Cytomegalovirus UL128 homolog mutants that form a pentameric complex produce virus with impaired epithelial and trophoblast cell tropism and altered pathogenicity in the guinea pig

Abstract

Guinea pig cytomegalovirus (GPCMV) encodes a homolog pentameric complex (PC) for specific cell tropism and congenital infection. In human cytomegalovirus, the PC is an important antibody neutralizing target and GPCMV studies will aid in the development of intervention strategies. Deletion mutants of the C-terminal domains of unique PC proteins (UL128, UL130 and UL131 homologs) were unable to form a PC in separate transient expression assays. Minor modifications to the UL128 homolog (GP129) C-terminal domain enabled PC formation but viruses encoding these mutants had altered tropism to renal and placental trophoblast cells. Mutation of the presumptive CC chemokine motif encoded by GP129 was investigated by alanine substitution of the CC motif (codons 26–27) and cysteines (codons 47 and 62). GP129 chemokine mutants formed PC but GP129 chemokine mutant viruses had reduced epitropism. A GP129 chemokine mutant virus pathogenicity study demonstrated reduced viral load to target organs but highly extended viremia.

Convalescent Immunity to Guinea Pig Cytomegalovirus Induces Limited Cross Strain Protection against Re-Infection but High-Level Protection against Congenital Disease

“…GPCMV TAMYC strain infection of REPI cells was verified by immunohistochemical (IHC) staining of infected monolayers for gB viral protein at 4 days post infection ( Figure 2 i, panels A–B). TAMYC strain virus easily replicates on other non-fibroblast cell lines established in the laboratory similar to 22122 strain, including trophoblast and amniotic sac cell lines [ 18 , 19 ] (data not shown). TAMYC strain virus growth on REPI cells was highly cell-associated with very little cell-released virus ( Figure 2 ii) in contrast to 22122 strain which was 50% cell-associated and 50% cell-released virus in REPI-derived virus from infected wells of a six-well plate [ 19 ] ( Figure 2 iii).…”

“…However, the model is not without specific shortcomings including reagents and assays. We have developed a number of novel cell lines and assays to enable evaluation of virus tropism and host immune responses [ 15 , 16 , 18 , 19 , 20 , 25 , 29 , 30 ].…”

“…The GPCMV homolog PC consists of gH/gL/GP129/GP131/GP133 [ 16 ]. Similar to HCMV, the GPCMV PC is essential for viral infection of non-fibroblast cell types [ 16 ] including placental cells leading to cCMV [ 16 , 18 , 19 ]. As with HCMV, the GPCMV viral glycoprotein complexes are important for neutralizing antibody targets [ 15 , 20 , 21 , 22 ].…”

MVA-Vectored Pentameric Complex (PC) and gB Vaccines Improve Pregnancy Outcome after Guinea Pig CMV Challenge, but Only gB Vaccine Reduces Vertical Transmission

“…Indeed, the GPCMV PC appears to play a critical role in entry into many, diverse cell types, since a GP129-133 deletion mutant demonstrates defects in both endothelial cell and fibroblast cell entry [38]. The GPCMV PC, in particular the GP129, also plays a role in macrophage-mediated dissemination of virus in vivo [59], possibly mediated through a putative CC chemokine function [54]. The GPCMV PC is also required for pathogenesis in experimentally challenged, non-pregnant animals [60].…”

“…Although GP131 and GP129 were not identified by mass spectrometry analysis, we were able to observe them via western blot analysis. Furthermore, it has been suggested that GP133 will not complex with the gH/gL heterodimer in the absence of the GP131 and GP129, due, in part, to the requirement of the C-terminus of GP131 and GP133 [54], although subcomplex formation cannot be ruled out [38]. Therefore, these results indirectly suggest that there is formation of a gpPC complex when these ORFs are expressed in the MVA vectored vaccine construct.…”