59). As HCMV mutants with mutations in ie1 and ie2 had not been described, we set out to generate such mutants through construction of recombinant viruses. We have previously reported the isolation and preliminary characterization of an HCMV ie1 mutant (RC303Acc) by reconstruction from cosmids derived from the Towne and Toledo strains (48). Here, we report the isolation and characterization of an ie1 mutant HCMV in a pure Towne strain background (CR208), derived by recombination between wild-type virus and plasmid DNA containing the selectable marker gene gpt. The two ie1 mutant viruses share a low-multiplicity growth defect in primary fibroblasts; however, viral gene expression during an attenuated low-multiplicity infection by CR208 was more extensive than was previously observed for RC303Acc, indicating that IE1 p72 function may have a strong impact at multiple stages of the virus life cycle.

	MATERIALS AND METHODS

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Plasmids. Plasmid DNAs were maintained and propagated in Escherichia coli DH5, except for gpt-containing plasmids, which were maintained and propagated as previously described in the E. coli gpt mutant strain WB-1 (22). Plasmid pON303G (13) contained the 7.4-kbp SalI IE fragment of HCMV Towne strain (AD169 equivalent nucleotides [nt] 168820 to 176220) (10) ligated into the SalI site of pGEM2 (Promega). pON303G was deleted between two NaeI sites to construct pON2535, which contained a 5.7-kbp viral NaeI-SalI fragment (AD169 equivalent nt 170512 to 176620). Exon 4 was deleted from pON303G by using endonuclease Bst1107I, which cuts at AccI sites in the flanking introns, to produce plasmid pON2551. A 2.6-kbp XhoI-ScaI fragment of pON303G (AD169 equivalent nt 170233 to 172863) was ligated between XhoI and EcoRV sites of pUC21 (79) to make plasmid pON2517. A 2.6-kbp SalI-BglII fragment of pON303G (AD169 equivalent nt 168820 to 171443) was cloned as an XbaI-BglII fragment into pBluescript SK+ (Stratagene) to produce plasmid pON2512. Plasmid pON2523 contained a 3-kbp EcoRI-XbaI fragment of pXbaE (78) ligated between EcoRI and XbaI sites of pUC21. Viral sequences in pON2523 commence at the EcoRI site in UL129 (AD169 equivalent nt 175524) and proceed away from the MIEP into Towne sequences not shared with AD169 (8). Plasmid pON308G (13) contained the 3.9-kbp ClaI IE fragment of HCMV (Towne) (AD169 equivalent nt 170811 to 174751) ligated into the AccI site of pGEM2.

View larger version (32K): [in this window] [in a new window]   FIG. 1.   (a) Genomic structures of Towne, RC2540, and CR208 viruses in the SalI fragment containing the IE locus, together with the structure of plasmid pRG208. The positions of ORFs UL121 and UL127 to UL130 are shown, together with those of the MIEP (shaded) and exons 1 to 5 of the major IE gene. The gpt expression cassette consists of the HSV-1 thymidine kinase gene promoter (tk), guanosine phosphoribosyl transferase-coding sequences (gpt) (shaded), and the simian virus 40 early polyadenylation signal (a). , HCMV sequences deleted from pRG208 and CR208. Restriction enzyme sites used in Southern blot analysis and for gpt insertion: A, AccI (Bst1107I); B, BamHI; C, ClaI; E, EcoRI; S, SalI; X, XhoI. Probe fragments are shown next to viral sequences as solid blocks. (b) Southern blot analysis of Towne and CR208 DNAs cut with the restriction enzymes shown and hybridized with an exon 4 probe or an exon 5 probe (see panel a). Molecular sizes of standards are shown to the left. (c) Southern blot analysis of Towne, CR208, and RC2540 DNAs cut with EcoRI and hybridized to probes from gpt, UL130, and UL128 and -129 (see panel a). Molecular sizes of standards are shown to the left.

Cell lines and culture. All cell lines were cultured in Dulbecco modified Eagle medium (GIBCO-BRL) supplemented with penicillin, streptomycin, nonessential amino acids, and 10% (vol/vol) NuSerum I (Collaborative Research). All procedures were carried out with cells cultured at 37°C and with 5% CO₂. The human fetal lung fibroblast (HFL) line GM05387 and the human foreskin fibroblast (HFF) line GM03468A were obtained from the National Institute of General Medical Sciences, Camden, N.J.

