Divergent effects of human cytomegalovirus and herpes simplex virus-1 on cellular metabolism.

Viruses rely on the metabolic network of the host cell to provide energy and macromolecular precursors to fuel viral replication. Here we used mass spectrometry to examine the impact of two related herpesviruses, human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1), on the metabolism of fibroblast and epithelial host cells. Each virus triggered strong metabolic changes that were conserved across different host cell types. The metabolic effects of the two viruses were, however, largely distinct. HCMV but not HSV-1 increased glycolytic flux. HCMV profoundly increased TCA compound levels and flow of two carbon units required for TCA cycle turning and fatty acid synthesis. HSV-1 increased anapleurotic influx to the TCA cycle through pyruvate carboxylase, feeding pyrimidine biosynthesis. Thus, these two related herpesviruses drive diverse host cells to execute distinct, virus-specific metabolic programs. Current drugs target nucleotide metabolism for treatment of both viruses. Although our results confirm that this is a robust target for HSV-1, therapeutic interventions at other points in metabolism might prove more effective for treatment of HCMV.

Experimental human cytomegalovirus latency in CD14+ monocytes.

CD14(+) monocytes are a reservoir for latent human cytomegalovirus, and virus replication is reactivated during their differentiation to macrophages or dendritic cells. It has not been clear whether the virus can establish latency upon direct infection of monocytes or whether it must first become quiescent in a progenitor cell that subsequently differentiates to generate a monocyte. We report that infection of primary human monocytes with a clinical strain of human cytomegalovirus exhibits the hallmarks of latency. We established conditions for culturing monocytes that prevent differentiation for at least 25 d, as evidenced by cell surface marker expression. Infection of these monocytes with the FIX clinical strain resulted in transient accumulation of many viral lytic RNAs and sustained expression of four previously described latency-associated transcripts. The amount of viral DNA remained constant after infection, and cell surface and total HLA-DR proteins were substantially reduced on a continuing basis after infection. When treated with cytokine mixtures that stimulate differentiation to a macrophage or dendritic cell phenotype, infected monocytes reactivated virus replication and produced infectious progeny. Treatment of infected monocytes with IL-6 alone also was sufficient for reactivation, and the particles produced after exposure to this cytokine were about fivefold more infectious than virions produced by other treatments. We propose that in vivo microenvironments influence not only the efficiency of reactivation but also the infectivity of the virions produced from latently infected monocytes.

Human cytomegalovirus UL69 protein facilitates translation by associating with the mRNA cap-binding complex and excluding 4EBP1.

4EBP1 is phosphorylated by the mTORC1 kinase. When mTORC1 activity is inhibited, hypophosphorylated 4EBP1 binds and sequesters eIF4E, a component of the mRNA cap-binding complex, and blocks translation. As a consequence, mTORC1 activity is needed to maintain active translation. The human cytomegalovirus pUL38 protein preserves mTORC1 activity, keeping most of the E4BP1 in the infected cell in a hyperphosphorylated, inactive state. Here we report that a second viral protein, pUL69, also antagonizes the activity of 4EBP1, but by a separate mechanism. pUL69 interacts directly with eIF4A1, an element of the cap-binding complex, and the poly(A)-binding protein, which binds to the complex. When pUL69 accumulates during infection with wild-type virus, 4EBP1 is excluded from the complex. However, 4EBP1 is present in the cap-binding complex after infection with a pUL69-deficient virus, coincident with reduced accumulation of several late virus-coded proteins. We propose that pUL69 supports translation in human cytomegalovirus-infected cells by excluding hypophosphorylated 4EBP1 from the cap-binding complex.

Functional genetic analysis of rhesus cytomegalovirus: Rh01 is an epithelial cell tropism factor.

Rhesus cytomegalovirus (RhCMV) is an emerging model for human cytomegalovirus (HCMV) pathogenesis that facilitates experimental CMV infection of a natural primate host closely related to humans. We have generated a library of RhCMV mutants with lesions in genes whose HCMV orthologues have been characterized as nonessential for replication in human fibroblasts, and we characterized their replication in rhesus fibroblasts and epithelial cells. The RhCMV mutants grew well in fibroblasts, as predicted by earlier studies with HCMV. However, mutations in four genes caused replication defects in rhesus retinal pigment epithelial cells: Rh01 (an HCMV TRL1 orthologue), Rh159 (HCMV UL148), Rh160 (HCMV UL132), and Rh203 (HCMV US22). Growth of the Rh01-deficient mutant was examined in detail. After entry into epithelial cells, the mutant expressed representative viral proteins, accumulated viral DNA, and generated infectious virus, but it failed to spread efficiently. We conclude that Rh01 is a cell tropism determinant that has the potential to dramatically affect virus spread and pathogenesis.

Reevaluation of human cytomegalovirus coding potential.

