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Program in Infectious Diseases and Immunity, Program in Comparative Biochemistry, School of Public Health, University of California, Berkeley, California 94720, USA
Reprint requests to: Fenyong Liu, Program in Infectious Diseases and Immunity, School of Public Health, 140 Warren Hall, University of California, Berkeley, CA 94720, USA; e-mail: liu_fy{at}uclink4.berkeley.edu; fax: (510) 643-9955.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Keywords: Gene therapy; ribozyme; RNase P; cytomegalovirus; herpesvirus; gene targeting
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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; Pass 2001). In addition to diseases that are known to be the results of HCMV infection such as AIDS-associated CMV retinitis and birth defect–related metal disorders, there are growing numbers of clinically important complications that are believed to be associated with HCMV, including atherosclerosis (Pass 2001). HCMV is a member of herpesvirus family that includes many medically important viruses such as herpes simplex virus (HSV) 1 and 2 (causative agents of cold sore and genital herpes, respectively), Epstein-Barr virus (found in patients with Burkitt’s lymphoma), and Kaposi’s sarcoma–associated herpesviruses (KSHV; Kieff and Rickinson 2001; Mocarski and Courcelle 2001; Moore and Chang 2001; Roizman and Knipe 2001). Continued development of effective antiviral compounds and approaches is central for the treatment and prevention of infections by HCMV, as well as other herpesviruses.
RNA enzymes are being developed as promising gene-targeting reagents to specifically cleave RNA sequences of choice (Sarver et al. 1990; Yu et al. 1993; Liu and Altman 1995; Lan et al. 1998; Guo et al. 2000). For example, both hammerhead and hairpin ribozymes have been shown to cleave viral mRNA sequences and inhibit viral replication in cells infected with human viruses, whereas a ribozyme derived from a group I intron has been used to repair mutant mRNAs in cells (Sarver et al. 1990; Yu et al. 1993; Lan et al. 1998; Wong-Staal et al. 1998; Rossi 2000). Thus, ribozymes can be used as a tool in both basic and clinical research, such as in studies of developmental processes and in antiviral gene therapy (Wong-Staal et al. 1998; Rossi 1999).
Ribonuclease P (RNase P) is a ribonucleoprotein complex responsible for the maturation of the 5' termini of tRNAs (Frank and Pace 1998; Altman and Kirsebom 1999). In bacteria, the RNase P holoenzyme contains a catalytic RNA subunit (Guerrier-Takada et al. 1983) and a small basic protein subunit (e.g., M1 RNA and C5 protein in Escherichia coli). One of the unique features of RNase P holoenzyme and its catalytic RNA is their ability to recognize the structures rather than the sequences of their substrates, which gives them the ability to hydrolyze different substrates. Thus, M1 ribozyme can cleave an mRNA substrate as long as the target sequence hybridizes with its complementary sequence (designated as external guide sequence or EGS) to form a complex resembling the portion of a tRNA molecule that includes the acceptor stem, the T-stem, the 3' CCA sequence, and the 5' leader sequence (Fig. 1A; Forster and Altman 1990). A sequence-specific ribozyme, M1GS RNA, can be constructed by covalently linking a guide sequence to the 3' terminus of M1 RNA (Fig. 1A; Frank et al. 1994; Liu and Altman 1995). RNase P ribozymes have been constructed and used to inhibit the expression of both cellular genes and genes of HSV-1 and HCMV (Cobaleda and Sanchez-Garcia 2000; Trang et al. 2000a,b). Moreover, a reduction of 1000-fold in HSV-1 growth and a reduction of 150-fold in HCMV replication were observed in cells that expressed ribozymes derived from the wild-type RNase P ribozyme sequence (Trang et al. 2000a,b). Thus, M1GS ribozyme represents a novel class of gene-targeting agents. Targeted cleavage of mRNA by RNase P ribozyme provides a unique approach to inactivate any RNA of known sequence expressed in vivo.
