Human cytomegalovirus deploys molecular mimicry to recruit VPS4A to sites of virus assembly.

The AAA-type ATPase VPS4 is recruited by proteins of the endosomal sorting complex required for transport III (ESCRT-III) to catalyse membrane constriction and membrane fission. VPS4A accumulates at the cytoplasmic viral assembly complex (cVAC) of cells infected with human cytomegalovirus (HCMV), the site where nascent virus particles obtain their membrane envelope. Here we show that VPS4A is recruited to the cVAC via interaction with pUL71. Sequence analysis, deep-learning structure prediction, molecular dynamics and mutagenic analysis identify a short peptide motif in the C-terminal region of pUL71 that is necessary and sufficient for the interaction with VPS4A. This motif is predicted to bind the same groove of the N-terminal VPS4A Microtubule-Interacting and Trafficking (MIT) domain as the Type 2 MIT-Interacting Motif (MIM2) of cellular ESCRT-III components, and this viral MIM2-like motif (vMIM2) is conserved across ß-herpesvirus pUL71 homologues. However, recruitment of VPS4A by pUL71 is dispensable for HCMV morphogenesis or replication and the function of the conserved vMIM2 during infection remains enigmatic. VPS4-recruitment via a vMIM2 represents a previously unknown mechanism of molecular mimicry in viruses, extending previous observations that herpesviruses encode proteins with structural and functional homology to cellular ESCRT-III components.

Altered plasma membrane abundance of the sulfatide-binding protein NF155 links glycosphingolipid imbalances to demyelination.

Myelin is a multilayered membrane that tightly wraps neuronal axons, enabling efficient, high-speed signal propagation. The axon and myelin sheath form tight contacts, mediated by specific plasma membrane proteins and lipids, and disruption of these contacts causes devastating demyelinating diseases. Using two cell-based models of demyelinating sphingolipidoses, we demonstrate that altered lipid metabolism changes the abundance of specific plasma membrane proteins. These altered membrane proteins have known roles in cell adhesion and signaling, with several implicated in neurological diseases. The cell surface abundance of the adhesion molecule neurofascin (NFASC), a protein critical for the maintenance of myelin-axon contacts, changes following disruption to sphingolipid metabolism. This provides a direct molecular link between altered lipid abundance and myelin stability. We show that the NFASC isoform NF155, but not NF186, interacts directly and specifically with the sphingolipid sulfatide via multiple binding sites and that this interaction requires the full-length extracellular domain of NF155. We demonstrate that NF155 adopts an S-shaped conformation and preferentially binds sulfatide-containing membranes in cis, with important implications for protein arrangement in the tight axon-myelin space. Our work links glycosphingolipid imbalances to disturbance of membrane protein abundance and demonstrates how this may be driven by direct protein-lipid interactions, providing a mechanistic framework to understand the pathogenesis of galactosphingolipidoses.

Non-native fold of the putative VPS39 zinc finger domain.

Background: The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is involved in regulating the fusion of late endosomes and autophagosomes with lysosomes in eukaryotes. The C-terminal regions of several HOPS components have been shown to be required for correct complex assembly, including the C-terminal really interesting new gene (RING) zinc finger domains of HOPS components VPS18 and VPS41. We sought to structurally characterise the putative C-terminal zinc finger domain of VPS39, which we hypothesised may be important for binding of VPS39 to cellular partners or to other HOPS components. Methods: We recombinantly expressed, purified and solved the crystal structure of the proposed zinc-binding region of VPS39. Results: In the structure, this region forms an anti-parallel ß-hairpin that is incorporated into a homotetrameric eight-stranded ß-barrel. However, the fold is stabilised by coordination of zinc ions by residues from the purification tag and an intramolecular disulphide bond between two predicted zinc ligands. Conclusions: We solved the structure of the VPS39 C-terminal domain adopting a non-native fold. Our work highlights the risk of non-native folds when purifying small zinc-containing domains with hexahistidine tags. However, the non-native structure we observe may have implications for rational protein design.