Virus culture and generation of recombinant HCMV strains. Cell-free virus stocks were prepared in HFL cells (for Towne, RC2540, CRQ208, and CR249) or ihfie1.3 cells (for CR208). Cells infected with approximately 0.01 PFU/cell were cultured until a cytopathic effect was apparent in 100% of cells and were then refed with fresh culture medium. After 5 days, the culture supernatant was passed through a 0.45-µm-pore-size filter, mixed 1:1 with a 9% (wt/vol) nonfat milk solution (autoclaved), and stored at 70°C.

Assays of viral growth. Virus plaque assays were performed on HFLs, HFFs, or ihfie1.3 cells. Virus samples were serially diluted in serum-free medium and absorbed in duplicate onto 10⁵ cells plated 16 to 24 h previously in 12-well dishes, and the inoculum was replaced after 1 h with culture medium supplemented with 0.16 mg of purified human gamma globulin per ml. After growth for 10 days, cells were fixed in 100% methanol (5 min) and stained with 0.04% (wt/vol) Giemsa stain in 10% methanol (20 min), and darkly staining foci or plaques were counted.

Analysis of viral DNA. Viral DNA was isolated from the cytoplasm of the same infected cells from which culture supernatant stocks were harvested. DNA was purified from proteinase K-digested cytoplasmic extracts (69), either by phenol extraction and isopropanol precipitation or by sodium iodide equilibrium density centrifugation (81).

Analysis of viral antigens and DNA replication. Hybridoma supernatants of mouse monoclonal antibodies p63-27 (55), SMX (55), and BS510 (64), which recognize IE1 p72, IE2 p86, and ppUL44, respectively, were the gifts of Bodo Plachter. Mouse monoclonal antibody CH160 ascites (16), which recognizes an amino-terminal epitope shared by IE1 p72 and IE2 p86, was the gift of Lenore Pereira. Mouse monoclonal antibody 2A6 ascites, specific for the pp65 virion phosphoprotein (57), was kindly provided by Maria Grazia Revello. Human convalescent serum from a patient recovering from primary HCMV infection was kindly provided by Mark Wills. Antibromodeoxyuridine (anti-BrdU) rat monoclonal antibody MAS-250 hybridoma supernatant and Texas red-linked goat anti-mouse immunoglobulin G (IgG) conjugate were purchased from Sera-Labs. Fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibody was purchased from Cappell.

	RESULTS

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Generation of an ie1 mutant virus. To complement a viral ie1 mutant, a permissive cell line expressing IE1 p72 was required. As human fibroblasts immortalized by the E6 and E7 genes of human papillomavirus type 16 support HCMV growth to high titers (14), the immortalized IE1-expressing cell line ihfie1.3 (48) was generated by the sequential transduction of an early-passage HFF culture with retroviral vectors expressing IE1 p72 and the human papillomavirus type 16 E6 and E7 products.

The ie1 mutant was replication deficient after low-multiplicity infection of primary human fibroblasts. Working stocks of the CR208 deletion mutant were grown and titrated in ihfie1.3 cells. Peak titers reached in the culture medium of infected cells were similar to those generally obtained from Towne virus grown in primary HFL cells, approximately 10⁷ PFU/ml. Prior to all subsequent experiments, CR208 and Towne stocks were titrated in parallel on ihfie1.3 cells. Thus, PFUs in comparative experiments were always defined on an ihfie1.3 background. Towne and CR208 plaques formed on ihfie1.3 cells had similar sizes and morphologies.

View larger version (29K): [in this window] [in a new window]   FIG. 2.   (a and b) Multiple-step growth curve analysis of Towne and CR208 viruses in HFL cells (a) and ihfie1.3 cells (b). The results are a time course of the total PFU of infectious virus present in 4 ml of culture supernatant at the indicated sampling times, as measured by plaque formation on ihfie1.3 cells. Experiments were performed with 2 × 105 cells at an input multiplicity of 0.1 PFU/cell. The results of a single experiment are shown; titers are the mean results from two parallel titrations. (c) Plot of titration data taken from a single plaque assay (Table 1), relating the plaque numbers formed on 2 × 105 HFFs or ihfie1.3 cells by various inocula of Towne or CR208 virus stocks. Log plaque number is plotted against log inoculum volume.

