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In vitro and in vivo characterization of a recombinant rhesus cytomegalovirus containing a complete genome.

ABSTRACT: Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the only available RhCMV clone that permits genetic modifications is based on the 68-1 strain which has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptations. As a result, 68-1 displays reduced viremia in RhCMV-naïve animals and limited shedding compared to non-clonal, low passage isolates. To overcome this limitation, we used sequence information from primary RhCMV isolates to construct a full-length (FL) RhCMV by repairing all mutations affecting open reading frames (ORFs) in the 68-1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve RM with the reconstituted virus resulted in significant viremia in the blood similar to primary isolates of RhCMV and furthermore led to high viral genome copy numbers in many tissues at day 14 post infection. In contrast, viral dissemination was greatly reduced upon deletion of genes also lacking in 68-1. Transcriptome analysis of infected tissues further revealed that chemokine-like genes deleted in 68-1 are among the most highly expressed viral transcripts both in vitro and in vivo consistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques.

SUBMITTER: Taher H

PROVIDER: S-EPMC7723282 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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In vitro and in vivo characterization of a recombinant rhesus cytomegalovirus containing a complete genome.

Taher Husam H Mahyari Eisa E Kreklywich Craig C Uebelhoer Luke S LS McArdle Matthew R MR Moström Matilda J MJ Bhusari Amruta A Nekorchuk Michael M E Xiaofei X Whitmer Travis T Scheef Elizabeth A EA Sprehe Lesli M LM Roberts Dawn L DL Hughes Colette M CM Jackson Kerianne A KA Selseth Andrea N AN Ventura Abigail B AB Cleveland-Rubeor Hillary C HC Yue Yujuan Y Schmidt Kimberli A KA Shao Jason J Edlefsen Paul T PT Smedley Jeremy J Kowalik Timothy F TF Stanton Richard J RJ Axthelm Michael K MK Estes Jacob D JD Hansen Scott G SG Kaur Amitinder A Barry Peter A PA Bimber Benjamin N BN Picker Louis J LJ Streblow Daniel N DN Früh Klaus K Malouli Daniel D

PLoS pathogens 20201124 11

Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the only available RhCMV clone that permits genetic modifications is bas ...[more]

PMID: 33232376

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Project description:Receptors recognizing the Fc part of immunoglobulin G (Fc?Rs) are key determinants in antibody-mediated immune responses. Members of the Herpesviridae interfere with this immune regulatory network by expressing viral Fc?Rs (vFc?Rs). Human cytomegalovirus (HCMV) encodes four distinct vFc?Rs that differ with respect to their IgG subtype specificity and their impact on antibody-mediated immune function in vitro The impact of vFc?Rs on HCMV pathogenesis and immunomodulation in vivo is not known. The closest evolutionary animal model of HCMV is rhesus CMV (RhCMV) infection of rhesus macaques. To enable the characterization of vFc?R function in this model, we studied IgG binding by RhCMV. We show that lysates of RhCMV-infected cells contain an IgG-binding protein of 30?kDa encoded by the gene Rh05 that is a predicted type I glycoprotein belonging to the RL11 gene family. Upon deletion of Rh05, IgG-Fc binding by RhCMV strain 68-1 is lost, whereas ectopic expression of Rh05 results in IgG binding to transfected cells consistent with Rh05 being a vFc?R. Using a set of reporter cell lines stably expressing human and rhesus Fc?Rs, we further demonstrate that Rh05 antagonizes host Fc?R activation. Compared to Rh05-intact RhCMV, RhCMV?Rh05 showed an increased activation of host Fc?R upon exposure of infected cells to IgG from RhCMV-seropositive animals, suggesting that Rh05 protects infected cells from opsonization and IgG-dependent activation of host Fc?Rs. However, antagonizing host Fc?R activation by Rh05 was not required for the establishment and maintenance of infection of RhCMV, even in a seropositive host, as shown by the induction of T cell responses to heterologous antigens expressed by RhCMV lacking the gene region encoding Rh05. In contrast to viral evasion of natural killer cells or T cell recognition, the evasion of antibody-mediated effects does not seem to be absolutely required for infection or reinfection. The identification of the first vFc?R that efficiently antagonizes host Fc?R activation in the RhCMV genome will thus permit more detailed studies of this immunomodulatory mechanism in promoting viral dissemination in the presence of natural or vaccine-induced humoral immunity.IMPORTANCE Rhesus cytomegalovirus (RhCMV) offers a unique model for studying human cytomegalovirus (HCMV) pathogenesis and vaccine development. RhCMV infection of nonhuman primates greatly broadened the understanding of mechanisms by which CMVs evade or reprogram T cell and natural killer cell responses in vivo However, the role of humoral immunity and viral modulation of anti-CMV antibodies has not been studied in this model. There is evidence from in vitro studies that HCMVs can evade humoral immunity. By gene mapping and with the help of a novel cell-based reporter assay system we characterized the first RhCMV encoded IgG-Fc? binding glycoprotein as a potent antagonist of rhesus Fc?R activation. We further demonstrate that, unlike evasion of T cell immunity, this viral Fc? receptor is not required to overcome anti-CMV immunity to establish secondary infections. These findings enable more detailed studies of the in vivo consequences of CMV evasion from IgG responses in nonhuman primate models.

Dataset Information

In vitro and in vivo characterization of a recombinant rhesus cytomegalovirus containing a complete genome.

Publications

In vitro and in vivo characterization of a recombinant rhesus cytomegalovirus containing a complete genome.

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