Virus-like particles (VLPs) have emerged as important and versatile architectures for chemical manipulation in the development of functional hybrid nanostructures. Here we have successfully demonstrated the site selective initiation of atom transfer radical polymerization (ATRP) reactions to form an addressable polymer constrained within the interior cavity of a VLP. This protein-polymer hybrid, of P22 and crosslinked poly(2-aminoethyl methacrylate), is potentially useful as a new high-density delivery vehicle for encapsulation and delivery of small molecule cargos. In particular, the encapsulated polymer can act as a scaffold for the attachment of primary amine reactive molecules of interest, such as a fluorescein dye or a Gd-DTPA MRI contrast agent. Using this approach, a significant increase in labeling density of the VLP, compared to previous modifications of VLPs, can be achieved. These results highlight the use of multimeric protein-polymer conjugates for their potential utility in the development of VLP-based MRI contrast agents with the possibility of loading other cargos.

The host cell factor cyclophilin A (CypA) interacts directly with the HIV-1 capsid and regulates viral infectivity. Although the crystal structure of CypA in complex with the N-terminal domain of the HIV-1 capsid protein (CA) has been known for nearly two decades, how CypA interacts with the viral capsid and modulates HIV-1 infectivity remains unclear. We determined the cryoEM structure of CypA in complex with the assembled HIV-1 capsid at 8-Å resolution. The structure exhibits a distinct CypA-binding pattern in which CypA selectively bridges the two CA hexamers along the direction of highest curvature. EM-guided all-atom molecular dynamics simulations and solid-state NMR further reveal that the CypA-binding pattern is achieved by single-CypA molecules simultaneously interacting with two CA subunits, in different hexamers, through a previously uncharacterized non-canonical interface. These results provide new insights into how CypA stabilizes the HIV-1 capsid and is recruited to facilitate HIV-1 infection.

Lentiviral DNA integration favors transcriptionally active chromatin. We previously showed that the interaction of human immunodeficiency virus type 1 (HIV-1) capsid with cleavage and polyadenylation specificity factor 6 (CPSF6) localizes viral preintegration complexes (PICs) to nuclear speckles for integration into transcriptionally active speckle-associated domains (SPADs). In the absence of the capsid-CPSF6 interaction, PICs uncharacteristically accumulate at the nuclear periphery and target heterochromatic lamina-associated domains (LADs) for integration. The integrase-binding protein lens epithelium-derived growth factor (LEDGF)/p75 in contrast to CPSF6 predominantly functions to direct HIV-1 integration to interior regions of transcription units. Though CPSF6 and LEDGF/p75 can reportedly interact with the capsid and integrase proteins of both primate and nonprimate lentiviruses, the extents to which these different viruses target SPADs versus LADs, as well as their dependencies on CPSF6 and LEDGF/p75 for integration targeting, are largely unknown. Here, we mapped 5,489,157 primate and nonprimate lentiviral integration sites in HEK293T and Jurkat T cells as well as derivative cells that were knocked out or knocked down for host factor expression. Despite marked preferences of all lentiviruses to target genes for integration, nonprimate lentiviruses only marginally favored SPADs, with corresponding upticks in LAD-proximal integration. While LEDGF/p75 knockout disrupted the intragenic integration profiles of all lentiviruses similarly, CPSF6 depletion specifically counteracted SPAD integration targeting by primate lentiviruses. CPSF6 correspondingly failed to appreciably interact with nonprimate lentiviral capsids. We conclude that primate lentiviral capsid proteins evolved to interact with CPSF6 to optimize PIC localization for integration into transcriptionally active SPADs. IMPORTANCE Integration is the defining step of the retroviral life cycle and underlies the inability to cure HIV/AIDS through the use of intensified antiviral therapy. The reservoir of latent, replication-competent proviruses that forms early during HIV infection reseeds viremia when patients discontinue medication. HIV cure research is accordingly focused on the factors that guide provirus formation and associated chromatin environments that regulate transcriptional reactivation, and studies of orthologous infectious agents such as nonprimate lentiviruses can inform basic principles of HIV biology. HIV-1 utilizes the integrase-binding protein LEDGF/p75 and the capsid interactor CPSF6 to target speckle-associated domains (SPADs) for integration. However, the extent to which these two host proteins regulate integration of other lentiviruses is largely unknown. Here, we mapped millions of retroviral integration sites in cell lines that were depleted for LEDGF/p75 and/or CPSF6. Our results reveal that primate lentiviruses uniquely target SPADs for integration in a CPSF6-dependent manner.

The phosphoprotein (P) is virally encoded by the Rhabdoviridae and Paramyxoviridae in the order Mononegavirales. P is a selfassociated oligomer and forms complexes with the large viral polymerase protein (L), the nucleocapsid protein (N), and the assembled nucleocapsid. P from different viruses has shown structural diversities even though their essential functions are the same. We systematically mapped the domains in mumps virus (MuV) P and investigated their interactions with nucleocapsidlike particles (NLPs). Similar to other P proteins, MuV P contains N-terminal, central, and C-terminal domains with flexible linkers between neighboring domains. By pulldown assays, we discovered that in addition to the previously proposed nucleocapsid binding domain (residues 343 to 391), the N-terminal region of MuV P (residues 1 to 194) could also bind NLPs. Further analysis of binding kinetics was conducted using surface plasmon resonance. This is the first observation that both the N-and C-terminal regions of a negative-strand RNA virus P are involved in binding the nucleocapsid. In addition, we defined the oligomerization domain (P OD ) of MuV P as residues 213 to 277 and determined its crystal structure. The tetrameric MuV P OD is formed by one pair of long parallel ␣-helices with another pair in opposite orientation. Unlike the parallel orientation of each ␣-helix in the tetramer of Sendai virus P OD , this represents a novel orientation of a P OD where both the N-and the C-terminal domains are at either end of the tetramer. This is consistent with the observation that both the N-and the C-terminal domains are involved in binding the nucleocapsid.T he phosphoprotein (P) is a multifunctional protein encoded in the genomes of negative-strand RNA viruses (NSVs) of the Rhabdoviridae and Paramyxoviridae in the order Mononegavirales. P performs several essential functions in virus replication. It is the cofactor in the viral RNA-dependent RNA polymerase (RdRp), a complex that also includes the virally encoded large (L) protein. L harbors the catalytic functions for RNA synthesis and mRNA capping. P also directly binds the nucleocapsid, the active template for viral RNA synthesis. Through this interaction, P delivers the RdRp to the nucleocapsid. In the absence of P, the RdRp cannot recognize the nucleocapsid or gain access to the viral genome. The domain responsible for P to bind the nucleocapsid, the phosphoprotein nucleocapsid binding domain (P NBD ), has been mapped to the C-terminal region for a number of NSVs, including vesicular stomatitis virus (VSV), rabies virus (RABV), measles virus, Sendai virus (SeV), Mokola virus, and mumps virus (MuV) (1-5). Threedimensional structures of this domain revealed a certain degree of structural homology among different viruses (5-9). The crystal structure of VSV P NBD in complex with a nucleocapsid-like particle (NLP) clearly showed that the P binding site in the nucleocapsid is formed by two neighboring nucleocapsid protein (N) subunits (10). The large extended loop in the C-lobe an...