Structural Models for Roseolovirus U20 And U21: Non-Classical MHC-I Like Proteins From HHV-6A, HHV-6B, and HHV-7

Abstract

Human roseolovirus U20 and U21 are type I membrane glycoproteins that have been implicated in immune evasion by interfering with recognition of classical and non-classical MHC proteins. U20 and U21 are predicted to be type I glycoproteins with extracytosolic immunoglobulin-like domains, but detailed structural information is lacking. AlphaFold and RoseTTAfold are next generation machine-learning-based prediction engines that recently have revolutionized the field of computational three-dimensional protein structure prediction. Here, we review the structural biology of viral immunoevasins and the current status of computational structure prediction algorithms. We use these computational tools to generate structural models for U20 and U21 proteins, which are predicted to adopt MHC-Ia-like folds with closed MHC platforms and immunoglobulin-like domains. We evaluate these structural models and place them within current understanding of the structural basis for viral immune evasion of T cell and natural killer cell recognition.

Keywords: MHC1b; human herpesvirus; immune recognition; immunoevasion; machine learning; major histocompatibility protein; natural killer cell ligand; structure prediction.

Figure 1

Phylogenetic relationships of viruses and viral immunoevasins discussed in this work. The complete genomic DNA sequences from HHV-6A (strain U1102), HHV-6B (strain Z29), HHV-7 (strain JI), HCMV (strain HAN-SCT17), and MCMV (strain N1) and poxviruses Cowpox (strain Brighton Red) and Yaba-like Disease Virus (strain Amano) were retrieved from the VIPR database (Table 1) and aligned using the MEGA11 software package. MEGA was then used to generate a maximum likelihood phylogeny reconstruction (1). Scale bar represents the probability of a nucleotide substitution at a given site, calculated for select betaherpesviruses. Viral MHC-like proteins that contribute to evasion of host T cell and NK cell responses and are discussed in this work are indicated next to the virus labels.

Figure 2

Viral evasion of T cell and NK cell recognition. Viral MHC-based immune evasion pathways employed by betaherpesviruses and poxviruses are shown. MHC-Ia refers to classical polymorphic peptide-binding class I MHC molecules, MHC-Ib refers to non-classical non-polymorphic class I MHC homologs that do not present peptides. cMHC-Ia and cMHC-Ib refer to cellular (host) protein, vMHC-Ia and vMHC-Ib refer to viral homologs. Other mechanisms employed by betaherpesviruses and poxvirus to evade T cell and NK cell recognition involve interference with antigen processing, transcriptional regulation, and innate immune evasion, but those pathways are not known to involve vMHC-I molecules and are not included here. (A) cMHC-Ia present peptides to CD8+ T cells, triggering induction of cytolytic pathways upon T cell receptor (TCR binding). (B) vMHC-Ib block this pathway by redirecting cMHC-Ia to proteasomes or lysosomes for destruction. (C) Inhibitory natural killer cell receptors (NKR) can recognize the loss of surface cMHC-Ia as part of the “missing-self” recognition system. vMHC-Ia combat this by engaging inhibitory NKR. (D) NK cells sense cellular stress using activating NKR to recognize stress-induced cMHC-Ib proteins. (E) vMHC-Ib block this pathway by redirecting cMHC-Ib to proteasomes or lysosomes for destruction. (F) vMHC-Ib can block activating NKRs by either masking them on the surface or by secreting vMHC-Ib variants that can engage activating NKR and competitively block recognition of cMHC-1b stress ligands. Wavy lines indicate glycosylphosphatidylinositol (GPI) membrane anchors used by some ULBP proteins instead of transmembrane helices. (G) Paradoxically, vMHC-Ib can engage activating NKR for reasons that remain poorly understood. (H) Another vMHC-Ib binds the inflammatory cytokine TNFα, sequestering it from productive engagement with TNF-receptors.

