Differential outcomes of human cytomegalovirus infection in primitive hematopoietic cell subpopulations

The cellular reservoir for latent human cytomegalovirus (HCMV) in the hematopoietic compartment, and the mechanisms governing a latent infection and reactivation from latency are unknown. Previous work has demonstrated that HCMV infects CD34+ progenitors and expresses a limited subset of viral genes. The outcome of HCMV infection may depend on the cell subpopulations infected within the heterogeneous CD34+ compartment. We compared HCMV infection in well-defined CD34+ cell subpopulations. HCMV infection inhibited hematopoietic colony formation from CD34+/CD38- but not CD34+/c-kit+ cells. CD34+/CD38- cells transiently expressed a large subset of HCMV genes that were not expressed in CD34+/c-kit+ cells or cells expressing more mature cell surface phenotypes. Although viral genomes were present in infected cells, viral gene expression was undetectable by 10 days after infection. Importantly, viral replication could be reactivated by coculture with permissive fibroblasts only from the CD34+/CD38- population. Strikingly, a subpopulation of CD34+/CD38- cells expressing a stem cell phenotype (lineage-/Thy-1+) supported a productive HCMV infection. These studies demonstrate that the outcome of HCMV infection in the hematopoietic compartment is dependent on the nature of the cell subpopulations infected and that CD34+/CD38- cells support an HCMV infection with the hallmarks of latency.     

Human cytomegalovirus gene expression during infection of primary hematopoietic progenitor cells: a model for latency

Human cytomegalovirus (HCMV) resides latently in hematopoietic cells of the bone marrow. Although viral genomes can be found in CD14+ monocytes and CD34+ progenitor cells, the primary reservoir for latent cytomegalovirus is unknown. We analyzed human hematopoietic subpopulations infected in vitro with a recombinant virus that expresses a green fluorescent protein marker gene. Although many hematopoietic cell subsets were infected in vitro, CD14+ monocytes and various CD34+ subpopulations were infected with the greatest efficiency. We have developed an in vitro system in which to study HCMV infection and latency in CD34+ cells cultured with irradiated stromal cells. Marker gene expression was substantially reduced by 4 days postinfection, and infectious virus was not made during the culture period. However, viral DNA sequences were maintained in infected CD34+ cells for >20 days in culture, and, importantly, virus replication could be reactivated by coculture with human fibroblasts. Using an HCMV gene array, we examined HCMV gene expression in CD34+ cells. The pattern of viral gene expression was distinct from that observed during productive or nonproductive infections. Some of these expressed viral genes may function in latency and are targets for further analysis. Altered gene expression in hematopoietic progenitors may be indicative of the nature and outcome of HCMV infection.     

Functional profiling of a human cytomegalovirus genome

Human cytomegalovirus (HCMV), a ubiquitous herpesvirus, causes a lifelong subclinical infection in healthy adults but leads to significant morbidity and mortality in neonates and immunocompromised individuals. Its ability to grow in different cell types is responsible for HCMV-associated diseases, including mental retardation and retinitis, and vascular disorders. To globally assess viral gene function for replication in cells, we determined the genomic sequence of a bacterial artificial chromosome (BAC)-based clone of HCMV Towne strain and used this information to delete each of its 162 unique ORFs and generate a collection of viral mutants. The growth of these mutants in different cultured cells was examined to systematically investigate the necessity of each ORF for replication. Our results showed that 45 ORFs are essential for viral replication in fibroblasts and 117 are nonessential. Some genes were found to be required for viral replication in retinal pigment epithelial cells and microvascular endothelial cells, but not in fibroblasts, indicating their role as tropism factors. Interestingly, several viral mutants grew 10- to 500-fold better than the parental strain in different cell types, suggesting that the deleted ORFs encode replication temperance or repressing functions. Thus, HCMV encodes supportive and suppressive growth regulators for optimizing its replication in human fibroblasts, epithelial, and endothelial cells. Suppression of viral replication by virus-encoded temperance factors represents a novel mechanism for regulating the growth of an animal virus, and may contribute to HCMV's optimal infection of different tissues and successful proliferation among the human population.     

