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Retroviruses assemble and bud from infected cells in an immature form and require proteolytic maturation for infectivity. The CA (capsid) domains of the Gag polyproteins assemble a protein lattice as a truncated sphere in the immature virion. Proteolytic cleavage of Gag induces dramatic structural rearrangements; a subset of cleaved CA subsequently assembles into the mature core, whose architecture varies among retroviruses. Murine leukemia virus (MLV) is the prototypical γ-retrovirus and serves as the basis of retroviral vectors, but the structure of the MLV CA layer is unknown. Here we have combined X-ray crystallography with cryoelectron tomography to determine the structures of immature and mature MLV CA layers within authentic viral particles. This reveals the structural changes associated with maturation, and, by comparison with HIV-1, uncovers conserved and variable features. In contrast to HIV-1, most MLV CA is used for assembly of the mature core, which adopts variable, multilayered morphologies and does not form a closed structure. Unlike in HIV-1, there is similarity between protein–protein interfaces in the immature MLV CA layer and those in the mature CA layer, and structural maturation of MLV could be achieved through domain rotations that largely maintain hexameric interactions. Nevertheless, the dramatic architectural change on maturation indicates that extensive disassembly and reassembly are required for mature core growth. The core morphology suggests that wrapping of the genome in CA sheets may be sufficient to protect the MLV ribonucleoprotein during cell entry.

HIV-1 Nef, a protein important for the development of AIDS, has well-characterized effects on host membrane trafficking and receptor downregulation. By an unidentified mechanism, Nef increases the intrinsic infectivity of HIV-1 virions in a host-cell-dependent manner. Here we identify the host transmembrane protein SERINC5, and to a lesser extent SERINC3, as a potent inhibitor of HIV-1 particle infectivity that is counteracted by Nef. SERINC5 localizes to the plasma membrane, where it is efficiently incorporated into budding HIV-1 virions and impairs subsequent virion penetration of susceptible target cells. Nef redirects SERINC5 to a Rab7-positive endosomal compartment and thereby excludes it from HIV-1 particles. The ability to counteract SERINC5 was conserved in Nef encoded by diverse primate immunodeficiency viruses, as well as in the structurally unrelated glycosylated Gag from murine leukaemia virus. These examples of functional conservation and convergent evolution emphasize the fundamental importance of SERINC5 as a potent anti-retroviral factor. The transmembrane protein SERINC5 is identified as a potent inhibitor of HIV-1 particle infectivity that is counteracted by Nef; Nef redirects SERINC5 from the plasma membrane to a Rab7-positive endosomal compartment, thus excluding it from HIV-1 particles, emphasizing the potential of SERINC5 as a potent anti-retroviral factor. SERINC5 is a natural antiretroviral agent In two separate papers, Massimo Pizzato and colleagues and Heinrich Göttlinger and colleagues identify previously unrecognized restriction factors for HIV-1. In the absence of the HIV-1 Nef protein, the multipass transmembrane proteins SERINC3 and SERINC5 become incorporated into assembling virions and profoundly block HIV-1 infection. The Nef protein, which is normally expressed by HIV-1, counteracts this activity by down-regulating SERINC3 and SERINC5 from the cell surface, thereby preventing their incorporation into virions. These findings identify SERINC5, and to a lesser extent SERINC3, as the agents responsible for the long-sought anti-HIV-1 activity that is overcome by Nef. This raises the possibility that SERINC5 might have potential as a basis for anti-HIV-1 therapeutics.

HIV-1 Nef and the unrelated mouse leukaemia virus glycosylated Gag (glycoGag) strongly enhance the infectivity of HIV-1 virions produced in certain cell types in a clathrin-dependent manner. Here we show that Nef and glycoGag prevent the incorporation of the multipass transmembrane proteins serine incorporator 3 (SERINC3) and SERINC5 into HIV-1 virions to an extent that correlates with infectivity enhancement. Silencing of both SERINC3 and SERINC5 precisely phenocopied the effects of Nef and glycoGag on HIV-1 infectivity. The infectivity of nef -deficient virions increased more than 100-fold when produced in double-knockout human CD4 + T cells that lack both SERINC3 and SERINC5 , and re-expression experiments confirmed that the absence of SERINC3 and SERINC5 accounted for the infectivity enhancement. Furthermore, SERINC3 and SERINC5 together restricted HIV-1 replication, and this restriction was evaded by Nef. SERINC3 and SERINC5 are highly expressed in primary human HIV-1 target cells, and inhibiting their downregulation by Nef is a potential strategy to combat HIV/AIDS. The transmembrane proteins SERINC3 and SERINC5 are identified as new restriction factors for HIV-1 replication; this restriction is counteracted by Nef and glycoGag, which prevent SERINC3 and SERINC5 from becoming incorporated into HIV-1 virions and from profoundly blocking HIV-1 infectivity, suggesting a potential new therapeutic strategy for immunodeficiency viruses. SERINC5 is a natural antiretroviral agent In two separate papers, Massimo Pizzato and colleagues and Heinrich Göttlinger and colleagues identify previously unrecognized restriction factors for HIV-1. In the absence of the HIV-1 Nef protein, the multipass transmembrane proteins SERINC3 and SERINC5 become incorporated into assembling virions and profoundly block HIV-1 infection. The Nef protein, which is normally expressed by HIV-1, counteracts this activity by down-regulating SERINC3 and SERINC5 from the cell surface, thereby preventing their incorporation into virions. These findings identify SERINC5, and to a lesser extent SERINC3, as the agents responsible for the long-sought anti-HIV-1 activity that is overcome by Nef. This raises the possibility that SERINC5 might have potential as a basis for anti-HIV-1 therapeutics.