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). AP and PR are two of the most conserved proteins found among all herpesviruses (Liu and Roizman 1991; Welch et al. 1991). Genetic and biochemical studies of HSV-1 AP and PR indicated that these two proteins are responsible for viral capsid assembly and are essential for HSV-1 replication (Preston et al. 1983; Gao et al. 1994; Matusick-Kumar et al. 1994). Therefore, the AP and the PR may serve as targets for novel drug development to combat all herpesviruses, including HCMV (Gibson et al. 1994).
The overlapping region of the AP and PR mRNAs is also an ideal target for M1GS targeting. This is because AP provides a stoichiometric function as the capsid scaffolding protein, whereas the proteolytic activity of PR is essential for capsid maturation (Preston et al. 1983; Gao et al. 1994; Gibson et al. 1994; Matusick-Kumar et al. 1994). Targeted cleavage of the common regions of AP and PR mRNA will achieve a greater antiviral effect, as it will shut down both the viral essential stoichiometric and enzymatic functions simultaneously. We showed that the constructed ribozyme cleaves the target mRNA sequence in vitro. Moreover, intracellular expression of the ribozyme using retroviral expression vectors leads to a significant inhibition of the expression of viral AP and PR. A reduction of 2000-fold in viral growth was observed in the ribozyme-expressing cells. Our study provides the direct evidence that RNase P ribozymes are highly effective in inhibiting HCMV gene expression and growth by targeting the mRNAs for AP and PR. These results also demonstrate the feasibility of developing highly effective RNase P ribozymes as a novel class of antiviral agents for gene therapy of infections caused by human viruses, including HCMV.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). M1GS RNA-mediated cleavage at the overlapping region of the PR and AP mRNAs is expected to block the expression of both mRNAs simultaneously and should lead to a significant reduction of HCMV growth.
Most mRNA species inside cells are associated with proteins and are present in a highly organized and folded conformation. Therefore, it is important to choose a targeted region that is accessible to binding of ribozymes in order to achieve efficient cleavage. By using dimethyl sulfate (DMS), we used an in vivo mapping approach (Ares and Igel 1990; Liu and Altman 1995; Zaug and Cech 1995) to determine the accessibility of the region of the AP mRNA in HCMV-infected cells. A position 58 nucleotides downstream of the AP translational initiation codon (Chee et al. 1990), was chosen as the cleavage site for M1GS RNA. This site appears to be one of the regions most accessible to DMS modification and, presumably, to ribozyme binding. Moreover, its flanking sequence exhibits several features that need to be present in order to interact with an M1GS ribozyme to achieve efficient cleavage. The interactions of these sequence elements with the M1GS ribozyme are critical for recognition and cleavage by the enzyme. These features include the requirement for a pyrimidine and a guanosine to be the nucleotide 3' and 5' adjacent to the site of cleavage, respectively (Liu and Altman 1996).
Ribozyme M1-AP was constructed by covalently linking the 3' terminus of M1 RNA with a guide sequence of 18 nucleotides that is complementary to the targeted AP mRNA sequence. The control ribozyme, C-AP, was also constructed in a similar way and included in the study. C-AP was derived from C102 RNA, a M1 mutant that contained several point mutations at the catalytic P4 domain and was at least 104-fold less active than was M1 RNA in cleaving a pre-tRNA (Kim et al. 1997). The DNA sequences coding for the M1GS ribozymes were generated by PCR using the DNA sequences for M1 RNA and C102 RNA as the templates and primers that contained the sequences complementary to the targeted region of AP mRNA. These DNA sequences were under the control of the promoter for T7 RNA polymerase, and M1GS RNAs were synthesized in vitro from these DNA sequences by T7 RNA polymerase. A substrate, ap40, which contained the targeted AP mRNA sequence of 40 nucleotides, was used (Fig. 1B
). In the absence of M1 RNAs (Fig. 2, lane 4), no cleavage of AP mRNA sequence was detected. Efficient cleavage of the substrate by M1-AP was observed (Fig. 2, lane 1). In contrast, cleavage by C-AP was barely detected (Fig. 2, lane 2).