Adenovirus E1A/E1B Transformed Amniotic Fluid Cells Support Human Cytomegalovirus Replication

The human cytomegalovirus (HCMV) replicates to high titers in primary human fibroblast cell cultures. A variety of primary human cells and some tumor-derived cell lines do also support permissive HCMV replication, yet at low levels. Cell lines established by transfection of the transforming functions of adenoviruses have been notoriously resistant to HCMV replication and progeny production. Here, we provide first-time evidence that a permanent cell line immortalized by adenovirus type 5 E1A and E1B (CAP) is supporting the full HCMV replication cycle and is releasing infectious progeny. The CAP cell line had previously been established from amniotic fluid cells which were likely derived from membranes of the developing fetus. These cells can be grown under serum-free conditions. HCMV efficiently penetrated CAP cells, expressed its immediate-early proteins and dispersed restrictive PML-bodies. Viral DNA replication was initiated and viral progeny became detectable by electron microscopy in CAP cells. Furthermore, infectious virus was released from CAP cells, yet to lower levels compared to fibroblasts. Subviral dense bodies were also secreted from CAP cells. The results show that E1A/E1B expression in transformed cells is not generally repressive to HCMV replication and that CAP cells may be a good substrate for dense body based vaccine production.     

Human immunodeficiency virus type 1 tat gene enhances human cytomegalovirus gene expression and viral replication.

Clonal lines of human rhabdomyosarcoma (RD) cells, constitutively expressing human immunodeficiency virus type 1 (HIV-1) tat gene (RD tat cell lines) showed enhanced expression of human cytomegalovirus (HCMV) immediate-early (IE) and late (L) proteins upon HCMV infection, as compared with control RD cells. One of the RD tat cell lines produced infectious HCMV. The RD-tat cell lines, following transfection with recombinant plasmids containing the full length of the HCMV-IE enhancer/promoter linked to the bacterial chloramphenicol acetyltransferase (CAT) gene, exhibited an increased CAT expression by the tat product. A chronically HIV-1-infected human T-lymphoid cell line, SupT1, superinfected with HCMV, expressed HCMV-IE proteins while the parental SupT1 cells infected with HCMV were negative. Parental SupT1 cells coinfected with HIV-1 and HCMV also expressed HCMV-IE proteins, indicating that HIV-1-encoded proteins exert a positive regulatory effect on HCMV expression.     

Mechanisms associated with the generation of biologically active human immunodeficiency virus type 1 particles from defective proviruses.

The human immunodeficiency virus (HIV) is the etiological agent of acquired immunodeficiency syndrome (AIDS). HIV exhibits extensive genetic diversity and it is apparent that an infected individual contains different populations of distinct viral strains, a large proportion of which has been found surprisingly to be defective for replication. A similar phenomenon has also been observed with some cell lines that are known to produce infectious viral particles but harbor defective proviral genomes. Here, we investigated the molecular basis of this phenomenon by cloning proviral genomes of HIV from a cell line that was capable of producing high titers of biologically active HIV particles that readily induced syncytia with CD4+ cell lines and peripheral blood lymphocytes. This cell line was found to contain five proviral genomes, all of which, when tested individually, failed to produce replication-competent viruses upon transfection into human cells. However, when a specific combination of two proviral genomes was used in such transfection studies, it was possible to obtain biologically active, replication-competent viral particles that infected and replicated in CD4+ cell lines and induced syncytia characteristic of HIV. Such a result may be due to homologous recombination between proviral DNAs occurring in cells after transfection and/or complementation of replication-defective proviral DNAs. The diploid nature of the viral RNA genome present in the viral particle may enable the persistence of defective HIV genomes.     

Construction of a recombinant human parvovirus B19: adeno-associated virus 2 (AAV) DNA inverted terminal repeats are functional in an AAV-B19 hybrid virus.

To facilitate genetic analysis of the human pathogenic parvovirus B19, we constructed a hybrid B19 viral genome in which the defective B19 inverted terminal repeats were replaced with the full-length inverted terminal repeats from a nonpathogenic human parvovirus, the adeno-associated virus 2 (AAV). The hybrid AAV-B19 genome was rescued from a recombinant plasmid and then the DNA was replicated upon transfection into adenovirus 2-infected human KB cells in the presence of AAV genes coding for proteins required for AAV DNA replication (AAV-Rep proteins). In addition, in the presence of AAV genes coding for the viral capsid proteins (AAV-Cap proteins), the rescued/replicated hybrid AAV-B19 genomes were packed into mature AAV progeny virions, which were subsequently released into culture supernatants. The recombinant AAV-B19 progeny virions were infectious for normal human bone marrow cells and strongly suppressed erythropoiesis in vitro. The availability of an infectious recombinant B19 virus should facilitate the mutational analysis of the viral genome, which, in turn, may yield information on individual viral gene functions in B19-induced pathogenesis. The hybrid AAV-B19 genome may also prove to be a useful vector for gene transfer in human bone marrow cells.     