Growth defects of CR208 were repaired by the restoration of exon 4 sequences: the large intron of the major IE gene and ORFs UL124 and UL125 are dispensable for lytic viral growth. To produce rescued derivatives of CR208, HFLs were transfected with linearized DNAs containing exon 4 and sequences flanking the exon 4 deletion in CR208. pON303G contained a wild-type IE locus. pRG249 lacked the large first intron of the major IE gene but was otherwise identical to pON303G. pGEM3ZF was included as a negative control. Transfected HFLs were then infected at a high multiplicity with CR208 virus. Progeny viruses from these recombinations were then used to infect primary HFL fibroblasts at a low multiplicity. Only stocks derived from the pON303G and pRG249 transfections gave rise to rapidly expanding foci of viral infection in this noncomplementing background. Viruses derived from these rapidly spreading infections were plaque purified and were termed CRQ208 and CR249, respectively. Figure 3a shows the expected genomic structures of CR208, CRQ208, and CR249. Figure 3b shows a Southern blot of XhoI digests of DNAs from these three viruses and the HCMV Towne strain. An exon 4 probe failed to recognize CR208 genomic DNA but detected fragments in Towne (5 kbp), CRQ208 (5 kbp), and CR249 (4.1 kbp). The same blot was hybridized to a probe containing 600 bp of the first intron of the IE gene but also containing 130 bp of exon 1. Fragments were then detected in the genomes of Towne (5 kbp), CR208 (3.6 kbp), and CRQ208 (5 kbp). The 4.1-kbp XhoI fragment of CR249 was recognized only very weakly, due to the deletion of 80% of the sequences homologous to the probe. Thus, the blot confirmed the expected structures of the rescued viruses shown in Fig. 3a. The failure to derive fully growth-competent viruses from the control pGEM3ZF+ recombination showed that the starting CR208 stocks were not contaminated to any detectable level by wild-type virus. The parallel demonstration of a coupled rescue and deletion in the isolation of CR249 provided additional evidence that CRQ208 was a bona fide rescued derivative of CR208.

View larger version (32K): [in this window] [in a new window]   FIG. 3.   (a) Genomic structures of viruses CR208, CRQ208, and CR249 in the SalI fragment containing the IE locus. The positions of ORFs UL121 and UL127 to UL130 are shown, together with those of the MIEP (shaded) and exons 1 to 5 of the major IE gene. , HCMV sequences deleted from CR208 and CR249. X, XhoI sites used in Southern blot analysis. Probe fragments used are shown next to viral sequences as solid blocks. (b) Southern blot analysis of CR208, CRQ208, and CR249 DNAs cut with XhoI and hybridized sequentially to an exon 4 probe and to an exon 1 plus intron 1 probe (see panel a). CR249 DNA was inadvertently underloaded in this experiment. Molecular sizes of standards are shown to the left.

View larger version (24K): [in this window] [in a new window]   FIG. 4.   Multiple-step growth curve analysis of Towne, CR208, CRQ208, and CR249 viruses in HFL cells (a) and ihfie1.3 cells (b). The results are a time course of the total PFU of infectious virus present in 4 ml of culture supernatant at the indicated sampling times, as measured by plaque formation on ihfie1.3 cells. Experiments were performed with 2 × 105 cells at an input multiplicity of 0.1 PFU/cell. The results of a single experiment are shown; titers are the mean results from two parallel titrations.

Full expression of viral antigens by CR208 after high-multiplicity infection of primary fibroblasts. An analysis of viral proteins produced during productive high-multiplicity infections of HFLs was undertaken. Figure 5a shows a Western blot comparing proteins from HFLs infected at 5 PFU/cell with Towne, CR208, CRQ208, and CR249 viruses. IE1 p72 and IE2 p86 were detected in parallel by using antibody CH160, which recognizes a shared epitope encoded in exon 2. CR208 clearly failed to express IE1 p72 at 24 and 72 h postinfection (hpi) but produced IE2 p86 at normal levels. The rescued CRQ208 and CR249 viruses showed patterns of IE antigen expression indistinguishable from that of the wild-type Towne virus. Figure 5b and c show Western blots of total viral antigens expressed during high-multiplicity infections (5 PFU/cell) of HFLs by Towne and CR208 viruses, respectively. Viral antigens were detected by using a human convalescent antiserum. CR208 produced a profile of viral antigens almost identical to that produced by wild-type virus. The accumulation of viral antigens was, however, slower in CR208-infected cells, demonstrating that although CR208 completed the lytic cycle after a high-multiplicity input, the kinetics of total viral protein production were delayed relative to those for wild-type virus. Furthermore, the cytopathic effect observed in cells infected at high multiplicity by CR208 was distinguishable from that in cells infected by Towne, with clearly reduced cell rounding.

