Subversion of cell cycle regulatory pathways.

Human cytomegalovirus (HCMV) has evolved numerous strategies to commandeer the host cell for producing viral progeny. The virus manipulates host cell cycle pathways from the early stages of infection to stimulate viral DNA replication at the expense of cellular DNA synthesis. At the same time, cell cycle checkpoints are by-passed, preventing apoptosis and allowing sufficient time for the assembly of infectious virus.

Human immunodeficiency virus infection of human-PBL-SCID mice.

Severe combined immunodeficient (SCID) mice reconstituted with human peripheral blood leukocytes (hu-PBL-SCID mice) have inducible human immune function and may be useful as a small animal model for acquired immunodeficiency syndrome (AIDS) research. Hu-PBL-SCID mice infected with human immunodeficiency virus-1 (HIV-1) contained virus that was recoverable by culture from the peritoneal cavity, spleen, peripheral blood, and lymph nodes for up to 16 weeks after infection; viral sequences were also detected by in situ hybridization and by amplification with the polymerase chain reaction (PCR). Mice could be infected with multiple strains of HIV-1, including LAV-1/Bru, IIIB, MN, SF2, and SF13. HIV-1 infection affected the concentration of human immunoglobulin and the number of CD4+ T cells in the mice. These results support the use of the hu-PBL-SCID mouse for studies of the pathogenesis and treatment of AIDS.

Sequences in human cytomegalovirus which hybridize with the avian retrovirus oncogene v-myc are G + C rich and do not hybridize with the human c-myc gene.

The degree of relatedness between previously identified cross-hybridizing regions within human cytomegalovirus strain AD169 and the avian retrovirus oncogene v-myc were investigated by nucleotide sequence comparison. We found that the homologous regions between the human cytomegalovirus genome and v-myc are limited to short G + C-rich regions in each genome and that the human cytomegalovirus genome shares little or no homology with the human c-myc gene.

In vivo and in vitro analysis of transcriptional activation mediated by the human cytomegalovirus major immediate-early proteins.

To define mechanistically how the human cytomegalovirus (HCMV) major immediate-early (IE) proteins induce early-gene transcription, the IE1 72-kDa protein, the IE2 55-kDa protein, and the IE2 86-kDa protein were analyzed for their ability to activate transcription from an HCMV early promoter in vivo and in vitro. In transient-expression assays in U373MG astrocytoma/glioblastoma and HeLa cells, only the IE2 86-kDa protein was able to activate the HCMV early promoter to high levels. In HeLa cells, the IE1 72-kDa protein was able to activate the promoter to a low but detectable level, and the level of promoter activity observed in response to the IE2 86-kDa protein was increased synergistically following cotransfection of the constructs expressing both IE proteins. To examine the interaction of the HCMV IE proteins with the RNA polymerase II transcription machinery, we assayed the ability of Escherichia coli-synthesized proteins to activate the HCMV early promoter in nuclear extracts prepared from U373MG cells, HeLa cells, and Drosophila embryos. The results of the in vitro experiments correlated well with those obtained in vivo. The basal activity of the promoter was minimal in both the HeLa and U373MG extracts but was stimulated 6- to 10-fold by the IE2 86-kDa protein. With a histone H1-deficient extract from Drosophila embryos, the HCMV early promoter was quite active and was stimulated two- to fourfold by the IE2 86-kDa protein. Addition of histone H1 at 1 molecule per 40 to 50 bp of DNA template significantly repressed basal transcription from this promoter. However, the IE2 86-kDa protein, but none of the other IE proteins, was able to counteract the H1-mediated repression and stimulate transcription at least 10- to 20-fold. The promoter specificity of the activation was demonstrated by the inability of the IE2 86-kDa protein to activate the Drosophila Krüppel promoter in either the presence or absence of histone H1. These results suggest that one mechanism of transcription activation by the IE2 86-kDa protein involves antirepression.

Exploitation of cellular signaling and regulatory pathways by human cytomegalovirus.