Human cytomegalovirus IE1 and IE2 proteins are mutagenic and mediate “hit-and-run” oncogenic transformation in cooperation with the adenovirus E1A proteins

Some epidemiological studies have suggested a possible link between human cytomegalovirus (HCMV) infection and various malignancies, and HCMV has been shown to transform cultured cells. However, viral DNA is not detected in most transformants, and the mechanism by which HCMV might contribute to oncogenesis has remained obscure. Here we show that the HCMV immediate early 1 and 2 genes can cooperate with the adenovirus E1A gene to generate transformed foci of primary baby rat kidney cells. HCMV gene expression is transient and viral DNA is not present in clonal cell lines derived from the transformed foci. We find that the HCMV immediate early proteins are mutagenic, and we propose that HCMV has the potential to contribute to oncogenesis through a “hit-and-run” mechanism, by inducing mutations in cellular genes.     

RNase P Ribozymes Inhibit the Replication of Human Cytomegalovirus by Targeting Essential Viral Capsid Proteins

An engineered RNase P-based ribozyme variant, which was generated using the in vitro selection procedure, was used to target the overlapping mRNA region of two proteins essential for human cytomegalovirus (HCMV) replication: capsid assembly protein (AP) and protease (PR). In vitro studies showed that the generated variant, V718-A, cleaved the target AP mRNA sequence efficiently and its activity was about 60-fold higher than that of wild type ribozyme M1-A. Furthermore, we observed a reduction of 98%–99% in AP/PR expression and an inhibition of 50,000 fold in viral growth in cells with V718-A, while a 75% reduction in AP/PR expression and a 500-fold inhibition in viral growth was found in cells with M1-A. Examination of the antiviral effects of the generated ribozyme on the HCMV replication cycle suggested that viral DNA encapsidation was inhibited and as a consequence, viral capsid assembly was blocked when the expression of AP and PR was inhibited by the ribozyme. Thus, our study indicates that the generated ribozyme variant is highly effective in inhibiting HCMV gene expression and blocking viral replication, and suggests that engineered RNase P ribozyme can be potentially developed as a promising gene-targeting agent for anti-HCMV therapy.     

A method for identifying the viral genes required for herpesvirus DNA replication.

Several laboratories have shown that transfected plasmid DNAs containing either of the two known origins of herpes simplex virus (HSV) DNA replication, oriS or oriL, are replicated in HSV-1-infected cells or in cells cotransfected with virion DNA. I have found that HSV-1 (KOS) DNA digested to completion with the restriction enzyme Xba I is as efficient as intact viral DNA in supporting the in vivo replication of cotransfected plasmids containing oriS. On the basis of this result, several of the Xba I restriction fragments of HSV-1 DNA were cloned into the plasmid vector pUC19, and combinations of cloned DNAs were tested for their ability to supply the trans-acting functions required for HSV origin-dependent replication. A combination of five cloned fragments of HSV-1 can supply all of the necessary functions: Xba I C (coordinates 0.074-0.294), Xba I F (coordinates 0.294-0.453), Xba I E (coordinates 0.453-0.641), Xba I D (coordinates 0.641-0.830), and EcoRI JK (coordinates 0.0-0.086; 0.830-0.865). Transient plasmid replication in this system is dependent on the presence of either oriS or oriL in cis. The plasmid containing Xba I F can be replaced by two smaller plasmids, one of which contains only the gene for the HSV-encoded DNA polymerase, and the other of which contains only the gene for the major DNA binding protein (ICP8). Thus, plasmid DNA replication in this system depends on two of the genes known from genetic studies to be essential for viral DNA replication in infected cells. This system defines a simple complementation assay for cloned fragments of HSV DNA that contain other genes involved in viral DNA replication and should lead to the rapid identification of all such genes.     

Human cytomegalovirus strain Toledo lacks a virus-encoded tropism factor required for infection of aortic endothelial cells.

Human cytomegalovirus (HCMV) strains were investigated to identify those with altered tropism for endothelial cells. In viral replication kinetics analysis, HCMV strain Toledo replicated poorly in aortic endothelial cells (AECs), and the virus count was reduced by 2-3 log units, in comparison with strain AD169. Virus entry at the cell surface for each strain was equivalent. However, immunofluorescence studies revealed a lack of immediate early viral antigen 72 expression, and direct blot hybridization failed to detect viral genomes in the nucleus of Toledo-infected AECs. Complementation assays were done to determine whether endothelial cell infectivity was dependent on a virus-encoded tropism factor. Pseudotype virus in endothelial cell cultures indicated that AD169 could provide trans factors to rescue Toledo during infection of endothelial cells. Collectively, these results show that a viral function provides an endothelial cell tropism factor for HCMV and plays a role during postentry infection events.     