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Efficient expression of the ribozymes in human cells
The DNA sequences coding for M1-AP and C-AP were cloned into retroviral vector LXSN and placed under the control of the small nuclear U6 RNA promoter, which has previously been shown to express M1GS RNA and other RNAs steadily (Das et al. 1988; Miller and Rosman 1989; Yuan et al. 1992; Liu and Altman 1995; Bertrand et al. 1997). This promoter is transcribed by RNA polymerase III, and its transcripts are highly expressed and primarily localized in the nucleus (Das et al. 1988; Yuan et al. 1992; Liu and Altman 1995; Bertrand et al. 1997). To construct cell lines that express M1GS ribozymes, amphotropic packaging cells (PA317; Miller and Rosman 1989) were transfected with LXSN-M1GS DNAs to produce retroviral vectors that contained the genes for M1GS RNA. Human U373MG cells were then infected with these vectors, and cells expressing the ribozymes were cloned.
An additional cell line, which expressed ribozyme M1-TK that targeted the mRNA for thymidine kinase (TK) of HSV-1 (Kilani et al. 2000), was also constructed. No cleavage of substrate ap40 by M1-TK was observed in vitro (Fig. 2, lane 3). We used this cell line to determine whether M1GS RNA with an incorrect guide sequence could target PR mRNA in tissue culture. The constructed lines and a control line in which cells were transfected with LXSN vector DNA alone were indistinguishable in terms of their growth and viability for up to 3 months (data not shown), suggesting that the expression of the ribozymes did not exhibit significant cytotoxicity.
We determined the level of M1GS RNA in each cell clone by using Northern analysis with a DNA probe that is complementary to M1 RNA. The M1GS RNAs were exclusively expressed in the nuclei as they were only detected in the nuclear but not the cytoplasmic RNA fractions (Fig. 3; data not shown). This is consistent with previous observations in our laboratory, as well as others, that the transcripts expressed by the U6 promoter are primarily localized in the nuclei (Yuan et al. 1992; Bertrand and Rossi 1995; Liu and Altman 1995; Good et al. 1997). The different levels of M1-AP expression between the two cloned cell lines (Fig. 3, cf. lanes 3 and 4) are presumably due to the incorporation of the LXSN-M1GS sequence into different locations of the host chromosome, and its expression is influenced by the flanking sequence at the insertion site. We only used the cell lines that expressed similar levels of these ribozymes for further studies in tissue culture.
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), was used as an internal control for the quantitation of expression of AP and PR mRNAs (Fig. 4). A reduction of ~85 ± 9% and 88 ± 9% (average of three experiments) in the expression level of PR and AP mRNA was observed in cells that expressed M1-AP, respectively (Table 1). In contrast, a reduction of <10% in the expression level of these two mRNAs was observed in cells that expressed C-AP or M1-TK. These results suggest that the significant reduction of AP mRNA expression in cells that expressed M1-AP was due to the targeted cleavage by the ribozyme. The low level of inhibition found in cells that expressed C-AP was presumably due to an antisense effect because C-AP exhibits similar binding affinity to the target sequence as does M1-AP but is catalytically inactive.
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). Another membrane was stained with a monoclonal antibody against human actin (anti-actin; Fig. 5A). The latter serves as an internal control for the quantitation of AP and PR protein expression. The expression of AP, PR, and actin was quantitated by using a chemiluminescent substrate for the antibody staining (Fig. 5). The results of three independent experiments are summarized in Table 1: A reduction of ~82% and 80% in the level of AP and PR proteins, respectively, was observed in cells that expressed M1-AP RNA. In contrast, a reduction of <10% was found in cells that expressed C-AP or M1-TK RNAs. The low level of reduction in the expression level of AP and PR proteins observed in cells that expressed C-AP was probably due to the antisense effect of the guide sequence.
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shows the results of the experiments. After 5 d postinfection, a reduction of at least 2000-fold in viral yield was observed in cells that expressed M1-AP (Fig. 6). No significant reduction was found in those that expressed the control ribozymes C-AP or M1-TK (Fig. 6; data not shown). These results suggest that M1GS-mediated targeting of viral PR and AP mRNAs effectively inhibits HCMV growth.