Human cytomegalovirus is a ubiquitous human pathogen that is the leading viral cause of birth defects. It also causes significant morbidity and mortality in both chemically and virally immunosuppressed individuals. Recent studies have begun to elucidate the interplay between this virus and its host cell on a molecular level. The interactions begin upon contact with the cell membrane, involve multiple processes including cell signaling, cell-cycle control and immune response mechanisms, and culminate in a productive infection.

Activation and regulation of human cytomegalovirus early genes.

The multiple pathogenic effects of human cytomegalovirus (HCMV) result from a complex interplay of viral gene products and induced and repressed cellular functions. HCMV immediate early and early gene expression clearly play a pivotal role in this scheme. I describe here work directed at elucidating the sequence requirements and trans-acting factors necessary for the activation and regulation of HCMV early genes. The focus is on three transcription units which are all activated at early times, but exemplify differential patterns of expression as the infection progresses, including: the major 2.7- and 1.2-kb transcripts encoded by the repeat bounding of the long unique segment of the genome, and a family of differentially spliced transcripts (originally designated the 2.2-kb RNAs-ORF UL112-113) which encode four proteins that are required in the transient complementation assay for HCMV DNA replication. Selected other early genes which illustrate alternative mechanisms of control are also discussed.

Pathogenesis of acute murine cytomegalovirus infection in resistant and susceptible strains of mice.

We have characterized the progress of acute murine cytomegalovirus (MCMV) infection in the spleen, liver, and salivary gland of susceptible (BALB/c) and resistant (C3H) strains of mice after intraperitoneal inoculation. Viral replication was analyzed by virus titration, infectious-center assays, and in situ cytohybridization with cloned subgenomic fragments of the MCMV genome. The most striking differences between strains were observed in the spleen. At 24 h postinfection (p.i.), both strains had a similar number of infected spleen cells. At 48 h p.i., BALB/c mice showed marked dissemination of the splenic infection which continued until 96 h p.i. In contrast, the number of infected C3H spleen cells did not increase from the 24-h level but declined later on. This early block in dissemination of MCMV infection in C3H mouse spleens was not a result of the H-2k haplotype, as BALB.K (H-2k) mice, which show an intermediate level of resistance to MCMV infection, exhibited dissemination of the infection between 24 and 48 h p.i., albeit at a reduced level. However, between 72 and 96 h p.i., we observed a decline in the number of infected spleen cells in BALB.K mice similar to that observed in C3H mice. We also demonstrated by Southern blot analysis of DNA from the infected spleen cells that the termini of the MCMV genome fuse after in vivo infection.

2.2-kilobase class of early transcripts encoded by cell-related sequences in human cytomegalovirus strain AD169.

In this report we describe the kinetics of appearance and fine mapping of a 2.2-kilobase (kb) class of transcripts arising from a region of the human cytomegalovirus genome which contains cell-related sequences. These transcripts are encoded by adjacent EcoRI fragments R and d (map units 0.682 to 0.713), located within the long unique segment of the genome. The 2.2-kb RNAs were first detected at 8h postinfection and appeared at comparable or slightly lower levels at 28 and 72 h postinfection. At late times (72 h) additional transcripts were detected with probes from this region. RNase, S1 nuclease, and exonuclease VII protection analyses of 8- and 28-h RNA indicated that the 2.2-kb RNAs had a complex spliced structure consisting of invariable 5' and internal exons and a heterogeneous 3' exon. The position of the 5' end of the RNA was determined with respect to the nucleotide sequence. Analysis of this sequence showed that the cell-related sequences were contained within a long open reading frame in the 5' exon.

Terminal structure and heterogeneity in human cytomegalovirus strain AD169.

