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Characterization of conserved region 2-deficient mutants of the cytomegalovirus egress protein pM53.

ABSTRACT: Dominant-negative (DN) mutants are powerful tools for studying essential protein-protein interactions. A systematic genetic screen of the essential murine cytomegalovirus (MCMV) protein pM53 identified the accumulation of inhibitory mutations within conserved region 2 (CR2) and CR4. The strong inhibitory potential of these CR4 mutants is characterized by a particular phenotype. The DN effect of the small insertion mutations in CR2 was too weak to analyze (M. Popa, Z. Ruzsics, M. Lötzerich, L. Dölken, C. Buser, P. Walther, and U. H. Koszinowski, J. Virol. 84:9035-9046, 2010); therefore, the present study describes the construction of M53 alleles lacking CR2 (either completely or partially) and subsequent examination of the DN effect on MCMV replication upon conditional expression. Overexpression of CR2-deficient pM53 inhibited virus production by about 10,000-fold. This was due to interference with capsid export from the nucleus and viral genome cleavage/packaging. In addition, the fate of the nuclear envelopment complex in the presence of DN pM53 overexpression was analyzed. The CR2 mutants were able to bind to pM50, albeit to a lesser extent than the wild-type protein, and relocalized the wild-type nuclear envelope complex in infected cells. Unlike the CR4 DN, the CR2 DN mutants did not affect the stability of pM50.

SUBMITTER: Pogoda M

PROVIDER: S-EPMC3497646 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Characterization of conserved region 2-deficient mutants of the cytomegalovirus egress protein pM53.

Pogoda Madlen M Bosse Jens B JB Wagner Felicia M FM Schauflinger Martin M Walther Paul P Koszinowski Ulrich H UH Ruzsics Zsolt Z

Journal of virology 20120919 23

Dominant-negative (DN) mutants are powerful tools for studying essential protein-protein interactions. A systematic genetic screen of the essential murine cytomegalovirus (MCMV) protein pM53 identified the accumulation of inhibitory mutations within conserved region 2 (CR2) and CR4. The strong inhibitory potential of these CR4 mutants is characterized by a particular phenotype. The DN effect of the small insertion mutations in CR2 was too weak to analyze (M. Popa, Z. Ruzsics, M. Lötzerich, L. Dö ...[more]

PMID: 22993161

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Project description:UnlabelledHuman cytomegalovirus (HCMV) pUL93 and pUL77 are both essential for virus growth, but their functions in the virus life cycle remain mostly unresolved. Homologs of pUL93 and pUL77 in herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV) are known to interact to form a complex at capsid vertices known as the capsid vertex-specific component (CVSC), which likely stabilizes nucleocapsids during virus maturation and also aids in nuclear egress. In herpesviruses, nucleocapsids assemble and partially mature in nuclear replication compartments and then travel to the inner nuclear membrane (INM) for nuclear egress. The factors governing the recruitment of nucleocapsids to the INM are not known. Kinetic analysis of pUL93 demonstrates that this protein is expressed late during infection and localizes primarily to the nucleus of infected cells. pUL93 associates with both virions and capsids and interacts with the components of the nuclear egress complex (NEC), namely, pUL50, pUL53, and pUL97, during infection. Also, multiple regions in pUL93 can independently interact with pUL77, which has been shown to help retain viral DNA during capsid assembly. These studies, combined with our earlier report of an essential role of pUL93 in viral DNA packaging, indicate that pUL93 serves as an important link between nucleocapsid maturation and nuclear egress.ImportanceHCMV causes life-threatening disease and disability in immunocompromised patients and congenitally infected newborns. In this study, we investigated the functions of HCMV essential tegument protein pUL93 and determined that it interacts with the components of the nuclear egress complex, namely, pUL50, pUL53, and pUL97. We also found that pUL93 specifically interacts with pUL77, which helps retain viral DNA during capsid assembly. Together, our data point toward an important role of pUL93 in linking virus maturation to nuclear egress. In addition to expanding our knowledge of the process of HCMV maturation, information from these studies will also be utilized to develop new antiviral therapies.