All retroviruses specifically package two copies of their genomes during virus assembly, a requirement for strand-transfer-mediated recombination during reverse transcription 1 , 2 . Genomic RNA exists in virions as dimers, and the overlap of RNA elements that promote dimerization and encapsidation suggests that these processes may be coupled 3 , 4 , 5 . Both processes are mediated by the nucleocapsid domain (NC) of the retroviral Gag polyprotein 3 . Here we show that dimerization-induced register shifts in base pairing within the Ψ-RNA packaging signal of Moloney murine leukaemia virus (MoMuLV) expose conserved UCUG elements that bind NC with high affinity (dissociation constant = 75 ± 12 nM). These elements are base-paired and do not bind NC in the monomeric RNA. The structure of the NC complex with a 101-nucleotide ‘core encapsidation’ segment of the MoMuLV Ψ site 6 reveals a network of interactions that promote sequence- and structure-specific binding by NC's single CCHC zinc knuckle. Our findings support a structural RNA switch mechanism for genome encapsidation, in which protein binding sites are sequestered by base pairing in the monomeric RNA and become exposed upon dimerization to promote packaging of a diploid genome.

Viral membrane fusion proteins of class I are trimers in which the protomeric unit is a complex of a surface subunit (SU) and a fusion active transmembrane subunit (TM). Here we have studied how the protomeric units of Moloney murine leukemia virus envelope protein (Env) are activated in relation to each other, sequentially or simultaneously. We followed the isomerization of the SU-TM disulfide and subsequent SU release from Env with biochemical methods and found that this early activation step occurred sequentially in the three protomers, generating two asymmetric oligomer intermediates according to the scheme (SU-TM)₃→(SU-TM)₂TM→(SU-TM)TM₂→TM₃. This was the case both when activation was triggered in vitro by depleting stabilizing Ca2+ from solubilized Env and when viral Env was receptor triggered on rat XC cells. In the latter case, the activation reaction was too fast for direct observation of the intermediates, but they could be caught by alkylation of the isomerization active thiol.

To prime reverse transcription of Moloney murine leukaemia virus, a transfer RNA molecule must bind two regions of the retroviral RNA, the primer binding site (PBS) and primer activation signal within the U5-PBS; here, the NMR structures of the U5-PBS RNA and tRNA primer are solved, with and without the retroviral nucleocapsid protein, which remodels these regions. Early steps in retroviral reverse transcription Retroviruses preferentially use specific host tRNAs as primers for the first step of reverse transcription. Moloney murine leukaemia virus (MLV) uses tRNA Pro , which binds to two regions of the retroviral RNA (U5-PBS and PAS). Victoria D'Souza and colleagues have solved NMR structures of the U5-PBS RNA and tRNA primer, with and without retroviral nucleocapsid protein, to understand how it remodels these regions as part of the priming process. In contrast to other ATP-independent nucleocapsid chaperones that bind many RNA sites with low affinity, this retroviral protein uses an entropy-driven capture-and-release remodelling mechanism where few sites are bound with high affinity. This results in local destabilization of the regions specifically required for the intermolecular interaction. To prime reverse transcription, retroviruses require annealing of a transfer RNA molecule to the U5 primer binding site (U5-PBS) region of the viral genome 1 , 2 . The residues essential for primer annealing are initially locked in intramolecular interactions 3 , 4 , 5 ; hence, annealing requires the chaperone activity of the retroviral nucleocapsid (NC) protein to facilitate structural rearrangements 6 . Here we show that, unlike classical chaperones, the Moloney murine leukaemia virus NC uses a unique mechanism for remodelling: it specifically targets multiple structured regions in both the U5-PBS and tRNA Pro primer that otherwise sequester residues necessary for annealing. This high-specificity and high-affinity binding by NC consequently liberates these sequestered residues—which are exactly complementary—for intermolecular interactions. Furthermore, NC utilizes a step-wise, entropy-driven mechanism to trigger both residue-specific destabilization and residue-specific release. Our structures of NC bound to U5-PBS and tRNA Pro reveal the structure-based mechanism for retroviral primer annealing and provide insights as to how ATP-independent chaperones can target specific RNAs amidst the cellular milieu of non-target RNAs.