We have characterized the heterogeneity occurring at the junction of the long (L) and short (S) segments and at the termini of the strain AD169 human cytomegalovirus (HCMV) genome by restriction endonuclease mapping and nucleotide sequence analyses. The HCMV a sequence was identified by its position at both termini and inverted orientation at the L-S junction. Heterogeneity at both termini and the L-S junction was generated by the presence of fused and tandem a sequences. Some S termini lacked an a sequence. In addition, near the L terminus and at the L-S junction there were a variable number of 217-base-pair (bp) XhoI fragments arranged in tandem. The 217-bp fragments consisted of a portion of the a and adjacent b sequences (in the L-segment repeat) bounded by the same direct repeats (DR1) found at the boundaries of the a sequence. A model for the generation of these heterogeneous fragments is presented. We also determined the sequence of seven cloned terminal fragments, five from the L terminus and two from the S terminus. All L termini contained identical terminal sequences ending with base 32 of a 33-bp DR1. The S termini differed from each other and from the L-segment termini. One S terminus lacked an a sequence and terminated within S-segment repeat (c) sequences. The second S terminus contained an a sequence and terminated with bases 20 to 33 of a 33-bp DR1. A comparison of the cloned L and S terminal sequences with cloned L-S junction sequences suggested that the termini contained 3' single base extensions which were removed during the cloning. We also show that the herpesvirus conserved sequence is in a similar position relative to the termini of HCMV and several other herpesviruses, thus adding further support for the role of the sequence in the maturation of viral DNA.

The human cytomegalovirus 2.7-kilobase RNA promoter contains a functional binding site for the adenovirus major late transcription factor.

We have examined the factors which influence the expression of a major 2.7-kilobase (kb) early transcript encoded by the long repeat of the human cytomegalovirus (HCMV) strain AD169 genome. Previously, by deletion analysis, we determined that the promoter for this early RNA consisted of multiple cis-acting elements (Klucher et al., J. Virol. 63:5334-5343, 1989). Using extracts prepared from HeLa cells as well as from infected and uninfected foreskin fibroblasts, we also obtained evidence for the interaction of a cellular factor with one of these elements. In this study, we have further defined the specificity and functional importance of this binding. On the basis of DNase I footprinting and methylation interference assays, we localized the site of interaction to a region (nucleotides -113 to -106 relative to the mRNA start site) which contains homology to the binding site for the adenovirus major late transcription factor (MLTF), also referred to as the upstream stimulatory factor (USF). The contact points of binding between the cellular factor and the guanine residues within this segment were consistent with the pattern of binding for USF/MLTF. Additionally, by using oligonucleotides containing the binding sites for USF/MLTF from the adenovirus major late promoter and the HCMV 2.7-kb RNA promoter as competitors in gel retardation assays, we were able to show that USF/MLTF bound to the two promoters with similar affinity. Correlation of the binding activity with in vivo functional importance was provided by mutagenesis and transient-expression assays. A point mutation within the HCMV USF/MLTF site lowered the affinity of binding 5- to 10-fold and decreased the inducible activity of the HCMV 2.7-kb RNA promoter by approximately 50%. Furthermore, the addition of the HCMV USF/MLTF site to a minimal 2.7-kb RNA promoter containing only the TATA sequence resulted in an increase in HCMV inducible transcriptional activity of 6- to 20-fold. However, the HCMV USF/MLTF site could not functionally substitute for the TATA sequence. These studies further support the idea that for maximal response to the HCMV infection, the 2.7-kb RNA promoter requires multiple cis-acting sequences, two of which include the binding sites for USF/MLTF and TFIID.

The use of DNA probes in studies of human cytomegalovirus.

Hybridization assays provide a sensitive and rapid means for studying the molecular biology of viral replication and for identifying viral nucleic acid in biological specimens. Such assays are attractive because the detection of virus does not require intact virions or concomitant viral protein synthesis, both of which may be absent in a latently infected cell or in a virus-associated tumor. For molecular and clinical studies on human cytomegalovirus (HCMV), we have cloned and characterized subgenomic EcoRI fragments representative of the entire genome of HCMV strain AD169. To study the epidemiology of HCMV infections and to identify the presence of HCMV nucleic acid in urine, blood, Kaposi's sarcoma, and other tissues, we have used various hybridization techniques, including DNA dot/slot-blot hybridization, Southern blot hybridization, and in situ cytohybridization. These studies demonstrate how cloned molecular probes can be used to study the molecular biology, pathogenesis, and treatment of viral infections.