Insights into herpesvirus assembly from the structure of the pUL7:pUL51 complex.

Herpesviruses acquire their membrane envelopes in the cytoplasm of infected cells via a molecular mechanism that remains unclear. Herpes simplex virus (HSV)-1 proteins pUL7 and pUL51 form a complex required for efficient virus envelopment. We show that interaction between homologues of pUL7 and pUL51 is conserved across human herpesviruses, as is their association with trans-Golgi membranes. We characterized the HSV-1 pUL7:pUL51 complex by solution scattering and chemical crosslinking, revealing a 1:2 complex that can form higher-order oligomers in solution, and we solved the crystal structure of the core pUL7:pUL51 heterodimer. While pUL7 adopts a previously-unseen compact fold, the helix-turn-helix conformation of pUL51 resembles the cellular endosomal complex required for transport (ESCRT)-III component CHMP4B and pUL51 forms ESCRT-III-like filaments, suggesting a direct role for pUL51 in promoting membrane scission during virus assembly. Our results provide a structural framework for understanding the role of the conserved pUL7:pUL51 complex in herpesvirus assembly.

Most people suffer from occasional cold sores, which are caused by the herpes simplex virus. This virus causes infections that last your entire life, but for the most part it lies dormant in your cells and reactivates only at times of stress. When it reactivates, the virus manipulates host cells to make new virus particles that may spread the infection to other people. Like many other viruses, herpes simplex viruses also steal jelly-like structures known as membranes from their host cells to form protective coats around new virus particles. In cells from humans and other animals, proteins belonging to a molecular machine known as ESCRT form filaments that bend and break membranes as the cells require. Many viruses hijack the ESCRT machinery to wrap membranes around new virus particles. However, herpes simplex viruses do not follow the usual rules for activating this machine. Instead, they rely on two viral proteins called pUL7 and pUL51 to hot-wire the ESCRT machinery. Previous studies have shown that these two proteins bind to each other, but it remained unclear how they work. Butt et al. used a combination of biochemical and biophysical techniques to solve the three-dimensional structures of pUL7 and pUL51 when bound to each other. The experiments determined that the structure of pUL51 resembles the structures of different components in the ESCRT machinery. Like the ESCRT proteins, pUL51 formed filaments, suggesting that pUL51 bends membranes in cells and that pUL7 blocks it from doing so until the time is right. Further experiments showed that the equivalents of pUL7 and pUL51 in other members of the herpes virus family also bind to each other in a similar way. These findings reveal that herpes simplex viruses and their close relatives have evolved a different strategy than many other viruses to steal membranes from host cells. Interfering with this mechanism may provide new avenues for designing drugs or improving vaccines against these viruses. The pUL7 and pUL51 proteins may also inspire new tools in biotechnology that could precisely control the shapes of biological membranes.

Propensity of a picornavirus polymerase to slip on potyvirus-derived transcriptional slippage sites.

The substitution rates of viral polymerases have been studied extensively. However less is known about the tendency of these enzymes to 'slip' during RNA synthesis to produce progeny RNAs with nucleotide insertions or deletions. We recently described the functional utilization of programmed polymerase slippage in the family Potyviridae. This slippage results in either an insertion or a substitution, depending on whether the RNA duplex realigns following the insertion. In this study we investigated whether this phenomenon is a conserved feature of superfamily I viral RdRps, by inserting a range of potyvirus-derived slip-prone sequences into a picornavirus, Theiler's murine encephalomyelitis virus (TMEV). Deep-sequencing analysis of viral transcripts indicates that the TMEV polymerase 'slips' at the sequences U6-7 and A6-7 to insert additional nucleotides. Such sequences are under-represented within picornaviral genomes, suggesting that slip-prone sequences create a fitness cost. Nonetheless, the TMEV insertional and substitutional spectrum differed from that previously determined for the potyvirus polymerase.