Many mammalian species express restriction factors that confer host resistance to retroviral infection. Here we show that HIV-1 sensitivity to restriction factors is modulated by cyclophilin A (CypA), a host cell protein that binds the HIV-1 capsid protein (CA). In certain nonhuman primate cells, the CA–CypA interaction is essential for restriction: HIV-1 infectivity is increased >100-fold by cyclosporin A (CsA), a competitive inhibitor of the interaction, or by an HIV-1 CA mutation that disrupts CypA binding. Conversely, disruption of CA–CypA interaction in human cells reveals that CypA protects HIV-1 from the Ref-1 restriction factor. These findings suggest that HIV-1 has co-opted a host cell protein to counteract restriction factors expressed by human cells and that this adaptation can confer sensitivity to restriction in unnatural hosts. Manipulation of HIV-1 CA recognition by restriction factors promises to advance animal models and new therapeutic strategies for HIV-1 and AIDS.

Retroviruses are the aetiological agents of a range of human diseases including AIDS and T-cell leukaemias. They follow complex life cycles, which are still only partly understood at the molecular level. Maturation of newly formed retroviral particles is an essential step in production of infectious virions, and requires proteolytic cleavage of Gag polyproteins in the immature particle to form the matrix, capsid and nucleocapsid proteins present in the mature virion 1 . Capsid proteins associate to form a dense viral core 2 that may be spherical, cylindrical or conical depending on the genus of the virus 3 . Nonetheless, these assemblies all appear to be composed of a lattice formed from hexagonal rings, each containing six capsid monomers 2 , 4 , 5 . Here, we describe the X-ray structure of an individual hexagonal assembly from N-tropic murine leukaemia virus (N-MLV). The interface between capsid monomers is generally polar, consistent with weak interactions within the hexamer. Similar architectures are probably crucial for the regulation of capsid assembly and disassembly in all retroviruses. Together, these observations provide new insights into retroviral uncoating and how cellular restriction factors may interfere with viral replication.

The membrane fusion activity of murine leukaemia virus Env is carried by the transmembrane (TM) and controlled by the peripheral (SU) subunit. We show here that all Env subunits of the virus form disulphide‐linked SU–TM complexes that can be disrupted by treatment with NP‐40, heat or urea, or by Ca 2+ depletion. Thiol mapping indicated that these conditions induced isomerization of the disulphide‐bond by activating a thiol group in a Cys‐X‐X‐Cys (CXXC) motif in SU. This resulted in dissociation of SU from the virus. The active thiol was hidden in uninduced virus but became accessible for alkylation by either Ca 2+ depletion or receptor binding. The alkylation inhibited isomerization, virus fusion and infection. DTT treatment of alkylated Env resulted in cleavage of the SU–TM disulphide‐bond and rescue of virus fusion. Further studies showed that virus fusion was specifically inhibited by high and enhanced by low concentrations of Ca 2+ . These results suggest that Env is stabilized by Ca 2+ and that receptor binding triggers a cascade of reactions involving Ca 2+ removal, CXXC‐thiol exposure, SU–TM disulphide‐bond isomerization and SU dissociation, which lead to fusion activation.

A single retroviral protein, Gag, is sufficient for virus particle assembly. While Gag is capable of specifically packaging the genomic RNA into the particle, this RNA species is unnecessary for particle assembly in vivo. In vitro, nucleic acids profoundly enhance the efficiency of assembly by recombinant Gag proteins, apparently by acting as \"scaffolding\" in the particle. To address the participation of RNA in retrovirus assembly in vivo, we analyzed murine leukemia virus particles that lack genomic RNA because of a deletion in the packaging signal of the viral RNA. We found that these particles contain cellular mRNA in place of genomic RNA. This result was particularly evident when Gag was expressed by using a Semliki Forest virus-derived vector: under these conditions, the Semliki Forest virus vector-directed mRNA became very abundant in the cells and was readily identified in the retroviral virus-like particles. Furthermore, we found that the retroviral cores were disrupted by treatment with RNase. Taken together, the data strongly suggest that RNA is a structural element in retrovirus particles.