Fusion of the termini of the murine cytomegalovirus genome after infection.

The genome of murine cytomegalovirus, extracted from extracellular virions, is a linear double-stranded DNA molecule ca. 240 kilobase pairs long. In our initial cloning of subgenomic fragments of the murine cytomegalovirus genome, we obtained a HindIII clone which contained fused HindIII-terminal fragments. By hybridizing this cloned DNA fragment to infected-cell DNA, we identified an intracellular restriction fragment which was the length of the sum of the two authentic termini. This fusion fragment was not present in virion DNA but could be detected as early as 2 h postinfection and reached its highest level shortly after the onset of DNA replication at 16 h postinfection. The prereplicative increase of fused ends was not inhibited by a level of phosphonoacetic acid which effectively shut off viral DNA synthesis, nor was the early conversion from free to fused ends prevented by inhibitors of protein or RNA synthesis. The results are consistent with the fused state of viral DNA being a replicative intermediate and precursor to DNA synthesis.

Replication of the murine cytomegalovirus genome: structure and role of the termini in the generation and cleavage of concatenates.

Following infection with murine cytomegalovirus (MCMV), the termini of the linear double-stranded DNA genome fuse to form circular or concatemeric forms which serve as replicative intermediates. To investigate the mechanisms involved in the generation and cleavage of the intracellular concatenates, we have used restriction endonuclease mapping and nucleotide sequence analyses to characterize the structure of the virion DNA termini and intracellular end-to-end fusion fragment. Four each of the cloned EcoRI X and EcoRI c terminal fragments were sequenced. All of the EcoRI X clones and three of the EcoRI c clones contained a 30-base-pair (bp) sequence which was directly repeated at the ends of the MCMV genome. The terminal sequence of the fourth EcoRI c clone began directly after the 30-bp direct repeat. The four EcoRI X clones also had minor length heterogeneity, differing in the number of GC bp at the terminus. Five fusion fragments were sequenced. Three of the fusion fragments contained both direct repeats, while two fusion fragments lacked one entire direct repeat. Direct analyses of the virion DNA and intracellular fusion fragments revealed that the clones accurately reflected the naturally occurring populations and that the relative proportion of fusion fragments containing only one direct repeat increased as the infection progressed. The data suggest that fusion of the termini arises by end-to-end ligation. We also show that adjacent to the MCMV termini are sequences highly conserved among the herpesviruses, and we discuss their potential role in the maturation of the viral genome.

Polyadenylic acid on poliovirus RNA. III. In vitro addition of polyadenylic acid to poliovirus RNAs.

A crude RNA polymerase preparation was made from HeLa cells infected for 3 h with poliovirus. All virus-specific RNA species labeled in vitro (35S RNA, replicative intermediate RNA [RI], and double-stranded RNA [dsRNA]) would bind to poly(U) filters and contained RNase-resistant stretches of poly(A) which could be analyzed by electrophoresis in polyacrylamide gels. After incubation for 45 min with [3-H]ATP in the presence of the other three nucleoside triphosphates, the labeled poly(A) on the RI and dsRNA migrated on gels as relatively homogenous peaks approximately 200 nucleotides in length. In contrast, the poly(A) from the 35S RNA had a heterogeneous size distribution ranging from 50 to 250 nucleotides. In the absence of UTP, CTP, and GTP, the size of the newly labeled poly(A) on the dsRNA and RI RNA was the same as it was in the presence of all four nucleoside triphosphates. However the poly(A) on the 35S RNA lacked the larger sequences seen when the other three nucleoside triphosphates were present. When [3-H]ATP was used as the label in infected and uninfected extracts, heterogeneous single-stranded RNA sedimenting at less than 28S was also labeled. This heterogeneous RNA probably represents HeLa cytoplasmic RNA to which small lengths of poly(A) (approximately 15 nucleotides) had been added. These results indicate that in the in vitro system poly(A) can be added to both newly synthesized and preexisting RNA molecules. Furthermore, an enzyme capable of terminal addition of poly(A) exists in both infected and uninfected extracts.

