Betaherpesvirus-conserved cytomegalovirus tegument protein ppUL32 (pp150) controls cytoplasmic events during virion maturation.

The UL32 gene of human cytomegalovirus (CMV) encodes a prominent betaherpesvirus-conserved virion tegument protein, called pp150 (basic phosphoprotein/ppUL32), that accumulates within a cytoplasmic inclusion adjacent to the nucleus at late times during infection. Using a UL32 deletion mutant (DeltaUL32-BAC) (where BAC is bacterial artificial chromosome), we demonstrate that pp150 is critical for virion maturation in the cytoplasmic compartment. Cotransfection of a pp150 expression plasmid with DeltaUL32-BAC DNA led to complementation of the replication defect with focus formation due to secondary spread. Deletion of the amino terminus of pp150 or disruption of the betaherpesvirus conserved regions, CR1 and CR2, revealed these regions to be critical for replication. In contrast, deletion of the carboxyl terminus only partially compromised maturation while disruption of glycosylation sites had no effect. An African green monkey CMV UL32 homolog complemented DeltaUL32-BAC replication but murine CMV M32 failed to complement, consistent with evolutionary divergence of rodent and primate cytomegaloviruses. Infection with DeltaUL32-BAC showed normal expression of all kinetic classes of viral genes and replication of viral DNA, with accumulation of viral DNA-containing particles in the cytoplasm; however, mutant virus did not spread to adjacent cells. In contrast to this block in virion infectivity, cell-to-cell transfer of pp65-containing particles was observed, suggesting that release of dense bodies continued in the absence of pp150. These observations demonstrate that pp150 is critical for virion egress, possibly at the stage of final envelopment.

Mitochondrial cell death suppressors carried by human and murine cytomegalovirus confer resistance to proteasome inhibitor-induced apoptosis.

Human cytomegalovirus carries a mitochondria-localized inhibitor of apoptosis (vMIA) that is conserved in primate cytomegaloviruses. We find that inactivating mutations within UL37x1, which encodes vMIA, do not substantially affect replication in TownevarATCC (Towne-BAC), a virus that carries a functional copy of the betaherpesvirus-conserved viral inhibitor of caspase 8 activation, the UL36 gene product. In Towne-BAC infection, vMIA reduces susceptibility of infected cells to intrinsic death induced by proteasome inhibition. vMIA is sufficient to confer resistance to proteasome inhibition when expressed independent of viral infection. Murine cytomegalovirus m38.5, whose position in the viral genome is analogous to UL37x1, exhibits mitochondrial association and functions in much the same manner as vMIA in inhibiting intrinsic cell death. This work suggests a common role for vMIA in rodent and primate cytomegaloviruses, modulating the threshold of virus-infected cells to intrinsic cell death.

Biosynthetic labeling of RNA with uracil phosphoribosyltransferase allows cell-specific microarray analysis of mRNA synthesis and decay.

Standard microarrays measure mRNA abundance, not mRNA synthesis, and therefore cannot identify the mechanisms that regulate gene expression. We have developed a method to overcome this limitation by using the salvage enzyme uracil phosphoribosyltransferase (UPRT) from the protozoan Toxoplasma gondii. T. gondii UPRT has been well characterized because of its application in monitoring parasite growth: mammals lack this enzyme activity and thus only the parasite incorporates (3)H-uracil into its nucleic acids. In this study we used RNA labeling by UPRT to determine the roles of mRNA synthesis and decay in the control of gene expression during T. gondii asexual development. We also used this approach to specifically label parasite RNA during a mouse infection and to incorporate thio-substituted uridines into the RNA of human cells engineered to express T. gondii UPRT, indicating that engineered UPRT expression will allow cell-specific analysis of gene expression in organisms other than T. gondii.

The promyelocytic leukemia protein does not mediate foamy virus latency in vitro.

Spumaviruses, commonly called foamy viruses, are complex retroviruses that establish life-long persistent infections in the absence of accompanying pathology. Depending upon cell type, infection of cells in tissue culture cells can result in either lytic replication, persistence, or latency. The cellular factors that mediate foamy virus (FV) latency are poorly understood. In this study we show that the only known inhibitor of FV replication, the promyelocytic leukemia protein (PML), which binds the FV transactivator (Tas), does not play an important role in FV latency in vitro. We found no significant differences in PML levels in cells that supported lytic replication compared to those that were latently infected. Furthermore, endogenous PML levels did not change following exposure to phorbol myristate acetate (PMA), which induces FV replication. We demonstrated that FV replication proceeded in the presence of substantial levels of PML, both in fully permissive cells and during reactivation of latent FV. Endogenous PML did not efficiently colocalize with Tas, even after upregulation by alpha interferon (IFN-alpha) treatment. IFN-alpha did, however, partially suppress the reactivation of latent FV by PMA. Finally, depletion of endogenous PML by small interfering RNA did not promote activation of FV in cells that responded to PMA treatment. Taken together, these data indicate that endogenous PML does not play an important role in mediating FV latency.

Reactivation of a complex retrovirus is controlled by a molecular switch and is inhibited by a viral protein.

Spumaviruses, commonly called foamy viruses (FV), are complex retroviruses that establish lifelong persistent infections without any accompanying pathologies. In tissue culture, cells can be either lytically or latently infected, depending on cell type. Regulation of FV replication is controlled by two promoters: the LTR and a second promoter within the env gene termed the internal promoter (IP). The IP directs expression of the transcriptional activator, Tas, and a second accessory protein, Bet, whose function has been elusive. In this study, we report that expression of exogenous Tas is sufficient to initiate a switch from latent to lytic replication. We also show that treatment with the phorbol ester phorbol 12-myristate 13-acetate (PMA) can lead to an increase in transcription from the IP, and that Bet protein expression abrogates this effect. Finally, we demonstrate that Bet expression severely limits the ability of PMA to activate transcription of latent FV genomes, and that replication of a Bet(-) virus is more easily activated than wild-type FV. Taken together, these data suggest that viral transcription is regulated by a sensitive switch, and that Bet functions as a negative regulator of basal IP activity.