Cytomegalovirus infection and trans-activation of HIV-1 and HIV-2 LTRs in human astrocytoma cells

Susceptibility of a human astrocytoma cell line to human cytomegalovirus (HCMV) infection was investigated. Infection of U-373MG astrocytoma cells with two strains of HCMV resulted in both production of extracellular, infectious virus and expression of immediate early and early antigens within 18 hours and late antigens after 72 hours of infection. The kinetics of infection in U-373MG cells were the same as in human diploid fibroblasts (MRC-5). Since HCMV and human immunodeficiency virus (HIV) have reportedly been found in astrocytic cells in vivo, we studied the possible interaction between HCMV and HIV long terminal repeat (LTR) elements in this cellular environment. HCMV infection transactivated the LTR of HIV-1 and HIV-2 to similar levels. Interestingly, transfection of these cells with infectious HIV-1 provirus did not result in expression of gag, env, or F proteins detectable by immunofluorescence. However, provirus gene expression was not completely silent, since it transactivated HIV-1 LTR. The level of this transactivation was similar to that seen following cotransfection with a tat expression vector. These results suggest that opportunistic infection with HCMV may reactivate latent HIV genomes in glial cells.     

Cloning human herpes virus 6A genome into bacterial artificial chromosomes and study of DNA replication intermediates

Cloning of large viral genomes into bacterial artificial chromosomes (BACs) facilitates analyses of viral functions and molecular mutagenesis. Previous derivations of viral BACs involved laborious recombinations within infected cells. We describe a single-step production of viral BACs by direct cloning of unit length genomes, derived from circular or head-to-tail concatemeric DNA replication intermediates. The BAC cloning is independent of intracellular recombinations and DNA packaging constraints. We introduced the 160-kb human herpes virus 6A (HHV-6A) genome into BACs by digesting the viral DNA replicative intermediates with the Sfil enzyme that cleaves the viral genome in a single site. The recombinant BACs contained also the puromycin selection gene, GFP, and LoxP sites flanking the BAC sequences. The HHV-6A-BAC vectors were retained stably in puromycin selected 293T cells. In the presence of irradiated helper virus, supplying most likely proteins enhancing gene expression they expressed early and late genes in SupT1 T cells. The method is especially attractive for viruses that replicate inefficiently and for viruses propagated in suspension cells. We have used the fact that the BAC cloning “freezes” the viral DNA replication intermediates to analyze their structure. The results revealed that HHV-6A-BACs contained a single direct repeat (DR) rather than a DR-DR sequence, predicted to arise by circularization of parental genomes with a DR at each terminus. HHV-6A DNA molecules prepared from the infected cells also contained DNA molecules with a single DR. Such forms were not previously described for HHV-6 DNA.     

Polyoma virus early-late switch: regulation of late RNA accumulation by DNA replication.

Early in infection of permissive mouse cells, messages from the early region of the polyoma virus genome accumulate preferentially over those from the late region. After initiation of DNA replication, the balance between early and late gene expression is reversed in favor of the late products. In previous work from our laboratory, we showed that viral early proteins do not activate the polyoma late promoter in the absence of DNA replication. Here we show that activation of the late genes in replication-incompetent viral genomes can occur if actively replicating genomes are present in the same cell. A low level of DNA replication, however, is insufficient to induce the early-late switch. Furthermore, replication-competent genomes that fail to accumulate late RNA molecules are defective in the transactivation of replication-incompetent genomes. We suggest that titration of an unknown diffusible factor(s) after DNA replication relieves the block to late RNA accumulation seen in the early phase, with most of this titration being attributable to late-strand RNA molecules themselves.     

A conditionally replicating HIV-1 vector interferes with wild-type HIV-1 replication and spread.

Defective-interfering viruses are known to modulate virus pathogenicity. We describe conditionally replicating HIV-1 (crHIV) vectors that interfere with wild-type HIV-1 (wt-HIV) replication and spread. crHIV vectors are defective-interfering HIV genomes that do not encode viral proteins and replicate only in the presence of wt-HIV helper virus. In cells that contain both wt-HIV and crHIV genomes, the latter are shown to have a selective advantage for packaging into progeny virions because they contain ribozymes that cleave wt-HIV RNA but not crHIV RNA. A crHIV vector containing a triple anti-U5 ribozyme significantly interferes with wt-HIV replication and spread. crHIV vectors are also shown to undergo the full viral replicative cycle after complementation with wt-HIV helper-virus. The application of defective interfering crHIV vectors may result in competition with wt-HIVs and decrease pathogenic viral loads in vivo.