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The early steps of HIV-1 infection, such as uncoating, reverse transcription, nuclear import, and transport to integration sites are incompletely understood. Here, we imaged nuclear entry and transport of HIV-1 replication complexes in cell lines, primary monocyte-derived macrophages (MDMs) and CD4 + T cells. We show that viral replication complexes traffic to and accumulate within nuclear speckles and that these steps precede the completion of viral DNA synthesis. HIV-1 transport to nuclear speckles is dependent on the interaction of the capsid proteins with host cleavage and polyadenylation specificity factor 6 (CPSF6), which is also required to stabilize the association of the viral replication complexes with nuclear speckles. Importantly, integration site analyses reveal a strong preference for HIV-1 to integrate into speckle-associated genomic domains. Collectively, our results demonstrate that nuclear speckles provide an architectural basis for nuclear homing of HIV-1 replication complexes and subsequent integration into associated genomic loci. Early steps of HIV infection of primary human cells remain poorly understood. Here, Francis et al. show that early viral replication complexes accumulate within nuclear speckles, in reliance on viral capsid/host CPSF6 interactions, and preferentially integrate in speckle-associated genomic domains.

Understanding HIV-1 persistence despite antiretroviral therapy (ART) is of paramount importance. Both single-genome sequencing (SGS) and integration site analysis (ISA) provide useful information regarding the structure of persistent HIV DNA populations; however, until recently, there was noway to link integration sites to their cognate proviral sequences. Here, we used multiple-displacement amplification (MDA) of cellular DNA diluted to a proviral endpoint to obtain full-length proviral sequences and their corresponding sites of integration. We applied this method to lymph node and peripheral blood mononuclear cells from 5 ART-treated donors to determine whether groups of identical subgenomic sequences in the 2 compartments are the result of clonal expansion of infected cells or a viral genetic bottleneck. We found that identical proviral sequences can result from both cellular expansion and viral genetic bottlenecks occurring prior to ART initiation and following ART failure. We identified an expanded T cell clone carrying an intact provirus that matched a variant previously detected by viral outgrowth assays and expanded clones with wild-type and drug-resistant defective proviruses. We also found 2 clones from 1 donor that carried identical proviruses except for nonoverlapping deletions, from which we could infer the sequence of the intact parental virus. Thus, MDA-SGS can be used for “viral reconstruction” to better understand intrapatient HIV-1 evolution and to determine the clonality and structure of proviruses within expanded clones, including those with drug-resistant mutations. Importantly, we demonstrate that identical sequences observed by standard SGS are not always sufficient to establish proviral clonality.

Background. Antiretroviral therapy (ART)-mediated immune reconstitution fails to restore the capacity of the immune system to spontaneously control human immunodeficiency virus (HIV) replication. Methods. A total of 23 HIV type 1 (HIV-1)-infected, virologically suppressed subjects receiving ART (CD4⁺ T-cell count, >450 cells/μL) were randomly assigned to have 180 μg/week (for arm A) or 90 μg/week (for arm B) of pegylated (Peg) interferon alfa-2a added to their current ART regimen. After 5 weeks, ART was interrupted, and Peg-interferon alfa-2a was continued for up to 12 weeks (the primary end point), with an option to continue to 24 weeks. End points included virologie failure (viral load, ≥400 copies/mL) and adverse events. Residual viral load and HIV-1 DNA integration were also assessed. Results. At week 12 of Peg-interferon alfa-2a monotherapy, viral suppression was observed in 9 of 20 subjects (45%), a significantly greater proportion than expected (arm A, P =. 0088; arm B, P =. 0010; combined arms, P< .0001). Over 24 weeks, both arms had lower proportions of subjects who had viral load, compared with the proportion of subjects in a historical control group (arm A, P =. 0046; arm B, P =.0011). Subjects who had a sustained viral load of <400 copies/mL had decreased levels of integrated HIV DNA (P =.0313) but increased residual viral loads (P =.0078), compared with subjects who experienced end-point failure. Conclusions. Peg-interferon alfa-2a immunotherapy resulted in control of HIV replication and decreased HIV-1 integration, supporting a role for immunomediated approaches in HIV suppression and/or eradication.

