Principles for studying in vivo attenuation of virus mutants: defining the role of the cytomegalovirus gH/gL/gO complex as a paradigm

Abstract

Initial virus entry into cells of host organs and subsequent spread of viral progeny between tissue cells are events fundamental to viral pathogenesis. Glycoprotein complexes inserted in the virion envelope are critically involved in the cell entry process. Here we review and discuss recent work that has shed light on the in vivo role of the trimeric glycoprotein complex gH/gL/gO of murine cytomegalovirus (mCMV) as a model to propose the role of the corresponding complex of human CMV, for which experimental studies in vivo are not feasible due to the host species specificity of CMVs and evident ethical constraints. A novel approach combining gO transcomplementation of a genetically gO-deficient virus and a mathematical log-linear regression analysis of the viral multiplication kinetics in host tissues revealed a critical role of mCMV gH/gL/gO only in first target cell entry of virions arriving with the circulation, whereas intra-tissue spread proceeded unaffected also in the absence of gH/gL/gO. These findings predict that targeting gO for an antiviral intervention may be of prophylactic value in preventing the seeding of virus to organs, but will likely fail to interfere with an established primary organ infection or with recurrent infection after virus reactivation from latency within tissue cells. The demonstration in the murine model of alternative gH/gL complexes gH/gL/gO and gH/gL/MCK-2, substituting one another in a redundant fashion for securing viral spread in tissues, has the medically interesting bearing that targeting the gH/gL core complex directly may be a promising approach to preventing primary, established, and recurrent CMV infections.

Reconstitution of CD8 T Cells Protective against Cytomegalovirus in a Mouse Model of Hematopoietic Cell Transplantation: Dynamics and Inessentiality of Epitope Immunodominance

“…Although the above described C-terminal replacements with Ala in the IDE peptide sequences are the minimal procedure required to achieve the intended immunological loss-of-presentation phenotype, one can never exclude an effect on protein folding and function. As explained in detail in recent reports ( 97 , 98 ), virus spreads and replicates exponentially over time in organs of an immunodepleted host, which leads to log-linear growth regression lines with the vDT as the growth constant that reveals replicative fitness. Unlike growth curves in cell culture, this in vivo approach has the advantage to incorporate all cell types that constitute the tissues of tested organs in the natural microanatomical context.…”

Role of PDGF receptor-α during human cytomegalovirus entry into fibroblasts

“…The viral and cellular determinants mediating entry into different cell types have been only partially defined. Two virus-encoded glycoprotein complexes on the virion envelope, a pentameric complex (gH/gL/pUL128-pUL130-pUL131A) and a trimeric complex (gH/gL/gO), are thought to control tropism (4,5). We refer to these complexes as "pentamer" and "trimer" and to viruses containing the trimer but lacking the pentameric complex as "trimer-only" virus in this paper.…”

Evaluating Human T-Cell Therapy of Cytomegalovirus Organ Disease in HLA-Transgenic Mice

“…To verify, in the first place, that integration of the NLV epitope into the IE2 protein does not attenuate the virus, which otherwise would render the model inapplicable for studying viral pathogenesis, we compared the in vivo growth kinetics of parental virus mCMV-Δm157 and its antigenicity variant mCMV-NLV in NSG/HHD mice. In selected organs relevant for viral pathogenesis, such as liver, spleen, and lungs ( Fig 1B ), the two viruses were found to replicate in a log-linear fashion with comparable doubling times within each organ (for the principle, see [ 36 , 37 ]), though with organ-typic numerical differences, with most aggressive growth in the lungs. In conclusion, insertion of the NLV epitope did not impair in vivo viral growth.…”

“…To demonstrate a histopathological correlate of mortality, we performed immunohistochemical (IHC) imaging for livers from the mock-transduction control group and the therapy group (corresponding to Fig 7B and 7b ) at the time of onset of mortality (day 20) in the control group ( Fig 8 ). Overview sections, identifying infected liver cells by their expression of intranuclear viral IE1 protein ( Fig 8a1 and 8b1 ; red staining), reveal extended plaque-like lesions from the cytopathogenic effect of virus replication in the control livers, whereas foci of infection were less frequent and less extended in the therapy group, though, as predictable from exponential (log-linear) growth ([ 36 , 37 ] and references therein) tissue infection is likely to close up to the control group with a few days delay explaining delayed onset of mortality in the therapy group. Higher-magnification 2-color IHC images show infected hepatocytes (iHc, red staining of IE1), which are distinctive by cytomorphology, infected endothelial cells (iEC, red staining of IE1 and black staining of CD31 antigen) ( Fig 8a2 and 8b2 ), and infected macrophages (iMΦ, red staining of IE1 and turquoise-green staining of F4/80 antigen) ( Fig 8a3 and 8b3 ).…”