View larger version (75K): [in this window] [in a new window]   FIG. 5.   (a) Western blot analysis of total cellular proteins, detecting IE1 and IE2 proteins produced in HFLs after 24 or 72 h of infection at 5 PFU/cell by Towne, CR208, CRQ208, or CR249 virus. Monoclonal antibody CH160, diluted 1:100, was used. uninf, uninfected-cell proteins. Molecular masses of standards are shown on the left. (b and c) Western blot analysis of time courses of viral proteins produced by Towne (b) and CR208 (c) after high-multiplicity infection (5 PFU/cell) of HFL cells. Viral antigens were detected specifically in total cellular proteins by using human antiserum, diluted 1:50, from a patient convalescing from primary HCMV infection. Molecular masses of standards are shown to the left of each panel.

View larger version (115K): [in this window] [in a new window]   FIG. 6.   Photomicrographs of immunofluorescence analysis of viral antigens expressed after high-multiplicity infection (5 PFU/cell) of HFFs by Towne (a and c) or CR208 (b and d). (a and b) Staining with IE1-specific antibody p63-27 (undiluted) at 24 hpi. (c and d) Staining with pp65-specific antibody 2A6, diluted 1:100, at 72 hpi. Color positive transparencies were digitized by using a Nikon Coolscan scanner, converted to greyscale using Adobe Photoshop software, and printed as a composite image by using Powerpoint software. Images within a pair were processed identically. The scale bar in panel a applies to all images.

Accumulation of the core viral DNA replication protein ppUL44 was defective after infection of primary fibroblasts at low multiplicity by CR208. To investigate the nature of the block to viral replication after low-multiplicity infection by CR208, HFLs were infected at various multiplicities and viral proteins were analyzed. The 50-kDa protein product of the UL44 ORF was chosen as a representative protein expressed at DE times. ppUL44 (ICP36) is the processivity factor for the viral DNA polymerase (17) and is necessary in assays of trans-acting factors required for viral DNA replication (52). Expression of antisense RNA to UL44 inhibits virus replication in otherwise permissive U373 astrocytoma cells, apparently by preventing viral DNA replication (58). Thus, although ppUL44 is maximally expressed at late times of infection and UL44 has been described as a late gene (20), DE expression of ppUL44 is very likely required for the progression of infection from the DE to the late phase. The ppUL44 protein is abundant during viral infection of cultured cells, and good specific antibodies are available (49, 64).

View larger version (65K): [in this window] [in a new window]   FIG. 7.   Western blot analysis of total cellular proteins, detecting IE1 p72, IE2 p86, and ppUL44 proteins produced in HFL cells over an infection time course. A mixture of monoclonal antibodies CH160 (diluted 1:100) and BS510 (diluted 1:25) was used to detect the three viral antigens simultaneously. Molecular masses of standards are shown to the sides. (a and b) Analysis performed after high-multiplicity infection (5 PFU/cell) by Towne (a) or CR208 (b) virus. The death of many Towne-infected cells at 144 h caused reduced protein loading compared to other lanes. (c and d) Analysis performed after low-multiplicity infection (0.2 PFU/cell) by Towne (c) or CR208 (d) virus.

View larger version (80K): [in this window] [in a new window]   FIG. 8.   Photomicrographs of immunofluorescence analysis of viral antigens expressed after different-multiplicity infections by Towne and CR208 at 48 hpi. (a to p) Immunostained cells; (a' to h') same respective fields visualized by Hoechst staining. (a, c, e, g, i, k, m, and o) Towne infection; (b, d, f, h, j, l, n, and p) CR208 infection. (a to d) HFFs infected at 5 PFU/cell and stained either for IE2 protein with antibody SMX diluted 1:2 (a and b) or for ppUL44 with antibody BS510 diluted 1:25 (c and d). (e to h) HFFs infected at 1 PFU/cell and stained either for IE2 (e and f) or for ppUL44 (g and h). (i to l) HFFs infected at 1 PFU/cell in the presence of PFA (200 µg/ml) and stained either for IE2 (i and j) or for ppUL44 (k and l). (m to p) ihfie1.3 cells stained for ppUL44 and infected at 5 PFU/cell (m and n) or 1 PFU/cell (o and p). Color positive transparencies were digitized as for Fig. 6 and printed as a composite image by using Quark Express software. All IE2-stained images were exposed and processed identically, except for panels i and j, which received a doubled exposure time. All ppUL44-stained images were exposed and processed identically, except for panels k and l, which received a doubled exposure time. The scale bar in panel d applies to all images.