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; Gibson et al. 1994; Matusick-Kumar et al. 1994), it has not been reported whether the HCMV AP is also essential for viral capsid assembly. Meanwhile, it is possible that the observed reduction of viral growth in the M1-AP-expressing cells is not necessarily due to specific M1GS RNA-mediated cleavage of AP and PR mRNAs but is due to other effects of the ribozyme on viral lytic replication that are unrelated to the consequence of the ribozyme cleavage or the inhibition of viral AP or PR expression, such as blocking the expression of viral IE genes.
To exclude these possibilities and further determine the antiviral mechanism of the M1GS-directed cleavage, two sets of experiments were carried out to investigate the step of the viral lytic cycles that is blocked in the cells that expressed M1-AP. First, the expression of other viral genes in the cells is examined. Inhibition of AP and PR expression is not expected to affect the expression of other viral genes, including IE (
), early (ß), and late (
) genes (Mocarski and Courcelle 2001), which are not regulated by the PR or the capsid AP as suggested in the studies of HSV-1 mutants (Preston et al. 1983; Matusick-Kumar et al. 1994). Northern analyses were carried out to determine the expression levels of the IE2 (an transcript) and US2 mRNA (a ß transcript; Fig. 4C,D). Moreover, we performed Western analyses to determine the expression level of viral protein UL44, a viral late (ß) protein, and UL83, a viral late () protein (Fig. 5D; Table 1). No significant difference in the expression level of these genes was observed in cells that expressed M1-AP, C-AP, or M1-TK (Table 1). These results suggest that M1-AP specifically inhibits the expression of AP and PR and does not affect overall viral gene expression. Moreover, our results are consistent with the notion that neither AP nor PR is required for the expression of most of viral genes.
In the second set of experiments, we investigated whether viral genomic replication, as well as capsid maturation, is affected in the cells that expressed M1-AP RNA. Total DNA was isolated from HCMV-infected cell lysates. The level of intracellular viral DNA was determined by PCR detection of HCMV IE1 sequence, using the level of ß-actin DNA as the internal control. Because HCMV only replicates in an episomal form and does not integrate its DNA into the host genome (Mocarski and Courcelle 2001), the amount of the intracellular viral DNA detected by the PCR assay represents the replication level of the viral genome. No significant difference in the level of viral DNA was found in cells that expressed M1-AP, C-AP, and M1-TK, suggesting that a reduction of AP and PR expression by the ribozyme does not affect the step of viral genome replication (Fig. 7, lanes 1–3). To examine viral capsid formation, the level of encapsidated viral DNA was assayed to determine the level of mature capsid assembled in the infected cells. DNA samples were isolated from HCMV-infected cell lysates that were treated with DNase I. The encapsidated viral DNAs will be resistant to DNase I digestion, whereas those that are not packaged in the capsid will be susceptible to degradation. The level of intracellular encapsidated viral DNA was determined by PCR detection of HCMV IE1 sequence. When assaying the DNA samples from cell lysates that were not treated with DNase I, we found no significant difference in the level of total intracellular (both encapsidated and uncapsidated) viral DNA in the M1GS-expressing cells (Fig. 7, lanes 1–3). When the DNase I–treated samples were assayed, however, the encapsidated DNA was hardly detected in the cells that expressed M1-AP (Fig. 7, lanes 4–6). These observations suggest that M1GS-mediated inhibition of AP and PR expression does not affect the replication of viral DNA but blocks the event(s) during or before packaging the viral genome into the capsids.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The activity of RNase P ribozymes appears to be specific. First, cells expressing the ribozymes appear to be healthy and indistinguishable from parental cells in term of cell growth and viability for up to three months (data not shown). Second, the inhibition of viral growth and capsid maturation seems to be due to the reduction of AP and PR, because the level of DNA encapsidation and the expression of AP and PR were found to be greatly decreased in cells expressing M1-AP and not in the control cells expressing C-AP or M1-TK (Figs. 4, 5, 7; data not shown). Third, expression of the ribozymes only inhibits the expression of the AP and PR mRNAs. We found no reduction in the expression levels of other viral genes examined (e.g., IE2, US2, and UL83) in M1GS-expressing cells (Figs. 4, 5; data not shown). Finally, the replication of viral genomic DNA does not appear to be affected by the expression of M1-AP and the subsequent reduction of AP and PR expression (Fig. 7). Together, these results support the belief that AP and PR are essential for capsid maturation and do not involve in viral gene expression and genome replication.
