Human cytomegalovirus (HCMV) infection leads to dysregulation of multiple cell cycle-regulatory proteins.In this study, we examined the effects of inhibition of cyclin-dependent kinase (cdk) activity on viral replication. With the drug Roscovitine, a specific inhibitor of cyclin-dependent kinases 1, 2, 5, 7, and 9, we have shown that during the first 6 h of infection, cyclin-dependent kinase-dependent events occurred that included the regulated processing and accumulation of the immediate-early (IE) UL122-123 transcripts and UL36-37 transcripts. Altered processing of UL122-123 led to a loss of IE1-72 and an increase in IE2-86. The ratio of spliced to unspliced UL37 transcripts also changed. These effects did not require de novo protein synthesis or degradation of proteins by the proteasome. Addition of Roscovitine at the beginning of the infection was also associated with inhibition of expression of selected viral early gene products, viral DNA replication, and late viral gene expression. When Roscovitine was added after the first 6 h of infection, the effects on IE gene expression were no longer observed and viral replication proceeded through the late phase, but viral titers were reduced. The reduction in viral titer was observed even when Roscovitine was first added at 48 h postinfection, indicating that cyclin-dependent kinase activity is required at both IE and late times. Flavopiridol, another specific inhibitor of cyclin-dependent kinases, had similar effects on IE and early gene expression. These results underscore the importance of accurate RNA processing and reiterate the significant role of cell cycle-regulatory factors in HCMV infection.Human cytomegalovirus (HCMV), a member of the family Herpesviridae, is the major viral cause of birth defects and is associated with significant morbidity in immunocompromised individuals (52). Its genome is 230 kbp and has the capacity to encode approximately 150 open reading frames (19,44). HCMV cell entry, gene expression, replication, and virion maturation are multilayered processes that require many viral as well as cellular factors during a productive infection. There are three major phases of viral gene expression. Viral immediateearly (IE) genes are the first to be expressed in an infected cell, and their transcription requires no de novo protein synthesis. A major site of IE transcription includes two genetic units, IE1 and IE2 (for review, see references 27 and 44) (Fig. 1B). The predominant IE RNA (IE1) consists of four exons; a single open reading frame (UL123) initiates in exon 2 and specifies a 72-kDa nuclear protein designated IE1-72. The major IE2 gene product, IE2-86 (open reading frame UL122), is an 86-kDa protein that is encoded by an alternatively spliced RNA that contains the first three exons of IE1 and a different terminal exon.IE1-72 and IE2-86 are essential transactivators of both early and late viral gene expression. Included in the early class are genes that are important for viral DNA replication. Some examples are the UL112-113 nuclear pho...
Using bacterial artificial chromosome (BAC) technology, we have constructed and characterized a human cytomegalovirus recombinant virus with a mutation in the exon specific for the major immediate-early region 2 (IE2) gene product. The resulting IE2 86-kDa protein (IE2 86) has an internal deletion of amino acids 136 to 290 and is fused at the carboxy terminus to enhanced green fluorescent protein (EGFP). The deletion also removes the promoter and initiator methionine for the p40 form of IE2 and initiator methionine for the p60 form of the protein, and therefore, these late gene products are not produced. The mutant virus IE2 86⌬SX-EGFP is viable but exhibits altered growth characteristics in tissue culture compared with a full-length wild-type (wt) IE2 86-EGFP virus or a revertant virus. When cells are infected with the mutant virus at a low multiplicity of infection (MOI), there is a marked delay in the production of infectious virus. This is associated with slower cell-to-cell spread of the virus. By immunofluorescence and Western blot analyses, we show that the early steps in the replication of the mutant virus are comparable to those for the wt. Although there is significantly less IE2 protein in the cells infected with the mutant, there is only a modest lag in the initial accumulation of IE1 72 and viral early proteins, and viral DNA replication proceeds normally. The mutation also has only a small effect on the synthesis of the viral major capsid protein. The most notable molecular defect in the mutant virus infection is that the steady-state levels of the pp65 (UL83) and pp28 (UL99) matrix proteins are greatly reduced. In the case of UL83, but not UL99, there is also a corresponding decrease in the amount of mRNA present in cells infected with the mutant virus.Human cytomegalovirus (HCMV) is a common pathogen that is the leading viral cause of birth defects (49). HCMV infection also results in significant morbidity and mortality in immunosuppressed individuals and may be one of the factors contributing to atherosclerosis and restenosis following coronary angioplasty (74).The survival of HCMV and its ability to establish both acute and latent infections depend on a complex set of interactions between the virus and the host cell machinery that optimize the environment for viral replication (3, 16). During the productive infection, there are three major phases of gene expression. The immediate-early (IE) genes are transcribed after viral entry and rely mainly on host factors for their expression, although some input virion proteins contribute to their activation. Early genes are synthesized prior to viral DNA replication, and their expression requires one or more viral IE gene products. Included in this early class are viral proteins required to "activate" the cell to a metabolic state most conducive for viral DNA synthesis as well as proteins involved in the replication process itself (for a review, see reference 16). Concurrent with these effects on cellular metabolism, viral DNA synthesis begins at ϳ18 h ...
There is a clear need for novel, effective therapeutic approaches to hemorrhagic fever due to filoviruses. Ebola virus hemorrhagic fever is associated with robust interferon (IFN)-α production, with plasma concentrations of IFN-α that greatly (60- to 100-fold) exceed those seen in other viral infections, but little IFN-β production. While all of the type I IFNs signal through the same receptor complex, both quantitative and qualitative differences in biological activity are observed after stimulation of the receptor complex with different type I IFNs. Taken together, this suggested potential for IFN-β therapy in filovirus infection. Here we show that early postexposure treatment with IFN-β significantly increased survival time of rhesus macaques infected with a lethal dose of Ebola virus, although it failed to alter mortality. Early treatment with IFN-β also significantly increased survival time after Marburg virus infection. IFN-β may have promise as an adjunctive postexposure therapy in filovirus infection.
The mechanisms of Ebola (EBOV) pathogenesis are only partially understood, but the dysregulation of normal host immune responses (including destruction of lymphocytes, increases in circulating cytokine levels, and development of coagulation abnormalities) is thought to play a major role. Accumulating evidence suggests that much of the observed pathology is not the direct result of virus-induced structural damage but rather is due to the release of soluble immune mediators from EBOV-infected cells. It is therefore essential to understand how the candidate therapeutic may be interrupting the disease process and/or targeting the infectious agent. To identify genetic signatures that are correlates of protection, we used a DNA microarray-based approach to compare the host genome-wide responses of EBOV-infected nonhuman primates (NHPs) responding to candidate therapeutics. We observed that, although the overall circulating immune response was similar in the presence and absence of coagulation inhibitors, surviving NHPs clustered together. Noticeable differences in coagulation-associated genes appeared to correlate with survival, which revealed a subset of distinctly differentially expressed genes, including chemokine ligand 8 (CCL8/MCP-2), that may provide possible targets for early-stage diagnostics or future therapeutics. These analyses will assist us in understanding the pathogenic mechanisms of EBOV infection and in identifying improved therapeutic strategies.
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