The Bio-Dictionary-based Gene Finder was used to reassess the coding potential of the AD169 laboratory strain of human cytomegalovirus and sequences in the Toledo strain that are missing in the laboratory strain of the virus. The gene-finder algorithm assesses the potential of an ORF to encode a protein based on matches to a database of amino acid patterns derived from a large collection of proteins. The algorithm was used to score all human cytomegalovirus ORFs with the potential to encode polypeptides >/=50 aa in length. As a further test for functionality, the genomes of the chimpanzee, rhesus, and murine cytomegaloviruses were searched for orthologues of the predicted human cytomegalovirus ORFs. The analysis indicates that 37 previously annotated ORFs ought to be discarded, and at least nine previously unrecognized ORFs with relatively strong coding potential should be added. Thus, the human cytomegalovirus genome appears to contain approximately 192 unique ORFs with the potential to encode a protein. Support for several of the predictions of our in silico analysis was obtained by sequencing several domains within a clinical isolate of human cytomegalovirus.

Construction of a rationally designed human cytomegalovirus variant encoding a temperature-sensitive immediate-early 2 protein.

We generated a set of cysteine-to-glycine mutations and screened them to identify a temperature-sensitive allele of the human cytomegalovirus UL122 gene, which encodes the immediate-early 2 transcriptional activating protein. The mutant allele contains a single base pair substitution at amino acid 510. In transcription activation assays, the mutant protein activated the simian virus 40 early and human cytomegalovirus UL112 promoters at 32.5 degrees C but not at 39.5 degrees C. We constructed a mutant virus, BTNtsUL122, in which the wild-type UL122 locus is substituted with the mutant allele. The mutant produced progeny at 32.5 degrees C but not at 39.5 degrees C. Although the mutant virus accumulated immediate-early transcripts and proteins at the nonpermissive temperature, it did not produce any early (UL44 and UL54) and late (UL82) transcripts and it did not replicate its DNA. The mutant's defect at the nonpermissive temperature results, at least in part, from the inability of the temperature-sensitive immediate-early 2 protein to activate early viral promoters, whose products are required for DNA replication and progression into the late phase of the virus growth cycle.

Murine cytomegalovirus m02 gene family protects against natural killer cell-mediated immune surveillance.

The murine cytomegalovirus m02 gene family encodes putative type I membrane glycoproteins named m02 through m16. A subset of these genes were fused to an epitope tag and cloned into an expression vector. In transfected and murine cytomegalovirus-infected cells, m02, m04, m05, m06, m07, m09, m10, and m12 localized to cytoplasmic structures near the nucleus, whereas m08 and m13 localized to a filamentous structure surrounding the nucleus. Substitution mutants lacking the m02 gene (SMsubm02) or the entire m02 gene family (SMsubm02-16) grew like their wild-type parent in cultured cells. However, whereas SMsubm02 was as pathogenic as the wild-type virus, SMsubm02-16 was markedly less virulent. SMsubm02-16 produced less infectious virus in most organs compared to wild-type virus in BALB/c and C57BL/6J mice, but it replicated to wild-type levels in the organs of immunodeficient gamma(c)/Rag2 mice, lacking multiple cell types including natural killer cells, and in C57BL/6J mice depleted of natural killer cells. These results argue that one or more members of the m02 gene family antagonize natural killer cell-mediated immune surveillance.

Inhibition of cyclooxygenase 2 blocks human cytomegalovirus replication.

Cyclooxygenase 2 (COX-2) mRNA, protein, and activity are transiently induced after infection of human fibroblasts with human cytomegalovirus. Prostaglandin E(2), the product of COX-2 activity, is transiently increased by a factor of >50 in cultures of virus-infected fibroblasts. Both specific (BMS-279652, 279654, and 279655) and nonspecific (indomethacin) COX-2 inhibitors can abrogate the virus-mediated induction of prostaglandin E(2) accumulation. Levels of COX-2 inhibitors that completely block the induction of COX-2 activity, but do not compromise cell viability, reduce the yield of human cytomegalovirus in human fibroblasts by a factor of >100. Importantly, the yield of infectious virus can be substantially restored by the addition of prostaglandin E(2) together with the inhibitory drug. This finding argues that elevated levels of prostaglandin E(2) are required for efficient replication of human cytomegalovirus in fibroblasts. COX-2 inhibitors block the accumulation of immediate-early 2 mRNA and protein, but have little effect on the levels of immediate-early 1 mRNA and protein. Viral DNA replication and the accumulation of some, but not all, early and late mRNAs are substantially blocked by COX-2 inhibitors. Elevated levels of prostaglandin E(2) apparently facilitate the production of immediate-early 2 protein. The failure to produce normal levels of this critical viral regulatory protein in the presence of COX-2 inhibitors might block normal progression beyond the immediate-early phase of human cytomegalovirus infection.

Coding potential of laboratory and clinical strains of human cytomegalovirus.

Six strains of human cytomegalovirus have been sequenced, including two laboratory strains (AD169 and Towne) that have been extensively passaged in fibroblasts and four clinical isolates that have been passaged to a limited extent in the laboratory (Toledo, FIX, PH, and TR). All of the sequenced viral genomes have been cloned as infectious bacterial artificial chromosomes. A total of 252 ORFs with the potential to encode proteins have been identified that are conserved in all four clinical isolates of the virus. Multiple sequence alignments revealed substantial variation in the amino acid sequences encoded by many of the conserved ORFs.