The initial experiments and the results from others (29
) indicated that the fully assembled envelope protein complexes of laboratory strain DBs lacking the PC already induced a distinct neutralizing antibody response against infection by a PC-positive strain. The PC had, however, been identified as a dominant target of the NT-abs response against HCMV. We thus designed experiments to analyze the impact of antibodies against the PC using side-by-side immunization with PC-positive and PC-negative DBs. To be able to directly compare the results, we decided to use a pair of viruses for DB production that only differed in PC expression. As a basis for this, we selected the Towne strain, which is a high-level DB producer. The Towne strain carries a double adenine insertion in the open reading frame (ORF) of the UL130 gene, leading to a frameshift (16
). The parental Towne strain is thus PC negative. To generate a Towne variant that expresses the PC, the mutated UL130 open reading frame in the BACmid pTowne (37
) was replaced by its functional homolog from the TB40/E strain, using the galK
negative-positive selection procedure (38
). The resulting BACmid was denominated pTowne-UL130rep (Fig. 2A
). We chose TB40/E UL130, as the expression of the PC by TB40/E appears to be conserved upon serial passaging in fibroblast cell cultures (C. Sinzger, unpublished data) (39
). Following reconstitution on HFF, virions and DBs of the novel strain Towne-UL130rep were purified by gradient ultracentrifugation. These particles were subjected to immunoblot analysis and probed with antibodies against pUL130, pUL128, and glycoprotein B (loading control, Fig. 2B
). Particles from the laboratory strains HB5, the parental strain Towne, and from TB40/E were carried along as a control. pUL130 and pUL128 were both detectable in virions and DBs of Towne-UL130rep. According to current knowledge, both proteins are inserted into the envelope of extracellular viral particles only in complex with gH/gL and UL131. This suggested that the PC was successfully restored in Towne-UL130rep. To formally prove the functionality of the complex, ARPE-19 cells were infected with Towne-UL130rep. Infection efficiency was compared to parallel cultures infected with the parental Towne strain or with TB40/E and was measured by counting immediate early protein 1 (IE1)-positive cells (Fig. 2C
). The median number of IE1-positive cells in the TB40/E-infected cultures was set to 100%. The infection rate of Towne-UL130rep was comparable to that of TB40/E. The parental Towne strain showed less than 20% IE1-positive cells, compared to the rate of positive cells, following TB40/E infection in that culture. Since the PC is considered to be important for the infection of epithelial cells, these results provided further evidence for successful reconstitution of the PC in Towne-UL130rep.
To further address this issue, ARPE-19 cells were tested for their capacity to support Towne-UL130rep DNA replication. Cells were infected with Towne or Towne-UL130rep, using identical input genome copy numbers. HFFs were infected in parallel (Fig. 2D
). Genome replication was assessed by quantitative PCR analysis. Towne-UL130rep genome replication was detectable in epithelial cells. No replication of the parental strain Towne was seen. Replication was detectable to high levels in HFF, with little difference between Towne and Towne-UL130rep. To address whether virus was released from Towne-UL130rep-infected epithelial cells and HFFs, culture supernatants were probed for viral genomes, again using quantitative PCR analysis (Fig. 2E
). Supernatants from Towne-UL130rep-infected epithelial cells contained increasing amounts of viral genomes over time, indicating that infectious virus was being released. In contrast, a decrease in the genome copy numbers was seen in Towne-infected ARPE-19 cultures. HFFs infected with either strain released equal genome copy numbers. Taken together, these data showed that the Towne strain had been functionally repaired in its capacity to replicate in epithelial cells and to release progeny from these cultures.
To analyze if the phenotype of Towne-UL130rep resembled that of other PC-positive HCMV strains, we investigated the cytopathogenic changes that were induced by that virus on HFFs, using immunofluorescence analyses. Restoration of the PC in the laboratory strain AD169 is associated with the appearance of multinucleated giant cells (35
). It is also a characteristic of the PC-positive strain TB40-E (C. Sinzger, unpublished data). To verify that Towne-UL130rep shared that phenotype, HFFs were infected for 8 days. Cells were then tested for the induction of cell-cell fusion by indirect immunofluorescence staining, using labeling with a pp65-specific monoclonal antibody for detection (Fig. 3
). Towne-UL130rep-infected HFFs indeed formed large multinucleated cells which were absent in Towne-infected cells. Substantial cell-cell fusion was also seen in cultures that were infected with TB40/E for a control. These results confirmed that the repair of UL130 in the HCMV strain Towne created a virus that was phenotypically indistinguishable from other PC-positive viruses.