SummaryHuman cytomegalovirus (HCMV) infection rewires host cell metabolism, up-regulating flux from glucose into acetyl-CoA to feed fatty acid metabolism, with saturated very long-chain fatty acids (VLFCA) required for production of infectious virion progeny. The human genome encodes seven elongase enzymes (ELOVL) that extend long chain fatty acids into VLCFA. Here we identify ELOVL7 as pivotal for HCMV infection. HCMV induces ELOVL7 by more than 150-fold. This induction is dependent on mTOR and SREBP-1. ELOVL7 knockdown or mTOR inhibition impairs HCMV-induced fatty acid elongation, HCMV particle release, and infectivity per particle. ELOVL7 overexpression enhances HCMV replication. During HCMV infection, mTOR activity is maintained by the viral protein pUL38. Expression of pUL38 is sufficient to induce ELOVL7, and pUL38-deficient virus is partially defective in ELOVL7 induction and fatty acid elongation. Thus, through its ability to modulate mTOR and SREBP-1, HCMV induces ELOVL7 to synthesize the saturated VLCFA required for efficient virus replication.

For a retrovirus such as HIV to be infectious, a properly formed capsid is needed; however, unusually among viruses, retrovirus capsids are highly variable in structure. According to the fullerene conjecture, they are composed of hexamers and pentamers of CA protein, with a capsid’s shape varying according to how the twelve pentamers are distributed and its size depending on the number of hexamers. Hexamers have been studied in planar and tubular arrays but the predicted pentamers have not been observed. Here we report cryo-electron microscopic analyses of two in vitro-assembled capsids of Rous sarcoma virus. Both are icosahedrally symmetric: one is composed of 12 pentamers; the other, of 12 pentamers and 20 hexamers. Fitting of atomic models of the two CA domains into the reconstructions shows three distinct inter-subunit interactions. These observations substantiate the fullerene conjecture, show how pentamers are accommodated at vertices, support the inference that nucleation is a crucial morphologic determinant, and imply that electrostatic interactions govern the differential assembly of pentamers and hexamers.

Human cytomegalovirus hijacks host cell metabolism, increasing the flux of carbon from glucose to malonyl-CoA, the committed precursor to fatty acid synthesis and elongation. Inhibition of acetyl-CoA carboxylase blocks the production of progeny virus. To probe further the role of fatty acid metabolism during infection, we performed an siRNA screen to identify host cell metabolic enzymes needed for the production of infectious cytomegalovirus progeny. The screen predicted that multiple long chain acyl-CoA synthetases and fatty acid elongases are needed during infection, and the levels of RNAs encoding several of these enzymes were upregulated by the virus. Roles for acyl-CoA synthetases and elongases during infection were confirmed by using small molecule antagonists. Consistent with a role for these enzymes, mass spectrometry-based fatty acid analysis with13C-labeling revealed that malonyl-CoA is consumed by elongases to produce very long chain fatty acids, generating an approximately 8-fold increase in C26-C34 fatty acid tails in infected cells. The virion envelope was yet further enriched in C26-C34 saturated fatty acids, and elongase inhibitors caused the production of virions with lower levels of these fatty acids and markedly reduced infectivity. These results reveal a dependence of cytomegalovirus on very long chain fatty acid metabolism.

During retroviral maturation, the CA protein undergoes dramatic structural changes and establishes unique intermolecular interfaces in the mature capsid shell that are different from those that existed in the immature precursor. The most conserved region of CA, the major homology region (MHR), has been implicated in both immature and mature assembly, although the precise contribution of the MHR residues to each event has been largely undefined. To test the roles of specific MHR residues in mature capsid assembly, an in vitro system was developed that allowed for the first-time formation of Rous sarcoma virus CA into structures resembling authentic capsids. The ability of CA to assemble organized structures was destroyed by substitutions of two conserved hydrophobic MHR residues and restored by second-site suppressors, demonstrating that these MHR residues are required for the proper assembly of mature capsids in addition to any role that these amino acids may play in immature particle assembly. The defect caused by the MHR mutations was identified as an early step in the capsid assembly process. The results provide strong evidence for a model in which the hydrophobic residues of the MHR control a conformational reorganization of CA that is needed to initiate capsid assembly and suggest that the formation of an interdomain interaction occurs early during maturation.The retroviral CA protein plays critical structural roles in each of the two distinct stages of virion assembly. When an immature particle is formed by the polymerization of the Gag polyprotein, the N-terminal CA domain (NTD) and C-terminal CA domain (CTD) embedded within Gag control packing and assembly (1,3,12,14,17,41,54,77,80). Subsequently, the processing of Gag by the viral protease initiates a maturation process in which the structural proteins MA (matrix), CA (capsid), and NC (nucleocapsid) are released from Gag. The free CA polymerizes as a capsid shell around the genomic RNA and NC protein, creating the core of the mature virion. The maturation events are complex and include disruption of the CA-CA interfaces that held the Gag proteins together, conformational changes within each domain of CA, and formation of new CA-CA interfaces of the mature capsid shell (6,11,12,24,31,44,52,53,58,74,(79)(80)(81).In spite of limited sequence similarity, the three-dimensional structure of mature CA is highly conserved among retroviruses and consists of the two mostly ␣-helical domains, NTD and CTD, connected by an interdomain linker (8,18,19,24,29,36,37). After maturation is completed, the final capsid shell consists of a lattice of CA hexamers, established by NTD-NTD interactions and linked by CTD-CTD dimerization (24,25,27,43,48,51,53,79). The dimer interface is formed by the dimerization helix, the second helix of the CTD. A third interface, an NTD-CTD interdomain interaction that forms during maturation, was originally predicted by a genetic study of the Alpharetrovirus Rous sarcoma virus (RSV) and subsequently confirmed and mapped by biochemical and st...