Complete Genome Sequence of a Human Cytomegalovirus Strain AD169 Bacterial Artificial Chromosome Clone

Ostermann, Eléonore; Spohn, Michael; Indenbirken, Daniela; Brune, Wolfram

doi:10.1128/genomea.00091-16

Cited by 7 publications

(6 citation statements)

References 13 publications

(12 reference statements)

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“…Wildtype HCMV strain TB40/E and its derivatives were reconstituted from TB40- BAC4 (27) or mutants thereof, and were grown on HFFT until 100% CPE was observed. For HCMV recombinants derived from AD169rv rUL131 , a BAC clone of HCMV strain AD169 (28, 29) repaired for UL131 (6, 24), viruses were amplified at low MOI in ARPE-19 cells until 100% cytopathic effect (CPE) was observed. Virus-containing culture supernatants were then subjected to centrifugation (1000 × g) for 10 min to pellet cellular debris.…”

Section: Methodsmentioning

confidence: 99%

The human cytomegalovirus protein UL116 interacts with the viral ER resident glycoprotein UL148 and promotes the incorporation of gH/gL complexes into virions

Siddiquey

Schultz

et al. 2020

Preprint

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Heterodimers of glycoproteins H (gH) and L (gL) comprise a basal element of the viral membrane fusion machinery conserved across herpesviruses. In human cytomegalovirus (HCMV), a glycoprotein encoded by UL116 noncovalently assembles onto gH at a position similar to that occupied by gL, forming a heterodimer that is incorporated into virions. However, physiological roles for UL116 or its complex with gH remain to be identified. Here, we show that UL116 promotes the expression of gH/gL complexes and is required for the efficient production of infectious cell-free virions. UL116-null mutants show a 10-fold defect in production of infectious cell-free virions from infected fibroblasts and epithelial cells. This defect is accompanied by reduced expression of the two disulfide-linked gH/gL complexes that play crucial roles in viral entry: the heterotrimer of gH/gL with glycoprotein O (gO) and the pentameric complex of gH/gL with UL128, UL130, and UL131. Furthermore, gH/UL116 complexes comprise a substantial constituent of virions since an abundant gH species not covalently linked to other glycoproteins, which has long been observed in the literature, is readily detected from wild-type but not UL116-null virions.Interestingly, UL116 co-immunoprecipitates with UL148, a viral ER resident glycoprotein previously shown to attenuate ER-associated degradation (ERAD) of gO, and we observe elevated levels of UL116 in UL148-null virions.Collectively, our findings suggest that UL116 may serve as a chaperone for gH to support the assembly, maturation, and incorporation of gH/gL complexes into virions.IMPORTANCEHCMV is a betaherpesvirus that causes dangerous opportunistic infections in immunocompromised patients, as well as in the immune-naive fetus and preterm infants. The potential of the virus to enter new host cells is governed in large part by two alternative viral glycoprotein H (gH) / glycoprotein L (gL) complexes that play important roles in entry: gH/gL/gO and gH/gL/UL128-131. A recently identified virion gH complex, comprised of gH bound to UL116, adds a new layer of complexity to the mechanisms that contribute to HCMV infectivity. Here, we show that UL116 promotes the expression of gH/gL complexes, and that UL116 interacts with the viral ER-resident glycoprotein UL148, a factor that supports the expression of gH/gL/gO. Overall, our results suggest that UL116 is a chaperone for gH. These findings have important implications for understanding of HCMV cell tropism as well as for the development of vaccines against the virus.

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Section: Methodsmentioning

confidence: 99%

The human cytomegalovirus protein UL116 interacts with the viral ER resident glycoprotein UL148 and promotes the incorporation of gH/gL complexes into virions

Siddiquey

Schultz

et al. 2020

Preprint

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“…The following HCMV strains were used: AD169-GFP (62), Toledo (63), VR1814 (64), BAC clones of AD169varATCC (65) and AD169varL (66), BAC clones of Towne (67), TB40/E (68), TR, PH (28), and FIX-GFP (69). Reconstitution of infectious HCMV from BAC DNA was done as described previously (31).…”

Section: Methodsmentioning

confidence: 99%

“…All modified BACs were examined by restriction fragment analysis and sequencing of the mutated region. The AD169 gB(275D) and TB40/E gB(275Y) BACs were sequence verified by Illumina sequencing essentially as described previously (65). The complete genome sequences of TB40-BAC4 (accession no.…”

Section: Methodsmentioning

confidence: 99%

Human cytomegalovirus glycoprotein B variants affect viral entry, cell fusion, and genome stability

Tang

Frascaroli

Lebbink

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Human cytomegalovirus (HCMV), like many other DNA viruses, can cause genome instability and activate a DNA damage response (DDR). Activation of ataxia-telangiectasia mutated (ATM), a kinase activated by DNA breaks, is a hallmark of the HCMV-induced DDR. Here we investigated the activation of caspase-2, an initiator caspase activated in response to DNA damage and supernumerary centrosomes. Of 7 HCMV strains tested, only strain AD169 activated caspase-2 in infected fibroblasts. Treatment with an ATM inhibitor or inactivation of PIDD or RAIDD inhibited caspase-2 activation, indicating that caspase-2 was activated by the PIDDosome. A set of chimeric HCMV strains was used to identify the genetic basis of this phenotype. Surprisingly, we found a single nucleotide polymorphism within the AD169 UL55 ORF, resulting in a D275Y amino acid exchange within glycoprotein B (gB), to be responsible for caspase-2 activation. As gB is an envelope glycoprotein required for fusion with host cell membranes, we tested whether gB(275Y) altered viral entry into fibroblasts. While entry of AD169 expressing gB(275D) proceeded slowly and could be blocked by a macropinocytosis inhibitor, entry of wild-type AD169 expressing gB(275Y) proceeded more rapidly, presumably by envelope fusion with the plasma membrane. Moreover, gB(275Y) caused the formation of syncytia with numerous centrosomes, suggesting that cell fusion triggered caspase-2 activation. These results suggest that gB variants with increased fusogenicity accelerate viral entry, cause cell fusion, and thereby compromise genome stability. They further suggest the ATM-PIDDosome-caspase-2 signaling axis alerts the cell of potentially dangerous cell fusion.

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“…AD169 is a well-characterized fibroblast-adapted laboratory strain that lacks the UL131 gene. ADrUL131 is an AD169 derivative in which UL131 is repaired [46][47][48] . PB plasma was collected longitudinally to measure HCMV-specific IgM levels.…”

Section: Immune-mediated Control Of Hcmv Infection In Blt-l Micementioning

confidence: 99%

Precision mouse models with expanded tropism for human pathogens

et al. 2019

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Complete Genome Sequence of a Human Cytomegalovirus Strain AD169 Bacterial Artificial Chromosome Clone

Cited by 7 publications

References 13 publications

The human cytomegalovirus protein UL116 interacts with the viral ER resident glycoprotein UL148 and promotes the incorporation of gH/gL complexes into virions

The human cytomegalovirus protein UL116 interacts with the viral ER resident glycoprotein UL148 and promotes the incorporation of gH/gL complexes into virions

Human cytomegalovirus glycoprotein B variants affect viral entry, cell fusion, and genome stability

Precision mouse models with expanded tropism for human pathogens

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