Expression levels of glycoprotein O (gO) vary between strains of human cytomegalovirus, influencing the assembly of gH/gL complexes and virion infectivity

Le, Zhang; Zhou, Momei; Stanton, Richard J.; Kamil, Jeremy P.; Ryckman, Brent J.

doi:10.1101/299222

Cited by 9 publications

(8 citation statements)

References 49 publications

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“…AdUL116myc was generated by subcloning the UL116_GGS_Myc sequence from pEF1_UL116_GGS_Myc plasmid to the AdMax pDC316(io) shuttle plasmid using the primers sDC116TBco and asDC116cocmyc (Table 1). AdUL115gL and AdGFP were described previously (21,35). All Ad vectors were propagated and titered by TCID50 on HEK293IQ cells.…”

Section: New Recombinant Hcmv Bacs and Plasmids For This Studymentioning

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: New Recombinant Hcmv Bacs and Plasmids For This Studymentioning

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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“…Zhang et al asserted that the differential tropism and infectivity of distinct strains is also function of the relative levels of Trimer, Pentamer and gH/gL carried by virons (18). This observation encouraged us to verify the levels of expression of the gH-based complexes proteins in absence of UL116 and/or UL148 both in virions and in infected cells.…”

Section: Expression Levels Of the Major Hcmv Envelope Proteins In Infmentioning

confidence: 99%

“…While differential expression of these receptors influences permissivity to HCMV infection, at least for fibroblasts and epithelial cell types the viral infectivity relies upon the presence of the two gH-based complexes. The relative abundance of Trimer and Pentamer complexes in virions is strain-specific and influence cell tropism and infectivity (18). How the formation of the two gH/gL complexes is regulated at the molecular level remains currently largely unknown although two recent reports identified two potential players.…”

Section: Introductionmentioning

confidence: 99%

The human cytomegalovirus UL116 glycoprotein is a chaperone to control gH-based complexes levels on virions

Vezzani

Amendola²,

Yü³

et al. 2020

Preprint

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Human cytomegalovirus (HCMV) relies in large part upon the viral membrane fusion glycoprotein B (gB) and two alternative gH/gL complexes, gH/gL/gO (Trimer) and the gH/gL/UL128/UL130/UL131A (Pentamer) to enter into cells. The relative amounts of the Trimer and Pentamer vary among HCMV strains and contribute to differences in cell tropism. Although the viral ER resident protein UL148 has been shown to interact with gH to facilitate gO incorporation, the mechanisms that favor the assembly and maturation of one complex over another remain poorly understood. HCMV virions also contain an alternative non-disulfide bound heterodimer comprised of gH and UL116 whose function remains unknown. Here, we show that disruption of HCMV gene UL116 causes infectivity defects of ~10-fold relative to wild-type virus and leads to reduced expression of both gH/gL complexes in virions. Furthermore, gH that is not covalently bound to other viral glycoproteins, which are readily detected in wild-type HCMV virions, become undetectable in the absence of UL116 suggesting that the gH/UL116 complex is abundant in virions. We find evidence that UL116 and UL148 interact during infection indicating that the two proteins might cooperate to regulate the abundance of HCMV gH complexes. Altogether, these results are consistent with a role of UL116 as a chaperone for gH during the assembly and maturation of gH complexes in infected cells.

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“…It is made The copyright holder for this preprint (which this version posted June 2, 2020. ; https://doi.org/10.1101/2020.06.02.129486 doi: bioRxiv preprint independent experiments. In HCMV, strain specific differences in tropism can arise from alterations in the levels of both the PRC and the gH/gL/gO trimeric receptor complex which can be caused by genetic sequence variations or altered mRNA expression levels of the proteins in each complex (46)(47)(48). Intriguingly, increased infection rates on epithelial cells have been reported for the PRC-repaired HCMV strain AD169 compared to PRC-intact low-passage isolates (49), a result very reminiscent of our data.…”

Section: In Vitro Characterization Of Fl-rhcmvmentioning

confidence: 99%

In vitroandin vivocharacterization of a recombinant rhesus cytomegalovirus containing a complete genome

Taher

Mahyari

Kreklywich

et al. 2020

Preprint

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In vitro and in vivo characterization of a recombinant1 rhesus cytomegalovirus containing a complete genome 2 3 Abstract (300 words) 42 Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species 43 specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models 44 using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has 45 been established as a representative model for infection of humans with HCMV due to the close 46 evolutionary relationships of both host and virus. However, the commonly used 68-1 strain of 47 RhCMV has been passaged in fibroblasts for decades resulting in multiple genomic changes due 48 to tissue culture adaptation that cause reduced viremia in RhCMV-naïve animals and limited 49 shedding compared to low passage isolates. Using sequence information from primary RhCMV 50 isolates we constructed a full-length (FL) RhCMV by repairing all presumed mutations in the 68-51 1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve 52 RM with the reconstituted virus resulted in significant replication in the blood similar to primary 53 isolates of RhCMV and furthermore led to extensive viremia in many tissues at day 14 post 54 infection. In contrast, viral dissemination and viremia was greatly reduced upon deletion of genes 55 also lacking in 68-1. Transcriptome analysis of infected tissues further revealed that chemokine-56 like genes deleted in 68-1 are among the most highly expressed viral transcripts both in vitro and 57 in vivo consistent with an important immunomodulatory function of the respective proteins. We 58 conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while 59 being amenable to genetic modifications through BAC recombineering techniques.60 61 4 Author Summary (150-200 word non-technical summary) 62 Human cytomegalovirus (HCMV) infections are generally asymptomatic in healthy 63 immunocompetent individuals, but HCMV can cause serious disease after congenital infection and 64 in individuals with immunocompromised immune systems. Since HCMV is highly species specific 65 and cannot productively infect immunocompetent laboratory animals, experimental infection of 66 rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a closely related 67 animal model for HCMV. By employing the unique ability of CMV to elicit robust and lasting 68 cellular immunity, this model has also been instrumental in developing novel CMV-based vaccines 69 against chronic and recurring infections with pathogens such as the human immunodeficiency 70 virus (HIV) and Mycobacterium tuberculosis (Mtb). However, most of this work was conducted 71 with derivatives of the 68-1 strain of RhCMV which has acquired multiple genomic alterations in 72 tissue culture. To model pathogenesis and immunology of clinical HCMV isolates we generated a 73 full-length (FL) RhCMV clone representative of low passage isolates. Infection of RhCMV-naïve 74 RM with FL-...

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Expression levels of glycoprotein O (gO) vary between strains of human cytomegalovirus, influencing the assembly of gH/gL complexes and virion infectivity

Cited by 9 publications

References 49 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

The human cytomegalovirus UL116 glycoprotein is a chaperone to control gH-based complexes levels on virions

In vitroandin vivocharacterization of a recombinant rhesus cytomegalovirus containing a complete genome

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