The pentameric complex drives immunologically covert cell–cell transmission of wild-type human cytomegalovirus

Abstract

Human cytomegalovirus (HCMV) strains that have been passaged in vitro rapidly acquire mutations that impact viral growth. These laboratory-adapted strains of HCMV generally exhibit restricted tropism, produce high levels of cell-free virus, and develop susceptibility to natural killer cells. To permit experimentation with a virus that retained a clinically relevant phenotype, we reconstructed a wild-type (WT) HCMV genome using bacterial artificial chromosome technology. Like clinical virus, this genome proved to be unstable in cell culture; however, propagation of intact virus was achieved by placing the RL13 and UL128 genes under conditional expression. In this study, we show that WT-HCMV produces extremely low titers of cell-free virus but can efficiently infect fibroblasts, epithelial, monocyte-derived dendritic, and Langerhans cells via direct cell-cell transmission. This process of cell-cell transfer required the UL128 locus, but not the RL13 gene, and was significantly less vulnerable to the disruptive effects of IFN, cellular restriction factors, and neutralizing antibodies compared with cell-free entry. Resistance to neutralizing antibodies was dependent on high-level expression of the pentameric gH/gL/gpUL128-131A complex, a feature of WT but not passaged strains of HCMV.virology | immune evasion | herpesvirus | HCMV | cell-cell spread H uman cytomegalovirus (HCMV) is a major cause of morbidity and mortality in the immunocompromised and the leading infectious cause of congenital malformation. As a result, a vaccine has been designated as a priority. However, basic studies of clinically relevant isolates that inform our understanding of the disease process are limited due to the rapid accumulation of genetic mutations during in vitro passage of HCMV. The same three genetic sites are reproducibly affected: the RL13 gene; the UL128 locus (UL128L), which comprises UL128, UL30, and UL131A; and the ∼15-kb U L /b′ gene region (1). Deletions in the U L /b′ region can affect tropism [UL148 (2)], latency [UL136 and UL138 (3-7)], and resistance to NK cells [UL141 (8-10), UL142 (11, 12), and UL135 (13)]. Disabling mutations in RL13 and UL128L independently contribute to the release of high-titer cell-free virus, whereas loss of UL128L restricts virus entry to fibroblasts alone by preventing assembly of a pentameric glycoprotein complex (gH/gL/pUL128/ pUL130/pUL131A) in the virion envelope (1,14,15).The rapid selection of mutations is a major obstacle to the propagation of genetically intact "clinical" strains of HCMV, which in turn has led to significant gaps in our understanding of the affiliated disease processes (16). In particular, HCMV is largely cell-associated in vivo (17), and clinical isolates exhibit a similar phenotype in vitro (17, 18), yet most studies in the field are based on laboratory strains that produce high titers of cell-free virus (19). As a consequence, little is known about the fundamental processes involved in the infectious spread of cell-associated HCMV.In this study, we used a system tha...

Specialization for cell-free or cell-to-cell spread of BAC-cloned HCMV strains is determined by factors beyond the UL128-131 and RL13 loci

“…While both types of antibodies can evidently block cell-free infection, the anti-UL130/131 appears to be able to inhibit the cell-associated mode, albeit less efficiently than they block cell-free infection. This interpretation is consistent with conclusions of Murrell et al that cell-associated spread of ME may be blocked by antibodies, but less potently (42).…”

“…However, as observed in fibroblasts, spread in epithelial cells was still highly resistant to neutralizing antibodies when gH/gL/UL128-131 was repressed. This was different than Murrell et al who concluded that spread of ME in epithelial cells was more sensitive to antibodies when gH/gL/UL128-131 was reduced (42). Again the discrepancy likely reflects that analysis of 21-day plaque size likely accentuates the effects of cell-free infectivity and masks the contribution of cell-associated spread.…”

“…The cell-associated nature of ME has been linked to the high expression levels of UL128-131 and the corollary poor infectivity of cell-free ME virions (24, 27, 40, 42). To address this in our quantitative system, we made use of the previously described tetracycline (Tet)-repression system developed by Stanton et al (40).…”

“…Thus, it seems that ME has a specific mechanism(s) to enhance cell-associated spread that does not depend on production of highly infectious virions. The mechanisms driving the highly efficient cell-associated spread of ME remain unclear, but have been suggested to involve the functions of gH/gL/UL128-131 and RL13 (24, 40, 42, 62).…”

“…While this basic observation can be explained by the respective low and high infectivity of ME and TB virions described above, it is not clear how strains compare in their ability to spread by cell-associated modes, nor are the genetic correlates of cell-associated spread well defined. Multiple reports have monitored foci size in the presence of neutralizing antibodies and attributed the cell-associated nature of ME to the expression of the UL128-131 and RL13 loci (24, 42). It seems clear that either gH/gL/gO or gH/gL/UL128-131 is required for cell-associated spread on fibroblasts, whereas gH/gL/UL128-131 seems to be required for cell-associated spread in epithelial or endothelial cells (24, 26, 43, 44).…”