Dominant Negative Mutants of the Murine Cytomegalovirus M53 Gene Block Nuclear Egress and Inhibit Capsid Maturation

ABSTRACT The alphaherpesvirus proteins UL31 and UL34 and their homologues in other herpesvirus subfamilies cooperate at the nuclear membrane in the export of nascent herpesvirus capsids. We studied the respective betaherpesvirus proteins M53 and M50 in mouse cytomegalovirus (MCMV). Recently, we established a random approach to identify dominant negative (DN) mutants of essential viral genes and isolated DN mutants of M50 (B. Rupp, Z. Ruzsics, C. Buser, B. Adler, P. Walther and U. H. Koszinowski, J. Virol 81:5508-5517). Here, we report the identification and phenotypic characterization of DN alleles of its partner, M53. While mutations in the middle of the M53 open reading frame (ORF) resulted in DN mutants inhibiting MCMV replication by ∼100-fold, mutations at the C terminus resulted in up to 1,000,000-fold inhibition of virus production. C-terminal DN mutants affected nuclear distribution and steady-state levels of the nuclear egress complex and completely blocked export of viral capsids. In addition, they induced a marked maturation defect of viral capsids, resulting in the accumulation of nuclear capsids with aberrant morphology. This was associated with a two-thirds reduction in the total amount of unit length genomes, indicating an accessory role for M53 in DNA packaging.

[1]  R. Roller,et al.  Analysis of a Charge Cluster Mutation of Herpes Simplex Virus Type 1 UL34 and Its Extragenic Suppressor Suggests a Novel Interaction between pUL34 and pUL31 That Is Necessary for Membrane Curvature around Capsids , 2010, Journal of Virology.

[2]  B. Klupp,et al.  Herpesvirus assembly: an update. , 2009, Virus research.

[3]  R. Park,et al.  Effects of Lamin A/C, Lamin B1, and Viral US3 Kinase Activity on Viral Infectivity, Virion Egress, and the Targeting of Herpes Simplex Virus UL34-Encoded Protein to the Inner Nuclear Membrane , 2008, Journal of Virology.

[4]  B. Klupp,et al.  Partial Functional Complementation of a Pseudorabies Virus UL25 Deletion Mutant by Herpes Simplex Virus Type 1 pUL25 Indicates Overlapping Functions of Alphaherpesvirus pUL25 Proteins , 2008, Journal of Virology.

[5]  R. Feederle,et al.  Deletion of Epstein-Barr Virus BFLF2 Leads to Impaired Viral DNA Packaging and Primary Egress as Well as to the Production of Defective Viral Particles , 2008, Journal of Virology.

[6]  U. Koszinowski,et al.  Natural Killer Cells Promote Early CD8 T Cell Responses against Cytomegalovirus , 2007, PLoS pathogens.

[7]  B. Trus,et al.  Allosteric signaling and a nuclear exit strategy: binding of UL25/UL17 heterodimers to DNA-Filled HSV-1 capsids. , 2007, Molecular cell.

[8]  Paul Walther,et al.  Random Screening for Dominant-Negative Mutants of the Cytomegalovirus Nuclear Egress Protein M50 , 2007, Journal of Virology.

[9]  P. Walther,et al.  Cytomegalovirus Primary Envelopment Occurs at Large Infoldings of the Inner Nuclear Membrane , 2006, Journal of Virology.

[10]  B. Klupp,et al.  The Capsid-Associated UL25 Protein of the Alphaherpesvirus Pseudorabies Virus Is Nonessential for Cleavage and Encapsidation of Genomic DNA but Is Required for Nuclear Egress of Capsids , 2006, Journal of Virology.

[11]  T. Mettenleiter,et al.  Egress of Alphaherpesviruses , 2006, Journal of Virology.

[12]  B. Berger,et al.  Herpesviral Protein Networks and Their Interaction with the Human Proteome , 2006, Science.

[13]  U. Koszinowski,et al.  Functional Domains of Murine Cytomegalovirus Nuclear Egress Protein M53/p38 , 2006, Journal of Virology.

[14]  R. Park,et al.  Herpes Simplex Virus Type 1 Infection Induces Activation and Recruitment of Protein Kinase C to the Nuclear Membrane and Increased Phosphorylation of Lamin B , 2006, Journal of Virology.

