Structure of human cytomegalovirus virion reveals host tRNA binding to capsid-associated tegument protein pp150

Abstract

Under the Baltimore nucleic acid-based virus classification scheme, the herpesvirus human cytomegalovirus (HCMV) is a Class I virus, meaning that it contains a double-stranded DNA genome-and no RNA. Here, we report sub-particle cryoEM reconstructions of HCMV virions at 2.9 Å resolution revealing structures resembling non-coding transfer RNAs (tRNAs) associated with the virion's capsid-bound tegument protein, pp150. Through deep sequencing, we show that these RNA sequences match human tRNAs, and we built atomic models using the most abundant tRNA species. Based on our models, tRNA recruitment is mediated by the electrostatic interactions between tRNA phosphate groups and the helix-loop-helix motif of HCMV pp150. The specificity of these interactions may explain the absence of such tRNA densities in murine cytomegalovirus and other human herpesviruses.

Fig. 1. CryoEM reconstruction of HCMV virion and identification of tRNA by sequencing.

a Central slices of 3D reconstructed HCMV virion (top) and sub-particle reconstruction of the boxed area (bottom) shows the relative locations of dsDNA genome, hexon channel, pp150nt, and L-shaped tRNA densities. b Icosahedral reconstruction of HCMV virion. Fivefold, threefold, and twofold axes are denoted by the pentagon, triangle, and oval shapes, respectively. The L-shaped tRNA density (cyan) was resolved. c CryoEM structure of L-shaped density. d, e Analysis of RNA molecules associated with HCMV virus on 15% TBE-urea PAGE. RNA was purified from isolated HCMV virions and separated on electrophoresis as described in the “Methods” section. The curly bracket indicates the size range of RNA that has been subjected to deep sequencing. Infection of MRC-5 cells with HCMV, virion and virion-bound RNA isolation, and electrophoresis analysis was done independently four times (n = 4). f Multi-map-corrected count number distribution of first 100 abundant human genomic tRNA (orange) and 22 mitochondrial tRNA (blue). g Gene sequence of the first 6 abundant human genomic tRNA, colored by the domains of tRNA. h de novo atomic model of tRNA-Glu-CTC-1-1 built based on the cryoEM density. Insets show ribbon-and-stick models in mesh density of the boxed regions. dsDNA double-stranded DNA (in a), pp150nt pp150 N-terminal domain (in a and b), nt nucleotides (in d).

Fig. 2. Interaction between tRNA and set-of-three pp150nt.

a Atomic model of tRNA and its three interacting pp150nts. Insets show the identified tRNA-pp150 interaction sites. b Upper panel: helix-loop-helix (blue) and capsid binding (magenta) motifs on a representative pp150nt model. Bottom panel: a pp150nt map surface colored by electrostatic potential superposed with bound tRNA backbone, shown as ribbon.

Fig. 3. Comparison between the pp150 and glutamyl-tRNA synthetase (GluRS) on their interactions with tRNA anticodon.

a Atomic model of tRNA and its three interacting pp150nts. Inset shows Arg 11 interaction with the anticodon region of tRNA. b Atomic model of GluRS and tRNA. Inset shows Arg358 interaction with anticodon.

Fig. 4. Distribution of tRNA on different triplexes.

a A facet of the icosahedron of HCMV reconstruction. b, Enlarged view of the region in the blue dashed box in a but displayed with a lower threshold. Ta’s tRNA-like density becomes visible at a lower threshold. No tRNA binds on Tc. σ represents the threshold used to display 3D structures. c Asymmetric reconstruction (e.g., C1) of the region in the red dashed box in a. Tf’s tRNA-like density becomes visible at a lower threshold. d–f Set-of-three pp150 shown as pipes and planks from different triplex areas are superimposed. tRNAs are shown as semi-transparent ribbons. Superimposition among set-of-three pp150 and their corresponding bound tRNA molecules on Tb, Td, and Te (d); on Ta, Tb, and Tf (e); and on Tb and Tc regions (f). The change in pp150 organization affects the ability of tRNA to bind to the pp150.