We showed that the M1GS RNAs introduced into cultured cells were stably expressed and localized primarily in the nuclei. The expression cassette we used to produce these M1GSs is driven by the promoter for small nuclear U6 RNA. This promoter has been extensively used to express functional RNAs and ribozymes for gene targeting applications, and the transcript from this promoter is quite stable and primarily localized in the nuclei (Das et al. 1988; Yuan et al. 1992; Liu and Altman 1995; Bertrand et al. 1997; Good et al. 1997). Nuclear localization of RNase P ribozymes may play a significant role in its efficacy in cell culture. This is because the regions of the catalytic M1 RNA domain in these M1GS RNAs, which may be homologous to those of the RNA subunit of human RNase P, are believed to interact with cellular proteins, including those associated with RNase P (Liu and Altman 1995; Altman and Kirsebom 1999). This hypothesis is consistent with previous observations that several "RNA chaperone" proteins have been shown to stimulate the activity of hammerhead and group I ribozymes by facilitating either folding of the ribozyme or dissociation of the product (Tsuchihashi et al. 1993; Bertrand and Rossi 1995). Further studies on potential interactions between M1GS RNAs and cellular proteins will provide insight into how a M1GS ribozyme functions in cultured cells.
HCMV is a member of the human herpesvirus family, which includes seven other different viruses such as HSV and Epstein-Barr virus (Kieff and Rickinson 2001; Mocarski and Courcelle 2001; Roizman and Knipe 2001). All these viruses can engage in lytic replication and establish latent infections. HCMV PR and capsid APs are believed to be involved in viral capsid maturation (Mocarski and Courcelle 2001). Their homologous proteins have been found in every other herpesviruses and are highly conserved among all herpesviruses, suggesting that these proteins may serve as the ideal targets for drug development for the treatment and prevention of the infections by HCMV and other herpesviruses. Our results presented in this study indicate that M1GS RNA-mediated inhibition of the expression of HCMV AP and PR leads to a significant (~2000-fold) reduction of viral growth, and are consistent with the notion that blocking of the expression of these two proteins should yield effective antiviral therapy. To further evaluate the anti-HCMV activity of M1GS RNA, the ribozymes can be delivered into the monocyte/macrophage-lineage cells in which HCMV is believed to establish latent infection (Mocarski and Courcelle 2001). These experiments will determine whether the ribozymes can abolish viral gene expression in these cells and prevent HCMV reactivation from latent infection into lytic replication.
The M1GS-based technology represents an attractive approach for gene inactivation because it generates catalytic and irreversible cleavage of the target RNA by using M1 RNA, a highly active RNA enzyme found in nature (Frank and Pace 1998; Altman and Kirsebom 1999). These properties, as well as the simple design of the guide sequence, make M1GS an attractive and unique gene-targeting agent that can be generally used for antiviral and other in vivo applications. Indeed, our laboratory has recently showed that M1GS RNA inhibited the expression of HCMV IE1/IE2 and HSV-1 ICP4 protein and the replication of HCMV and HSV-1 (Trang et al. 2000a,b). The levels of inhibition of AP and PR expression observed in this study are similar to those of inhibition of the expression of HSV-1 ICP4 protein and HCMV IE1/IE2. However, the level of inhibition (2000-fold) of HCMV growth by M1-AP is twofold better than that of inhibition of HSV-1 replication and at least 10-fold higher than that by the ribozyme against IE1/IE2 mRNAs (Trang et al. 2000a,b). Thus, the efficacy of the ribozyme in inhibiting viral growth might be dependent on the nature of the gene target. Our results also suggest that targeting both viral regulatory and stoichiometric functions may yield better antiviral efficacy, as AP and PR serve as regulatory and structural proteins during viral propagation, whereas IE1/IE2 and ICP4, the viral major transcription factors (Mocarski and Courcelle 2001; Roizman and Knipe 2001), encode only the regulatory functions. Further studies on determining the best viral target for the ribozymes, as well as increasing the activity and efficacy of the M1GS ribozymes, will significantly facilitate the development of more active and effective ribozymes for both in vitro and in vivo applications.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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; Kilani et al. 2000). Mutant ribozyme C102 contains several point mutations (e.g., A347C348 
C347U348, C353C354C355G356 G353G354A355U356) at the catalytic domain (P4 helix; Kim et al. 1997). The DNA sequences that encode ribozymes M1-AP and C-AP were constructed by PCR using the DNA sequences of the M1 and C102 ribozymes as the templates and oligonucleotides AF25 and M1AP2 (5'-CCCGCTCGAGAAAAAATGGTGTGGCAGGTGCGTCGCCGT GTGGAATTGTG-3') as 5' and 3' primers, respectively.