[15]  J. Harper,et al.  Identification and Functional Evaluation of Cellular and Viral Factors Involved in the Alteration of Nuclear Architecture during Herpes Simplex Virus 1 Infection , 2005, Journal of Virology.

[16]  L. Frati,et al.  Characterization and Intracellular Localization of the Epstein-Barr Virus Protein BFLF2: Interactions with BFRF1 and with the Nuclear Lamina , 2005, Journal of Virology.

[17]  J. Baines,et al.  Identification of an Essential Domain in the Herpes Simplex Virus 1 UL34 Protein That Is Necessary and Sufficient To Interact with UL31 Protein , 2005, Journal of Virology.

[18]  L. Frati,et al.  BFRF1 of Epstein-Barr Virus Is Essential for Efficient Primary Viral Envelopment and Egress , 2005, Journal of Virology.

[19]  U. Koszinowski,et al.  Conditional Cytomegalovirus Replication In Vitro and In Vivo , 2005, Journal of Virology.

[20]  Y. Kawaguchi,et al.  Cell lines that support replication of a novel herpes simplex virus 1 UL31 deletion mutant can properly target UL34 protein to the nuclear rim in the absence of UL31. , 2004, Virology.

[21]  U. Koszinowski,et al.  Comprehensive Mutational Analysis of a Herpesvirus Gene in the Viral Genome Context Reveals a Region Essential for Virus Replication , 2004, Journal of Virology.

[22]  S. Jonjić,et al.  Gain of Virulence Caused by Loss of a Gene in Murine Cytomegalovirus , 2004, Journal of Virology.

[23]  J. Baines,et al.  Conformational Changes in the Nuclear Lamina Induced by Herpes Simplex Virus Type 1 Require Genes UL31 and UL34 , 2004, Journal of Virology.

[24]  L. Hutt-Fletcher,et al.  The Epstein-Barr virus BFRF1 and BFLF2 proteins interact and coexpression alters their cellular localization. , 2004, Virology.

[25]  V. Štolc,et al.  Functional profiling of a human cytomegalovirus genome , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[26]  T. Shenk,et al.  Functional map of human cytomegalovirus AD169 defined by global mutational analysis , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[27]  R. Roller,et al.  Effects of Charged Cluster Mutations on the Function of Herpes Simplex Virus Type 1 UL34 Protein , 2003, Journal of Virology.

[28]  U. Koszinowski,et al.  Role of Murine Cytomegalovirus US22 Gene Family Members in Replication in Macrophages , 2003, Journal of Virology.

[29]  J. Haas,et al.  Cytomegalovirus Recruitment of Cellular Kinases to Dissolve the Nuclear Lamina , 2002, Science.

[30]  E. Bogner.  Human cytomegalovirus terminase as a target for antiviral chemotherapy , 2002, Reviews in medical virology.

[31]  B. Klupp,et al.  The Interacting UL31 and UL34 Gene Products of Pseudorabies Virus Are Involved in Egress from the Host-Cell Nucleus and Represent Components of Primary Enveloped but Not Mature Virions , 2002, Journal of Virology.

[32]  B. Klupp,et al.  Egress of Alphaherpesviruses: Comparative Ultrastructural Study , 2001, Journal of Virology.

[33]  B. Klupp,et al.  Primary Envelopment of Pseudorabies Virus at the Nuclear Membrane Requires the UL34 Gene Product , 2000, Journal of Virology.

[34]  B. Roizman,et al.  The essential protein encoded by the UL31 gene of herpes simplex virus 1 depends for its stability on the presence of UL34 protein. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[35]  R. Roller,et al.  Herpes Simplex Virus Type 1 UL34 Gene Product Is Required for Viral Envelopment , 2000, Journal of Virology.

[36]  W. Hammerschmidt,et al.  Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[37]  C. Van Sant,et al.  The null mutant of the U(L)31 gene of herpes simplex virus 1: construction and phenotype in infected cells , 1997, Journal of virology.

[38]  B. Barrell,et al.  Analysis of the complete DNA sequence of murine cytomegalovirus , 1996, Journal of virology.

[39]  I. Herskowitz.  Functional inactivation of genes by dominant negative mutations , 1987, Nature.

[40]  S. Jonjić,et al.  Interstitial murine cytomegalovirus pneumonia after irradiation: characterization of cells that limit viral replication during established infection of the lungs , 1985, Journal of virology.

[41]  W. Wackernagel,et al.  Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. , 1995, Gene.