Viral DNA replication compartments were not formed after low-multiplicity infection by CR208. Our analysis of the ppUL44 antigen showed that the defect in the CR208 life cycle after a low-multiplicity input correlated with a deficiency in ppUL44 accumulation at DE times. As ppUL44 is a core DNA replication complex protein, which is very likely required for viral DNA replication, our results predict that viral DNA replication may not occur after a low-multiplicity infection by the ie1 mutant virus. To establish this, and to investigate the fate of individual CR208-infected cells over a longer time course, HFFs were infected at a low multiplicity (0.05 PFU/cell) with Towne or CR208 virus. The cells were then analyzed over a time course by combined staining for IE2 protein and for the incorporation of the thymidine analog BrdU into viral DNA replication structures.

View larger version (58K): [in this window] [in a new window]   FIG. 9.   Time course immunofluorescence experiment, showing the progression of infection in individual cells after the low-multiplicity inoculation (0.05 PFU/cell) of HFFs with Towne (a, c, e, g, i, and k) or CR208 (b, d, f, h, j, and l). (a and b) Fixation at 24 hpi; IE2 staining (antibody SMX diluted 1:2). (c and d) Fixation at 48 hpi; IE2 staining. (e and f) BrdU pulse and fixation at 72 hpi; IE2 staining. (g and h) BrdU pulse and fixation at 144 hpi; IE2 staining. (i and j) Same fields as panels e and f, respectively, with parallel BrdU uptake staining (antibody MAS-250 diluted 1:2). (k and l) Same fields as panels g and h, respectively, with parallel BrdU uptake staining. Color transparency images were processed as for Fig. 8. All IE2-stained images were exposed and processed identically. All BrdU-stained images were exposed and processed identically. The scale bar in panel a applies to all images.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

We have isolated a recombinant HCMV (CR208) from which all unique coding sequences for the abundant nuclear IE1 p72 protein were deleted. IE1 protein was provided in trans in a complementing immortalized fibroblast line, demonstrating the general applicability of these methods to the isolation of HCMV mutants with deletions in essential viral genes.

IE1 functions were not required for lytic replication upon infection of primary fibroblasts at a high multiplicity, although there were indications that the cascade of viral gene expression was slower in the absence of IE1, and there were also alterations in the viral cytopathic effect. Thus, the most abundant viral IE protein was not absolutely essential for virus replication in cultured fibroblasts. However, after infection at low input multiplicities, replication of the ie1 mutant virus exhibited a marked deficiency. The phenotype of CR208 during plating and growth assays suggested that IE1 p72 function was essential when single virions initiated infection. After low-multiplicity infection, the mutant virus life cycle was apparently stalled between the IE and DE phases, with synthesis of the major regulatory protein IE2 p86 but with defective accumulation of the core DNA replication protein ppUL44 and a failure to establish viral DNA replication compartments. The failure to accumulate ppUL44 in the absence of IE1 p72 was independent of viral DNA replication and occurred in the presence of the inhibitor PFA. The faulty accumulation of ppUL44 observed at 24 and 48 hpi may have contributed to the failure of the formation of viral DNA replication compartments in mutant-infected cells. All of the phenotypic differences observed in viral replication, ppUL44 accumulation, and establishment of DNA replication compartments were corrected in two independently derived rescued derivatives of CR208. The defects were also corrected by the provision of the IE1 protein in trans in the complementing ihfie1.3 cell line. Thus, the observed multiplicity-dependent defects in growth and ppUL44 accumulation by the ie1 mutant CR208 were directly related to the inability to code for IE1 p72.

The previously reported ie1 null mutant RC303Acc (48) exhibited growth properties similar to those of CR208 when growth in HFFs was compared to that in ihfie1.3 cells. Because RC303Acc exhibited a striking multiplicity-dependent expression of IE2 p86, the growth defect was attributed to a defect in IE1 p72-mediated autoregulation (48). This virus also exhibited a reduced expression of other viral antigens, but these effects correlated with the reduced expression of IE2 p86. Expression of IE2 p86 by CR208 appears to be less dependent upon multiplicity (Fig. 7 and 8), a characteristic which might be due to the differences in the genetic backgrounds used to construct RC303Acc and CR208. CR208, characterized here, is purely Towne based, whereas RC303Acc is a chimera of the Towne and Toledo strains. The most relevant differences between these two mutants are probably in the region upstream of the ie1-ie2 enhancer, where the Toledo strain carries a 15-kbp segment that includes a major rearrangement of sequences (8). This difference may account for the different impact of the absence of IE1 p72 on levels of ie2 expression. Irrespective of these differences, or of the possible impact of IE1 p72 on autoregulation in certain strains of virus or under certain circumstances, the phenotype of CR208 demonstrates that IE1 p72 function is important even under conditions when IE2 p86 is synthesized and that the impact of IE1 p72 therefore goes well beyond the IE phase of infection.