Polyadenylic acid on poliovirus RNA. II. poly(A) on intracellular RNAs.

The content, size, and mechanism of synthesis of 3'-terminal poly(A) on the various intracellular species of poliovirus RNA have been examined. All viral RNA species bound to poly(U) filters and contained RNase-resistant stretches of poly(A) which could be analyzed by electrophoresis in polyacrylamide gels. At 3 h after infection, the poly(A) on virion RNA, relicative intermediate RNA, polyribosomal RNA, and total cytoplasmic 35S RNA was heterogeneous in size with an average length of 75 nucleotides. By 6 h after infection many of the intracellular RNA's had poly(A) of over 150 nucleotides in length, but the poly(A) in virion RNA did not increase in size suggesting that the amount of poly(A) which can be encapsidated is limited. At all times, the double-stranded poliovirus RNA molecules had poly(A) of 150 to 200 nucleotides. Investigation of the kinetics of poly(A) appearance in the replicative intermediate and in finished 35S molecules indicated that poly(A) is the last portion of the 35S RNA to be synthesized; no nascent poly(A) could be detected in the replicative intermediate. Although this result indicates that poliovirus RNA is synthesized 5' leads to 3' like other RNA's, it also suggests that much of the poly(A) found in the replicative intermediate is an artifact possibly arising from the binding of finished 35S RNA molecules to the replicative intermediate during extraction. The addition of poly(A) to 35S RNA molecules was not sensitive to guanidene.

Requirement of 3'-terminal poly(adenylic acid) for the infectivity of poliovirus RNA.

Ribonuclease H (EC 3.1.4.34) has been used to remove selectively much of the 3'-terminal poly(adenylic acid) [poly(A)] from poliovirus RNA by treating the RNA with the enzyme in the presence of poly(dT). Over 80% of the poly(A) could be removed and the residuum was found as oligo(A) stretches attached to many or all of the viral RNA molecules. Reduction of the size of the poly(A) markedly decreased the specific infectivity of poliovirus RNA, indicating that poly(A) is necessary to the infectivity of the RNA. The virions in plaques deriving from infection with treated RNA have a normal amount and size of poly(A), indicating that mechanisms exist in infected cells to regenerate normal length poly(A) from truncated poly(A).

Extracellular signal-regulated kinase activity is sustained early during human cytomegalovirus infection.

Expression of many early viral genes during human cytomegalovirus (HCMV) infection is dependent on cellular transcription factors. Several immediate-early and early viral promoters contain DNA binding sites for cellular factors such as CREB, AP-1, serum response factor, and Elk-1, and these transcription factors can be activated by phosphorylation via the cellular mitogen-activated protein kinase (MAPK) signal transduction cascade. To determine if the extracellular signal-regulated MAPKs, ERK1 and ERK2, play a role in transcription factor activation during infection, we tested for ERK activity during viral infection. We found that HCMV infection resulted in the maintenance of previously activated ERK1 and ERK2 by a mechanism which appears to involve the inhibition of a cellular phosphatase activity. ERK phosphorylation and activity were sustained for at least 8 h after infection, whereas in mock-infected cells, ERK activity steadily declined by 1 h postinfection. The activity of at least one cellular substrate of the ERKs, the protein kinase RSK1, was also maintained during this period. UV inactivation experiments suggested that viral gene expression was required for sustained ERK activity. In turn, activation of the ERKs appeared to be important for viral gene expression, as evidenced by the observed decrease in the transcriptional activity of the HCMV UL112-113 promoter during infection in the presence of the MEK inhibitor PD98059. These data suggest that HCMV utilizes cellular signal transduction pathways to activate viral or cellular transcription factors involved in the control of early viral gene expression and DNA replication.