Stimulatory effects of human cytomegalovirus tegument protein pp71 lead to increased expression of CCL2 (monocyte chemotactic protein-1) during infection.

Human cytomegalovirus (CMV) is the most common infectious cause of congenital birth defects in developed countries. Studies of infected amniotic fluid and placentae show CMV infection leads to a pro-inflammatory shift in cytokine profiles with implications for pathogenesis of foetal disease. ELISA, immunofluorescence and real-time-PCR assays were used to investigate CCL2 (monocyte chemotactic protein-1) and TNF-α changes following CMV infection of human fibroblasts, as well as following transient expression of CMV gene products in HeLa cells. Infection of human fibroblasts with CMV AD169 resulted in increased cytoplasmic and extracellular expression of CCL2 during early stages of infection, followed by marked downregulation of the chemokine at late times. Induction of CCL2 was not observed with CMV clinical strain Merlin, consistent with the postulated immune-evasion potential of this genetically intact WT strain. Comparison between live and UV-irradiated virus infections showed that changes in CCL2 levels were a direct response to active CMV replication. There were no significant changes in TNF-α expression during a parallel time-course of CMV infection. In transient transfection assays, overexpression of CMV tegument protein pp71 resulted in intracellular and extracellular upregulation of CCL2 protein. mRNA analysis showed that pp71-induced elevation in CCL2 was mediated through transcriptional upregulation. The data showed that CMV-induced upregulation of CCL2 during early stages of infection was mediated, at least in part, by stimulation of viral pp71, which may contribute to viral pathogenesis through enhanced virus dissemination.

Proteomic analysis of the multimeric nuclear egress complex of human cytomegalovirus.

Herpesviral capsids are assembled in the host cell nucleus before being translocated into the cytoplasm for further maturation. The crossing of the nuclear envelope represents a major event that requires the formation of the nuclear egress complex (NEC). Previous studies demonstrated that human cytomegalovirus (HCMV) proteins pUL50 and pUL53, as well as their homologs in all members of Herpesviridae, interact with each other at the nuclear envelope and form the heterodimeric core of the NEC. In order to characterize further the viral and cellular protein content of the multimeric NEC, the native complex was isolated from HCMV-infected human primary fibroblasts at various time points and analyzed using quantitative proteomics. Previously postulated components of the HCMV-specific NEC, as well as novel potential NEC-associated proteins such as emerin, were identified. In this regard, interaction and colocalization between emerin and pUL50 were confirmed by coimmunoprecipitation and confocal microscopy analyses, respectively. A functional validation of viral and cellular NEC constituents was achieved through siRNA-mediated knockdown experiments. The important role of emerin in NEC functionality was demonstrated by a reduction of viral replication when emerin expression was down-regulated. Moreover, under such conditions, reduced production of viral proteins and deregulation of viral late cytoplasmic maturation were observed. Combined, these data prove the functional importance of emerin as an NEC component, associated with pUL50, pUL53, pUL97, p32/gC1qR, and further regulatory proteins. Summarized, our findings provide the first proteomics-based characterization and functional validation of the HCMV-specific multimeric NEC.

The cytomegalovirus egress proteins pUL50 and pUL53 are translocated to the nuclear envelope through two distinct modes of nuclear import.

The nucleocytoplasmic export of cytomegaloviral capsids is regulated by formation of a multi-component nuclear egress complex (NEC), essentially based on viral proteins pUL50 and pUL53. In this study, the generation of recombinant human cytomegaloviruses, expressing tagged versions of pUL50 and pUL53, enabled the investigation of NEC formation in infected primary fibroblasts. For these recombinant viruses, a wild-type-like mode of pUL50-pUL53 interaction and recruitment of both proteins to the nuclear envelope could be demonstrated. Importantly, pUL50 was translocated from an initial cytoplasmic distribution to the nuclear rim, whereas pUL53 accumulated in the nucleus before attaining overall rim colocalization with pUL50. Specified experimental settings illustrated that pUL50 and pUL53 were subject to different pathways of intracellular trafficking. Importantly, a novel nuclear localization signal (NLS) could be identified and functionally verified for pUL53 (amino acids 18-27), whereas no NLS was present in pUL50. Analysis of amino acid replacement mutants further illustrated the differential modes of nuclear import of the two essential viral egress proteins. Taken together, our findings suggest a combination of classical nuclear import (pUL53) and interaction-mediated recruitment (pUL50) as the driving forces for core NEC formation and viral nuclear egress.