Figure 3

Structural aspects of classical and non-classical MHC immunoevasion. Crystal structures of cMHC-I (orange) and vMHC-I (green) in complex with cellular binding partners (Table 2). (A) cMHC-Ia protein HLA-A2 bound to inhibitory NKR LIR-1A (PDB: 1P7Q). (B) viral cMHC-Ia homolog UL18 from HCMV bound to inhibitory NKR LIR-1A (PDB: 3D2U). (C) cMHC-Ib ULBP3 bound to the activating NKR NKG2D (cyan) (PDB: 1KCG). (D) ULBP homolog CPXV018 from cowpox virus interacting with activating NKR NKG2D (cyan) (PDB: 4PDC). (E) m152, a vMHC-Ib protein bound to cMHC-Ib RAE-1γ, promoting retention and eventual degradation (PDB: 4G59). (F) vMHC-Ib UL16 from HCMV bound to cMHC-Ib MICB (PDB: 2WY3 and 1JE6). (G) Viral Ig-domain protein US2 from HCMV binding cMHC-Ia HLA-A2, targeting it for degradation (PDB: 1IM3). (H) Viral Ig-domain protein CPXV203 from cowpox virus binding cMHC-Ia HLA-A2, targeting it for degradation (PDB: 4HKJ). (I) cMHC-Ia protein H-2Kb bound to inhibitory NKR Ly49C (38CK). (J) cMHC-Ia protein H-2Dd bound to inhibitory NKR Ly49A (PDB: 1QO3). (K) vMHC-Ib m157 from HCMV interacting with activating NKR Ly49H (PDB: 4JO8). (L) vMHC-Ib 2L protein from YDL virus interacting with cytokine TNFα (PDB:3IT8).

Figure 4

Sequence homology of U20 and U22 from HHV-6A, HHV-6B, HHV-7. The amino acid sequences for U20 and U21 from HHV-6A, -6B, and -7 were retrieved from the Uniprot database (Table 3). They were then aligned using Clustal Omega (49). Top: Sequence identity matrix for U20 and U21 from HHV-6A, HHV-6B, and HHV-6. Bottom Left: Alignment of U20 sequences from HHV-6A (strain U1102), HHV-6B (Z29) and HHV-7 (JI). Amino acids are colored by type. Identities are indicated by an asterisk and similarities are indicated with a colon. Bottom Right: Alignment of U21 sequences from these same strains, colored and annotated as in B.

Figure 5

Evolution of U20 structure prediction. (A) Structure of HLA-A2, a classical MHC-Ia protein that adopts the canonical MHC fold. MHC α1 and α2 domains in the MHC platform and α3 immunoglobulin-like domain colored by secondary structure for comparison with panel B, with the MHC-associated non-polymorphic small subunit β2-microglobulin shown in green and bound peptide shown in yellow. (B) Secondary structure prediction from JPRED (48, 53), figure reproduced with permission from reference (48). (C–F), Three-dimensional structure predictions from (C), Phyre², (D), I-TASSER, (E), AlphaFold, and (F), RoseTTAfold. Top panels show ribbon diagrams colored by linear protein sequence; bottom panels show tube diagrams colored by modeling confidence.

Figure 6

Roseolovirus-specific U20-U26 gene cluster. (A) Schematic diagram of HHV-6B gene organization, modified from (52). The U20-U26 cluster of genes specific to roseolovirus is indicated in blue. (B) AlphaFold predictions for members of the HHV-6B U20-U26 gene cluster.

Figure 7

AlphaFold and RoseTTAfold structures for U20 and U21. AlphaFold structure predictions for U20 from HHV-6A (A), HHV-6B (B), and HHV-7 (C), and for U21 from HHV-6A (D), HHV-6B (E), and HHV-7 (F). RoseTTAfold structure predictions for U20 from HHV-6A (G), HHV-6B (H), and HHV-7 (I), and for U21 from HHV-6A (J), HHV-6B (K), and HHV-7 (L). The same sequences used in the alignments were processed through SignalP to identify signal sequences and TMHMM to identify transmembrane domains (73, 74). The sequences were then truncated to reflect the extracellular portion of the protein before use for structure prediction. For Phyre2, sequences were submitted for analysis via the Phyre2 server utilizing the “Intensive” modeling mode (57). For I-TASSER, we provided a protein sequence and specified no constraints or template exclusions (59). For AlphaFold we used the AlphaFold2 Advanced Script hosted by Google Colab (23). We used the default settings, specifically utilizing de novo generation of multisequence alignments with mmseqs2. We generated 5 models for each prediction with 1 ensemble, 3 recycles, a tolerance of 0, and 1 random seed. For RoseTTAfold we used the Robetta server and predicted on a single sequence using the “RoseTTAFold” method with no additional constraints (24). Both AlphaFold2 and RoseTTAFold generate several structural models from each modeling run. Figures were generated using the top-scoring model. For AlphaFold and RoseTTAfold, the top five scoring models from each run were examine for consistency with the model presented. Scale bars representing the confidence intervals are shown on a linear scale, RMSD for Phye2 and RoseTTA, TM-score for ITASSER, and pLDDT for AlphaFold. (A-L) Predicted structural models and previously determined crystal structures were visualized and figures were prepared using the Pymol molecular graphics program (75). Ribbon and tube diagrams colored as in Figure 5. Asterisks indicate features highlighted in text.