Cauliflower mosaic virus (CaMV) gene VI encodes a multifunctional protein (P6) involved in the translation of viral RNA, the formation of inclusion bodies, and the determination of host range. Arabidopsis thaliana ecotype Tsu-0 prevents the systemic spread of most CaMV isolates, including CM1841. However, CaMV isolate W260 overcomes this resistance. In this paper, the N-terminal 110 amino acids of P6 (termed D1) were identified as the resistance-breaking region. D1 also bound full-length P6. Furthermore, binding of W260 D1 to P6 induced higher β-galactosidase activity and better leucine-independent growth in the yeast two-hybrid system than its CM1841 counterpart. Thus, W260 may evade Tsu-0 resistance by mediating P6 self-association in a manner different from that of CM1841. Because Tsu-0 resistance prevents virus movement, interaction of P6 with P1 (CaMV movement protein) was investigated. Both yeast two-hybrid analyses and maltose-binding protein pull-down experiments show that P6 interacts with P1. Although neither half of P1 interacts with P6, the N-terminus of P6 binds P1. Interestingly, D1 by itself does not interact with P1, indicating that different portions of the P6 N-terminus are involved in different activities. The P1-P6 interactions suggest a role for P6 in virus transport, possibly by regulating P1 tubule formation or the assembly of movement complexes.

Immunoglobulins are only moderately effective for the treatment of human cytomegalovirus (HCMV) infections, possibly due to ineffectiveness against cell-associated virus spread. To overcome this limitation, we aimed to identify individuals with exceptional antibodies in their plasma that can efficiently block the cell-associated spread of HCMV. A Gaussia luciferase-secreting mutant of the cell-associated HCMV strain Merlin was generated, and luciferase activity evaluated as a readout for the extent of cell-associated focal spread. This reporter virus-based assay was then applied to screen plasma samples from 8400 HCMV-seropositive individuals for their inhibitory effect, including direct-acting antiviral drugs as positive controls. None of the plasmas reduced virus spread to the level of these controls. Even the top-scoring samples that partially reduced luciferase activity in the screening assay failed to inhibit focal growth when reevaluated with a more accurate, immunofluorescence-based assay. Selected sera with high neutralizing capacity against free viruses were analyzed separately, and none of them prevented the focal spread of three recent clinical HCMV isolates nor reduced the number of particles transmitted, as demonstrated with a fluorescent Merlin mutant. We concluded that donors with cell-to-cell-spread-inhibiting plasma are nonexistent or extremely rare, emphasizing cell-associated spread as a highly efficient immune escape mechanism of HCMV.

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The development of a vaccine against human cytomegalovirus infection (HCMV) is a high-priority medical goal. The viral pentameric protein complex consisting of glycoprotein H (gH)/gL/UL128-131A (PC) is considered to be an important vaccine component. Its relevance to the induction of a protective antibody response is, however, still a matter of debate. We addressed this issue by using subviral dense bodies (DBs) of HCMV. DBs are exceptionally immunogenic. Laboratory HCMV strain DBs harbor important neutralizing antibody targets, like the glycoproteins B, H, L, M, and N, but they are devoid of the PC. To be able to directly compare the impact of the PC on the levels of neutralizing antibody (NT-abs) responses, a PC-positive variant of the HCMV laboratory strain Towne was established by bacterial artificial chromosome (BAC) mutagenesis (Towne-UL130rep). This strain synthesized PC-positive DBs upon infection of fibroblasts. These DBs were used in side-by-side immunizations with PC-negative Towne DBs. Mouse and rabbit sera were tested to address the impact of the PC on DB immunogenicity. The neutralizing antibody response to PC-positive DBs was superior to that of PC-negative DBs, as tested on fibroblasts, epithelial cells, and endothelial cells and for both animal species used. The experiments revealed the potential of the PC to enhance the antibody response against HCMV. Of particular interest was the finding that PC-positive DBs induced an antibody response that blocked the infection of fibroblasts by a PC-positive viral strain more efficiently than sera following immunizations with PC-negative particles. IMPORTANCE Infections with the human cytomegalovirus (HCMV) may cause severe and even life-threatening disease manifestations in newborns and immunosuppressed individuals. Several strategies for the development of a vaccine against this virus are currently pursued. A critical question in this respect refers to the antigenic composition of a successful vaccine. Using a subviral particle vaccine candidate, we show here that one protein complex of HCMV, termed the pentameric complex (PC), enhances the neutralizing antibody response against viral infection of different cell types. We further show for the first time that this not only relates to the infection of epithelial or endothelial cells; the presence of the PC in the particles also enhanced the neutralizing antibody response against the infection of fibroblasts by HCMV. Together, these findings argue in favor of including the PC in strategies for HCMV vaccine development.

To improve the potency of anti-human cytomegalovirus (HCMV) immunoglobulin preparations, we intended to find elite neutralizers among 9000 HCMV-seropositive blood donors. We identified the top 2.6% neutralizers by use of high-throughput screening and further analyzed the 80 neutralizers with the most effective plasma for strain-independent activity. Of those, 58 had broad neutralizing activity against various HCMV strains and hence were regarded as elite neutralizers. All elite neutralizers were then analyzed to determine their effect on individual virus particles during entry. Most had plasma specimens that preferentially inhibited viral penetration, whereas 2 had exceptional plasma specimens that prevented adsorption of virus to cells. Furthermore, the neutralizing capacity of plasma samples from 3 randomly chosen elite neutralizers was up to 10-fold higher than that for commercial immunoglobulins. In a retrospective analysis of 6 selected donors, anti-HCMV neutralization titers in repeated donations were constantly high over 5 years. In conclusion, plasma samples from elite-neutralizing donors can be considered to improve antibody-based treatment of HCMV infections.