A consideration of the characterized activities of IE1 p72 suggests several additional mechanisms which alone or in combination could account for the low-multiplicity growth and expression defects of CR208. First, and most consistent with the data presented here, cooperation between IE1 p72 and IE2 p86 to activate HCMV DE and late promoters has been well documented in transient-assay experiments (9, 35, 43, 45, 73). Further analysis showed that the UL44 promoter and those of other core replication genes require accessory functions IRS1/TRS1, UL112-UL113, and UL36-UL38 in addition to IE2 p86 and IE1 p72 to be fully activated (30, 71). The absence of IE1 p72 during infection could thus lead directly to reduced activation of the UL44 promoter, as observed in these transient assays, or may result in reduced levels of the accessory transactivators. Either mechanism could account for the observed defects in ppUL44 accumulation during CR208 infection. Our data cannot exclude the additional possibility that IE1 p72 up-regulates ppUL44 levels by a posttranscriptional mechanism. Regardless of their exact cause, insufficient levels of ppUL44 or any critical replication protein might result in the attenuation of CR208 infection prior to DNA replication.

Another possibility is that IE1, either alone or acting with other viral functions, sustains the host cell through viral infection. IE1 p72 and IE2 p86 have been attributed antiapoptotic effects (85), and IE1 p72 was the stronger of the two. While we did not directly evaluate apoptosis in this study, it is possible that cells infected nonproductively with CR208 are lost through apoptosis. However, a large population of CR208-infected cells containing IE2 but not ppUL44 was observed at both 24 and 48 hpi. The apparent stability of this abortively infected cell population argues that the observed failure in ppUL44 accumulation occurs prior to any apoptotic events.

Finally, expression of ie2 products mediates a shutoff of ie1-ie2 gene expression via crs (12, 41, 53) that may be accentuated in the absence of IE1 p72. Shutoff of the MIEP may thus occur more readily in cells infected with CR208. However, many CR208-infected cells accumulated significant amounts of the major regulatory protein IE2 p86 without accumulating ppUL44, while wild-type-infected cells containing similar amounts of IE2 p86 had also accumulated ppUL44 (Fig. 8). This observation suggests that even if MIEP shutoff is increased in CR208 infections, the absence of IE1 p72 probably impacts directly on downstream gene expression also.

We do not anticipate that altered ppUL44 levels are the sole mediator of the loss of replicative potential in the absence of IE1 p72. As suggested above, levels of accessory viral transactivators may also be reduced during CR208 infection, as may levels of other viral DE products. The levels during CR208 infection of a wider range of viral IE and DE gene products are currently being investigated. We also do not yet know which features of high-multiplicity infection by CR208 bypass the requirement for IE1 function in ppUL44 accumulation and productive infection. These effects could potentially be mediated either by an increased dosage of the viral genomic template, by an increased dosage of virion transactivator proteins, or by some other stimulatory signal delivered to cells upon infection with HCMV.

The growth phenotype of the ie1 mutants in primary fibroblastslytic growth at high multiplicities, a low plating efficiency, small plaques, and a nonlinear relationship between inoculum size and plaque numberis strikingly similar to the growth phenotype of mutants of HSV that are unable to express the IE regulatory protein ICP0 or Vmw110 (61, 77). Other similarities between the two proteins include their synergistic transactivating functions in combination with the major transcriptional transactivators IE2 p86 and ICP4 (18, 45, 50, 73) and the abilities of both proteins to target and modify PML-associated nuclear domain 10 (ND10) bodies (1, 19, 34, 37). It has also been observed that HCMV coinfection can supply functions which complement the low-multiplicity plating defect of an HSV-1 ICP0 mutant (76), although the HCMV gene products responsible have not been identified. It is attractive to speculate that HCMV IE1 and HSV-1 ICP0, although apparently not homologs, may have evolved to perform fundamentally similar functions in these two distantly related herpesviruses.