Cytomegalovirus pp65 limits dissemination but is dispensable for persistence
Daniel Malouli, … , Louis J. Picker, Klaus Früh
Daniel Malouli, … , Louis J. Picker, Klaus Früh
Published April 1, 2014
Citation Information: J Clin Invest. 2014;124(5):1928-1944. https://doi.org/10.1172/JCI67420.
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Research Article Virology

Cytomegalovirus pp65 limits dissemination but is dispensable for persistence

Abstract

The most abundantly produced virion protein in human cytomegalovirus (HCMV) is the immunodominant phosphoprotein 65 (pp65), which is frequently included in CMV vaccines. Although it is nonessential for in vitro CMV growth, pp65 displays immunomodulatory functions that support a potential role in primary and/or persistent infection. To determine the contribution of pp65 to CMV infection and immunity, we generated a rhesus CMV lacking both pp65 orthologs (RhCMVΔpp65ab). While deletion of pp65ab slightly reduced growth in vitro and increased defective particle formation, the protein composition of secreted virions was largely unchanged. Interestingly, pp65 was not required for primary and persistent infection in animals. Immune responses induced by RhCMVΔpp65ab did not prevent reinfection with rhesus CMV; however, reinfection with RhCMVΔUS2-11, which lacks viral-encoded MHC-I antigen presentation inhibitors, was prevented. Unexpectedly, induction of pp65b-specific T cells alone did not protect against RhCMVΔUS2-11 challenge, suggesting that T cells targeting multiple CMV antigens are required for protection. However, pp65-specific immunity was crucial for controlling viral dissemination during primary infection, as indicated by the marked increase of RhCMVΔpp65ab genome copies in CMV-naive, but not CMV-immune, animals. Our data provide rationale for inclusion of pp65 into CMV vaccines but also demonstrate that pp65-induced T cell responses alone do not recapitulate the protective effect of natural infection.

Authors

Daniel Malouli, Scott G. Hansen, Ernesto S. Nakayasu, Emily E. Marshall, Colette M. Hughes, Abigail B. Ventura, Roxanne M. Gilbride, Matthew S. Lewis, Guangwu Xu, Craig Kreklywich, Nathan Whizin, Miranda Fischer, Alfred W. Legasse, Kasinath Viswanathan, Don Siess, David G. Camp II, Michael K. Axthelm, Christoph Kahl, Victor R. DeFilippis, Richard D. Smith, Daniel N. Streblow, Louis J. Picker, Klaus Früh

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Figure 2

Intact and defective viral particles are secreted from fibroblasts infected with Δpp65ab.

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Intact and defective viral particles are secreted from fibroblasts infec...
(A) Image of a Nycodenz gradient loaded with RhCMVΔpp65ab, and electron microscope images of virions (top image) and defective particles (bottom image) contained in the visible bands of the gradient. (B) Electron microscope image of purified RhCMVΔpp65ab virions showing the purity of the sample. (C) Purified RhCMV WT and Δpp65ab virions were lysed, and 10 μg protein was electrophoretically separated using NuPAGE MOPS gradient gels and visualized by Coomassie blue staining. (D) Western blots of 5 μg gradient-purified RhCMV 68-1 WT and viral mutant Δpp65ab stained for RhCMV pp65a, pp65b, or a RhCMV-specific antibody. (E) Various electron microscopy images of purified WT and Δpp65ab virions were taken, and the diameters of virions, capsids, and the tegument were determined in multiple images and magnifications (WT, n = 39; Δpp65ab, n = 45). The mean diameters with their respective SDs are shown, and Student’s t tests were performed to determine the P values. Scale bars: 100 nm.