Universität zu Köln

Borst, Eva Maria, Bauerfeind, Rudolf, Binz, Anne, Stephan, Thomas Min, Neuber, Sebastian, Wagner, Karen, Steinbrueck, Lars, Sodeik, Beate, Rovis, Tihana Lenac, Jonjic, Stipan ORCID: 0000-0001-5003-3108 and Messerle, Martin ORCID: 0000-0002-1227-3933 (2016). The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation. J. Virol., 90 (13). S. 5860 - 5876. WASHINGTON: AMER SOC MICROBIOLOGY. ISSN 1098-5514

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Abstract

Several essential viral proteins are proposed to participate in genome encapsidation of human cytomegalovirus ( HCMV), among them pUL77 and pUL93, which remain largely uncharacterized. To gain insight into their properties, we generated an HCMV mutant expressing a pUL77-monomeric enhanced green fluorescent protein (mGFP) fusion protein and a pUL93-specific antibody. Immunoblotting demonstrated that both proteins are incorporated into capsids and virions. Conversely to data suggesting internal translation initiation sites within the UL93 open reading frame (ORF), we provide evidence that pUL93 synthesis commences at the first start codon. In infected cells, pUL77-mGFP was found in nuclear replication compartments and dot-like structures, colocalizing with capsid proteins. Immunogold labeling of nuclear capsids revealed that pUL77 is present on A, B, and C capsids. Pulldown of pUL77-mGFP revealed copurification of pUL93, indicating interaction between these proteins, which still occurred when capsid formation was prevented. Correct subnuclear distribution of pUL77-mGFP required pUL93 as well as the major capsid protein ( and thus probably the presence of capsids), but not the tegument protein pp150 or the encapsidation protein pUL52, demonstrating that pUL77 nuclear targeting occurs independently of the formation of DNA-filled capsids. When pUL77 or pUL93 was missing, generation of unit-length genomes was not observed, and only empty B capsids were produced. Taken together, these results show that pUL77 and pUL93 are capsid constituents needed for HCMV genome encapsidation. Therefore, the task of pUL77 seems to differ from that of its alphaherpesvirus orthologue pUL25, which exerts its function subsequent to genome cleavage-packaging. IMPORTANCE The essential HCMV proteins pUL77 and pUL93 were suggested to be involved in viral genome cleavage-packaging but are poorly characterized both biochemically and functionally. By producing a monoclonal antibody against pUL93 and generating an HCMV mutant in which pUL77 is fused to a fluorescent protein, we show that pUL77 and pUL93 are capsid constituents, with pUL77 being similarly abundant on all capsid types. Each protein is required for genome encapsidation, as the absence of either pUL77 or pUL93 results in a genome packaging defect with the formation of empty capsids only. This distinguishes pUL77 from its alphaherpesvirus orthologue pUL25, which is enriched on DNA-filled capsids and exerts its function after the viral DNA is packaged. Our data for the first time describe an HCMV mutant with a fluorescent capsid and provide insight into the roles of pUL77 and pUL93, thus contributing to a better understanding of the HCMV encapsidation network.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Borst, Eva Maria UNSPECIFIED UNSPECIFIED UNSPECIFIED Bauerfeind, Rudolf UNSPECIFIED UNSPECIFIED UNSPECIFIED Binz, Anne UNSPECIFIED UNSPECIFIED UNSPECIFIED Stephan, Thomas Min UNSPECIFIED UNSPECIFIED UNSPECIFIED Neuber, Sebastian UNSPECIFIED UNSPECIFIED UNSPECIFIED Wagner, Karen UNSPECIFIED UNSPECIFIED UNSPECIFIED Steinbrueck, Lars UNSPECIFIED UNSPECIFIED UNSPECIFIED Sodeik, Beate UNSPECIFIED UNSPECIFIED UNSPECIFIED Rovis, Tihana Lenac UNSPECIFIED UNSPECIFIED UNSPECIFIED Jonjic, Stipan UNSPECIFIED orcid.org/0000-0001-5003-3108 UNSPECIFIED Messerle, Martin UNSPECIFIED orcid.org/0000-0002-1227-3933 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-271209
DOI:	10.1128/JVI.00384-16
Journal or Publication Title:	J. Virol.
Volume:	90
Number:	13
Page Range:	S. 5860 - 5876
Date:	2016
Publisher:	AMER SOC MICROBIOLOGY
Place of Publication:	WASHINGTON
ISSN:	1098-5514
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language HERPES-SIMPLEX-VIRUS; DNA-PACKAGING PROTEIN; GREEN FLUORESCENT PROTEIN; TERMINASE SUBUNITS PUL56; DISULFIDE BOND FORMATION; PSEUDORABIES VIRUS; NUCLEAR EGRESS; CAPSID PROTEIN; UL25 PROTEIN; TEGUMENT PROTEINS Multiple languages Virology Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/27120