Optimized recombinant dense bodies of human cytomegalovirus efficiently prime virus specific lymphocytes and neutralizing antibodies without the addition of adjuvant

Becke, Sabine; Aue, Steffi; Thomas, Doris; Schader, Sina I.; Podlech, Jürgen; Bopp, Tobias; Sedmak, Tina; Wolfrum, Uwe; Plachter, Bodo; Reyda, Sabine

doi:10.1016/j.vaccine.2010.07.016

Cited by 23 publications

(33 citation statements)

References 45 publications

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Section: Discussionmentioning

confidence: 87%

“…This insertion was responsible for the phenotypic alterations of RV-VM1, as they were lost upon reversion. Insertion of the same sequence in other regions of pp65 or fusion of a larger fragment of the IE1 protein to the very C terminus did not lead to the same phenotypic changes of the resulting viruses (Mersseman et al, 2008a, b;Becke et al, 2010).One hallmark of the infection with RV-VM1 was the dissolution of NIBs and the formation of nuclear LGS. Similar nuclear structures had been reported to be formed Protein kinase inhibitors were added to the RV-HB5-samples shown in lanes 11 and 12, 15 h before harvesting the cells.…”

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confidence: 94%

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Modification of the major tegument protein pp65 of human cytomegalovirus inhibits virus growth and leads to the enhancement of a protein complex with pUL69 and pUL97 in infected cells

Becke

Fabre-Mersseman

Aue

et al. 2010

Journal of General Virology

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The tegument protein pp65 of human cytomegalovirus (HCMV) is abundant in lytically infected human foreskin fibroblasts (HFF), as well as in virions and subviral dense bodies (DB). Despite this, we showed previously that pp65 is dispensable for growth in HFF. In the process of refining a DB-based vaccine candidate, different HCMV mutants were generated, expressing a dominant HLA-A2-presented peptide of the IE1 protein fused to pp65. One of the mutant viruses (RV-VM1) surprisingly showed marked impairment in virus release from HFF. We hypothesized that analysis of the phenotypic alterations of RV-VM1 would provide insight into the functions of pp65, poorly defined thus far. RV-VM1 infection resulted in nuclear retention of the fusion protein and reorganization of nuclear inclusion bodies. Coimmunoprecipitation experiments suggested that wild-type (wt) pp65 and pp65-VM1 were substrates of the viral pUL97 kinase in vitro and formed a complex with the viral RNA-export protein pUL69 and with pUL97 in lysates of infected cells. No evidence for an impairment of pUL97 within this complex was found. However, RV-VM1 replication in infected cells was resistant to a pUL97 inhibitor, and pUL97 inhibitors mimicked the mutant in terms of pp65 being retained in the nucleus. The results suggest that the life cycle of RV-VM1 was impeded at the stages of early-late transcription, RNA export or capsid maturation. wt-pp65 may play a role at these stages of infection, and complex formation with pUL69 and pUL97 may be important for that function. INTRODUCTIONHuman cytomegalovirus (HCMV) is a ubiquitous pathogen that severely affects individuals with impaired or immature immune-defence functions (Griffiths et al., 2009;Mocarski et al., 2007). The components of the HCMV tegument have recently attracted considerable attention, as these proteins carry functions important for various stages of the virus replication cycle (reviewed by Kalejta, 2008). The phosphoprotein pp65 (ppUL83) has long been known as an abundant tegument constituent (Roby & Gibson, 1986), yet it is dispensable for HCMV growth in human foreskin fibroblast (HFF) cell cultures (Schmolke et al., 1995b).pp65 is an important target antigen of cellular and humoral immune responses (Beninga et al., 1995; McLaughlinTaylor et al., 1994; Plachter et al., 1990;Wills et al., 1996). It would thus be reasonable to assume that modification or deletion of the protein would be favourable for the virus. However, the sequence of pp65 is highly conserved in laboratory strains and clinical isolates (Dolan et al., 2004;Pande et al., 1991) and lack of pp65 expression in such strains has never been reported. Indeed, detection of pp65 has been used as a reliable diagnostic marker for acute HCMV infection in transplant recipients for over 15 years (Grefte et al., 1992a, b Initial studies described an association of pp65 with serine/ threonine kinase activity (Britt & Auger, 1986;Somogyi et al., 1990). Subsequent analyses identified the cellular Polo-like kinase 1 (Gallina et al., 1999) and the vi...

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Section: Discussionmentioning

confidence: 87%

mentioning

confidence: 94%

Modification of the major tegument protein pp65 of human cytomegalovirus inhibits virus growth and leads to the enhancement of a protein complex with pUL69 and pUL97 in infected cells

Becke

Fabre-Mersseman

Aue

et al. 2010

Journal of General Virology

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“…To determine if this was truly an effect of the pp65 loss or due to contamination with DBs, HCMV strains RV-SB2 and RV-VM1 were analyzed. Both strains fail to produce DBs (33,34). RV-SB2 is also deficient in pp65 packaging into virions, whereas RV-VM1 virions contain normal amounts of the tegument protein (Fig.…”