Cleavage and binding analysis
M1GS RNAs and the ap40 mRNA substrate were synthesized in vitro by T7 RNA polymerase (Promega Inc.), following the manufacturer’s recommendations, and further purified on 8% polyacrylamide gels containing 8 M urea. Subsequently, the M1GS RNAs were mixed with the [32P]-labeled mRNA substrate. The cleavage reactions were carried out in a volume of 10 µL for 30 min at 37°C in buffer A (50 mM Tris at pH 7.5, 100 mM NH4Cl, and 100 mM MgCl2; Kilani et al. 2000). Cleavage products were separated in denaturing gels and quantitated with a STORM840 phosphorimager (Molecular Dynamics). The procedures to measure the equilibrium Kd of the M1GS-ap40 complexes were modified from Pyle et al (1990) and have been described previously (Trang et al. 2000b). The values of Kd obtained were the average of three experiments.
Viruses, cells, and antibodies
Human primary foreskin fibroblasts (HFFs), astrocytoma U373MG cells, and PA317 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum. The propagation of HCMV (AD169) in HFFs and U373MG cells was carried out as described previously (Trang et al. 2000b). The monoclonal antibodies c1201, c1202, c1203, and c1205, which react with HCMV proteins gB, UL44, IE1/IE2, and UL83, were purchased from Goodwin Institute for Cancer Research. The anti-rabbit polyclonal antibodies against HCMV PR and capsid AP were kindly provided by Annette Meyer of Parke-Davis Pharmaceutical Research Institute of Warner Lambert. The monoclonal antibody against human actin was purchased from Sigma.
Construction of ribozyme-expressing cells
The protocols for the construction of U373MG cells expressing different ribozymes were modified from Miller and Rosman (1989) and have been described previously (Liu and Altman 1995). In brief, amphotropic PA317 cells were transfected with retroviral vector DNAs (LXSN-M1-AP and LXSN-C-AP) with the aid of a mammalian transfection kit purchased from GIBCO BRL. Forty-eight hours posttransfection, culture supernatants that contained retroviral vectors were collected and used to infect U373MG cells. At 48–72 h postinfection, cells were incubated in culture medium that contained 600 µg/mL neomycin. Cells were subsequently selected in the presence of neomycin for 2 wk, and neomycin-resistant cells were cloned.
For Northern analyses of the expression of the ribozymes, both nuclear and cytoplasmic RNA fractions from M1GS-expressing cells were isolated as described previously (Kawa et al. 1998). The RNA fractions were separated in a 2.5% agarose gel that contained formaldehyde, transferred to a nitrocellulose membrane, hybridized with the [32P]-radiolabeled DNA probe that contained the DNA sequence coding for M1 RNA, and finally analyzed with a STORM840 phosphorimager. The radiolabeled DNA probe used to detect M1GS RNAs was synthesized from plasmid pFL117, by using a random primed labeling kit (Boehringer Manheim).
Viral infection and assays for viral gene expression and growth
T-25 flasks of cells (~106 cells) were either mock-infected or infected with HCMV as described previously (Trang et al. 2000b). The MOI is specified as that in the Results section. The infected cells were incubated for 8 to 72 h, and viral mRNAs or proteins were isolated as described previously (Liu and Altman 1995). To measure the levels of viral IE transcripts, some of the cells were also treated with 100 µg/mL cycloheximide prior to and during infection.