Project description:The regulation of the nucleocytoplasmic release of herpesviral capsids is defined by the process of nuclear egress. Due to their large size, nuclear capsids are unable to traverse via nuclear pores, so that herpesviruses evolved to develop a vesicular transport pathway mediating their transition through both leaflets of the nuclear membrane. This process involves regulatory proteins, which support the local distortion of the nuclear envelope. For human cytomegalovirus (HCMV), the nuclear egress complex (NEC) is determined by the pUL50-pUL53 core that initiates multicomponent assembly with NEC-associated proteins and capsids. Hereby, pUL50 serves as a multi-interacting determinant that recruits several viral and cellular factors by direct and indirect contacts. Recently, we generated an ORF-UL50-deleted recombinant HCMV in pUL50-complementing cells and obtained first indications of putative additional functions of pUL50. In this study, we produced purified ΔUL50 particles under both complementing (ΔUL50C) and non-complementing (ΔUL50N) conditions and performed a phenotypical characterization. Findings were as follows: (i) ΔUL50N particle preparations exhibited a clear replicative defect in qPCR-based infection kinetics compared to ΔUL50C particles; (ii) immuno-EM analysis of ΔUL50C did not reveal major changes in nuclear distribution of pUL53 and lamin A/C; (iii) mass spectrometry-based quantitative proteomics showed a large concordance of protein contents in the NIEP fractions of ΔUL50C and ΔUL50N particles, but virion fraction was close to the detection limit for ΔUL50N; (iv) confocal imaging of viral marker proteins of immediate early (IE) and later phases of ΔUL50N infection indicated a very low number of cells showing an onset of viral lytic protein expression; and, finally (v) quantitative measurements of encapsidated genomes provided evidence for a substantial reduction in the DNA contents in ΔUL50N compared to ΔUL50C particles. In summary, the results point to a complex and important regulatory role of the HCMV nuclear egress protein pUL50 in the maturation of infectious virus.

Project description:WD repeat-containing protein 5 (WDR5) is essential for assembling the VISA-associated complex to induce a type I interferon antiviral response to Sendai virus infection. However, the roles of WDR5 in DNA virus infections are not well described. Here, we report that human cytomegalovirus exploits WDR5 to facilitate capsid nuclear egress. Overexpression of WDR5 in fibroblasts slightly enhanced the infectious virus yield. However, WDR5 knockdown dramatically reduced infectious virus titers with only a small decrease in viral genome replication or gene expression. Further investigation of late steps of viral replication found that WDR5 knockdown significantly impaired formation of the viral nuclear egress complex and induced substantially fewer infoldings of the inner nuclear membrane. In addition, fewer capsids were associated with these infoldings, and there were fewer capsids in the cytoplasm. Restoration of WDR5 partially reversed these effects. These results suggest that WDR5 knockdown impairs the nuclear egress of capsids, which in turn decreases virus titers. These findings reveal an important role for a host factor whose function(s) is usurped by a viral pathogen to promote efficient replication. Thus, WDR5 represents an interesting regulatory mechanism and a potential antiviral target.IMPORTANCE Human cytomegalovirus (HCMV) has a large (∼235-kb) genome with over 170 open reading frames and exploits numerous cellular factors to facilitate its replication. HCMV infection increases protein levels of WD repeat-containing protein 5 (WDR5) during infection, overexpression of WDR5 enhances viral replication, and knockdown of WDR5 dramatically attenuates viral replication. Our results indicate that WDR5 promotes the nuclear egress of viral capsids, the depletion of WDR5 resulting in a significant decrease in production of infectious virions. This is the first report that WDR5 favors HCMV, a DNA virus, replication and highlights a novel target for antiviral therapy.

Dataset Information

Characterization of conserved region 2-deficient mutants of the cytomegalovirus egress protein pM53.

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Characterization of conserved region 2-deficient mutants of the cytomegalovirus egress protein pM53.

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