Section: Purification Of Hcmv Virions For Proteomic Analysismentioning

confidence: 99%

“…The pp65neg strain RV-KB14 was generated by inserting a tetracycline resistance cassette into the UL83 (pp65) ORF of pAD/cre (the BAC clone used for reconstitution of RV-BADwt), thereby deleting the pp65-coding region except for 152 5= base pairs of the ORF (32). The mutants RV-SB2 and RV-VM1 were generated by inserting the nonapeptide TMYGGISLL from the immediate-early 1 (IE1) protein of HCMV at position S101 or R387, respectively, of pp65, using galK-mediated recombination on the BACmid pHB5 (33,34). All viruses were characterized previously with respect to their genomic structures and biological properties.…”

Section: Cells and Virusesmentioning

confidence: 99%

“…RV-KB14 (28,32), which fail to produce subviral DBs; and additionally, two viral mutants, RV-SB2 and RV-VM1, in which pp65 was modified by insertion of a nonapeptide sequence at positions S101 and R387, respectively (33,34). DB formation is also abrogated in HFF infected with RV-SB2 and RV-VM1 (33,34). RV-VM1-infected HFF display wt pp65 steady-state protein levels (33), whereas RV-SB2 (a pp65low strain)-infected cells show a marked reduction in pp65 levels in relation to other virion proteins (Fig.…”

Section: Purification Of Hcmv Virions For Proteomic Analysismentioning

confidence: 99%

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The Tegument Protein pp65 of Human Cytomegalovirus Acts as an Optional Scaffold Protein That Optimizes Protein Uploading into Viral Particles

Reyda

Navarro

Gebauer

et al. 2014

J Virol

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The mechanisms that lead to the tegumentation of herpesviral particles are only poorly defined. The phosphoprotein 65 (pp65) is the most abundant constituent of the virion tegument of human cytomegalovirus (HCMV). It is, however, nonessential for virion formation. This seeming discrepancy has not met with a satisfactory explanation regarding the role of pp65 in HCMV particle morphogenesis. Here, we addressed the question of how the overall tegument composition of the HCMV virion depended on pp65 and how the lack of pp65 influenced the packaging of particular tegument proteins. To investigate this, we analyzed the proteomes of pp65-positive (pp65pos) and pp65-negative (pp65neg) virions by label-free quantitative mass spectrometry and determined the relative abundances of tegument proteins. Surprisingly, only pUL35 was elevated in pp65neg virions. As the abundance of pUL35 in the HCMV tegument is low, it is unlikely that it replaced pp65 as a structural component in pp65neg virions. A subset of proteins, including the third most abundant tegument protein, pUL25, as well as pUL43, pUL45, and pUL71, were reduced in pp65neg or pp65low virions, indicating that the packaging of these proteins was related to pp65. The levels of tegument components, like pp28 and the capsid-associated tegument proteins pp150, pUL48, and pUL47, were unaffected by the lack of pp65. Our analyses demonstrate that deletion of pp65 is not compensated for by other viral proteins in the process of virion tegumentation. The results are concordant with a model of pp65 serving as an optional scaffold protein that facilitates protein upload into the outer tegument of HCMV particles. IMPORTANCEThe assembly of the tegument of herpesviruses is only poorly understood. Particular proteins, like HCMV pp65, are abundant tegument constituents. pp65 is thus considered to play a major role in tegument assembly in the process of virion morphogenesis. We show here that deletion of the pp65 gene leads to reduced packaging of a subset of viral proteins, indicating that pp65 acts as an optional scaffold protein mediating protein upload into the tegument.

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Synthetic DNA Approach to Cytomegalovirus Vaccine/Immune Therapy

Villarreal

Shedlock

et al. 2015

Advances in Experimental Medicine and Biology

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There is no licensed vaccine or cure for human cytomegalovirus (CMV), a ubiquitous β-herpes virus that infects 60–95 % of adults worldwide. Infection is a major cause of congenital abnormalities in newborns, contributes to development of childhood cerebral palsy and medulloblastoma, can result in severe disease in immunocompromised patients, and is a major impediment during successful organ transplantation. While CMV has been increasingly associated with numerous inflammatory diseases and cancers, only recently has it been correlated with increased risk of heart disease in adults, the number-one killer in the USA. These data, among others, suggest that subclinical CMV infection, or microinfection, in healthy individuals may play more of a causative role than an epiphenomenon in development of CMV-associated pathologies. Due to the myriad of diseases and complications associated with CMV, an efficacious vaccine would be highly valuable in reducing human morbidity and mortality as well as saving billions of dollars in annual health-care costs and disability adjusted life years (DALY) in the developing world. Therefore, the development of a safe efficacious CMV vaccine or immune therapy is paramount to the public health. This review aims to provide a brief overview on aspects of CMV infection and disease and focuses on current vaccine strategies. The use of new synthetic DNA vaccines might offer one such approach to this difficult problem.

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Optimized recombinant dense bodies of human cytomegalovirus efficiently prime virus specific lymphocytes and neutralizing antibodies without the addition of adjuvant

Cited by 23 publications

References 45 publications

Modification of the major tegument protein pp65 of human cytomegalovirus inhibits virus growth and leads to the enhancement of a protein complex with pUL69 and pUL97 in infected cells

Modification of the major tegument protein pp65 of human cytomegalovirus inhibits virus growth and leads to the enhancement of a protein complex with pUL69 and pUL97 in infected cells

The Tegument Protein pp65 of Human Cytomegalovirus Acts as an Optional Scaffold Protein That Optimizes Protein Uploading into Viral Particles

Synthetic DNA Approach to Cytomegalovirus Vaccine/Immune Therapy

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