The RNA fractions were separated in 1% agarose gels that contained formaldehyde, transferred to a nitrocellulose membrane, hybridized with the [32P]-radiolabeled DNA probes that contained the HCMV or human ß-actin DNA sequences, and analyzed with a STORM840 Phosphorimager. The DNA probes used to detect M1GS RNAs, human ß-actin mRNA, HCMV IE 5-kb RNA transcript, IE2 mRNA, US2 mRNA, and PR and AP mRNA were synthesized from plasmids pFL117, pß-actin RNA, pCig27, pIE2, pCig38, and pUL80, respectively. For Western analyses, we separated the polypeptides from cell lysates on either SDS/7.5% polyacrylamide gels or SDS/9% polyacrylamide gels cross-linked with N,N''methylenebisacrylamide. We then transferred the separated polypeptides electrically to nitrocellulose membranes. We stained the membranes by using the antibodies against HCMV proteins and human actin in the presence of a chemiluminescent substrate (Amersham Inc.), and analyzed the stained membranes with a STORM840 phosphorimager. Quantitation was performed in the linear range of RNA and protein detection.
To determine the level of the inhibition of viral growth, 5 x 105 cells were either mocked infected or infected with HCMV at an MOI of one to five. The infection was carried out by incubating cells with DMEM in the absence or presence of viruses for 90 min at 37°C, and then with fresh DMEM for different periods of time (Trang et al. 2000b). The cells and medium were harvested at 1, 2, 3, 4, 5, 6, and 7 d postinfection, and viral stocks were prepared by adding 10% skim milk followed by sonication. The titers of the viral stocks were determined by performing plaque assays on HFFs (Trang et al. 2000b). The values obtained were the average from triplicate experiments.
Assaying the level of viral genome replication
The 5 x 105 cells grown on six-well plates were mock-infected or infected with HCMV. After a 1.5-h incubation at 37°C, the inoculum was removed, and the cells were further incubated and harvested at 48–96 h postinfection. Total and encapsidated (DNase I–treated) DNAs were isolated essentially as described (Gao et al. 1994; Matusick-Kumar et al. 1994) and used as the PCR DNA templates.
Viral DNA was detected by PCR amplification of the viral IE1/IE2 sequence, using human ß-actin sequence as the internal control. The 5' and 3' primers were CMV3 (5'-CCAAGCGGCCTC TGATAACCAAGCC-3') and CMV4 (5'-CAGCACCATCCTCC TCTTCCTCTGG-3'), respectively (Demmler et al. 1988), whereas those used to amplify the actin sequence were Actin5 (5'-TGAC GGGGTCACCCACACTGTGCCCATCTA-3') and Actin3 (5'-CTAGAAGCATTGCGGTGGCAGATGGAGGG-3'), respectively (Daftarian et al. 1996). The PCR reaction consisted of 20 cycles with denaturation for 1 min at 94°C, followed by primer annealing for 1 min at 47°C and extension for 1 min at 72°C. The last cycle was again an extension for 10 min at 72°C. PCR cycles and other conditions were optimized to assure that the amplification was within the linear range.
We carried out the PCR reactions in the presence of -[32P]-dCTP. The radiolabeled DNA samples were separated on polyacrylamide gels and then scanned with a STORM840 phosphorimager. We also generated a standard (dilution) curve by amplifying different dilutions of the template DNA. The plot of counts for both HCMV and ß-actin versus dilutions of DNA did not reach a plateau for the saturation curve (data not shown) under the conditions described above, indicating that quantitation of viral DNA could be accomplished. Moreover, we observed that the ratio of viral DNA to ß-actin remained constant with respect to each DNA dilution in the standard curve, suggesting that the assay is adequately accurate and reproducible. The PCR results were derived from three independent experiments.
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ACKNOWLEDGMENTS |
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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Footnotes |
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Received September 8, 2003; accepted November 7, 2003.
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