Viral 'fossils' in the genome DNA derived from endogenous retroviruses is a common ancestral feature in mammalian genomes. Until now retroviruses have been the only group of viruses known to have left a fossil record of this type, but now elements derived from Borna-like N (EBLN) sequences have been found in the genomes of humans, non-human primates, rodents and a species of ground squirrel. Bornaviruses are non-segmented, negative-strand RNA viruses that replicate in the nucleus of infected cells. In primates, the elements are very old, formed more than 40 million years ago, while squirrel EBLN sequences are a more recent introduction. The conservation of open reading frames of primate EBLNs, as well as their expression as mRNA, implies that they may function as a source of genetic novelty in their host. Until now, retroviruses have been the only group of viruses known to have left a fossil record, in the form of endogenous proviruses; those elements make up approximately 8% of the human genome. Elements homologous to the nucleoprotein gene of the non-retroviral bornavirus are now shown to exist in the genomes of several mammalian species; the results give insights into the role of bornavirus as a source of genetic novelty to its host. Retroviruses are the only group of viruses known to have left a fossil record, in the form of endogenous proviruses, and approximately 8% of the human genome is made up of these elements 1 , 2 . Although many other viruses, including non-retroviral RNA viruses, are known to generate DNA forms of their own genomes during replication 3 , 4 , 5 , none has been found as DNA in the germline of animals. Bornaviruses, a genus of non-segmented, negative-sense RNA virus, are unique among RNA viruses in that they establish persistent infection in the cell nucleus 6 , 7 , 8 . Here we show that elements homologous to the nucleoprotein (N) gene of bornavirus exist in the genomes of several mammalian species, including humans, non-human primates, rodents and elephants. These sequences have been designated endogenous Borna-like N (EBLN) elements. Some of the primate EBLNs contain an intact open reading frame (ORF) and are expressed as mRNA. Phylogenetic analyses showed that EBLNs seem to have been generated by different insertional events in each specific animal family. Furthermore, the EBLN of a ground squirrel was formed by a recent integration event, whereas those in primates must have been formed more than 40 million years ago. We also show that the N mRNA of a current mammalian bornavirus, Borna disease virus (BDV), can form EBLN-like elements in the genomes of persistently infected cultured cells. Our results provide the first evidence for endogenization of non-retroviral virus-derived elements in mammalian genomes and give novel insights not only into generation of endogenous elements, but also into a role of bornavirus as a source of genetic novelty in its host.

Interaction between HIV-1 integrase and the cellular cofactor LEDGF/p75 is important for viral integration. Newly designed small molecules that block this interaction inhibit HIV replication, illustrating the potential of viral–host protein-protein interaction inhibitors. Lens epithelium–derived growth factor (LEDGF/p75) is a cellular cofactor of HIV-1 integrase that promotes viral integration by tethering the preintegration complex to the chromatin. By virtue of its crucial role in the early steps of HIV replication, the interaction between LEDGF/p75 and integrase represents an attractive target for antiviral therapy. We have rationally designed a series of 2-(quinolin-3-yl)acetic acid derivatives (LEDGINs) that act as potent inhibitors of the LEDGF/p75-integrase interaction and HIV-1 replication at submicromolar concentration by blocking the integration step. A 1.84-Å resolution crystal structure corroborates the binding of the inhibitor in the LEDGF/p75-binding pocket of integrase. Together with the lack of cross-resistance with two clinical integrase inhibitors, these findings define the 2-(quinolin-3-yl)acetic acid derivatives as the first genuine allosteric HIV-1 integrase inhibitors. Our work demonstrates the feasibility of rational design of small molecules inhibiting the protein-protein interaction between a viral protein and a cellular host factor.

The selection of chromosomal targets for retroviral integration varies markedly, tracking with the genus of the retrovirus, suggestive of targeting by binding to cellular factors. γ-Retroviral murine leukemia virus (MLV) DNA integration into the host genome is favored at transcription start sites, but the underlying mechanism for this preference is unknown. Here, we have identified bromodomain and extraterminal domain (BET) proteins (Brd2, -3, -4) as cellular-binding partners of MLV integrase. We show that purified recombinant Brd4(1-720) binds with high affinity to MLV integrase and stimulates correct concerted integration in vitro. JQ-1, a small molecule that selectively inhibits interactions of BET proteins with modified histone sites impaired MLV but not HIV-1 integration in infected cells. Comparison of the distribution of BET protein-binding sites analyzed using ChIP-Seq data and MLV-integration sites revealed significant positive correlations. Antagonism of BET proteins, via JQ-1 treatment or RNA interference, reduced MLV-integration frequencies at transcription start sites. These findings elucidate the importance of BET proteins for MLV integration efficiency and targeting and provide a route to developing safer MLV-based vectors for human gene therapy.

Eradication of HIV-1 with highly active antiretroviral therapy (HAART) is not possible due to the persistence of long-lived, latently infected resting memory CD4⁺ T cells. We now show that HIV-1 latency can be established in resting CD4⁺ T cells infected with HIV-1 after exposure to ligands for CCR7 (CCL19), CXCR3 (CXCL9 and CXCL10), and CCR6 (CCL20) but not in unactivated CD4⁺ T cells. The mechanism did not involve cell activation or significant changes in gene expression, but was associated with rapid dephosphorylation of cofilin and changes in filamentous actin. Incubation with chemokine before infection led to efficient HIV-1 nuclear localization and integration and this was inhibited by the actin stabilizer jasplakinolide. We propose a unique pathway for establishment of latency by direct HIV-1 infection of resting CD4⁺ T cells during normal chemokine-directed recirculation of CD4⁺ T cells between blood and tissue.

HIV-1 cores enter the nucleus and undergo capsid disassembly (uncoating) near their integration site. Although most viral cores are localized to nuclear speckles (NSs), the spatial relationship between the uncoating site and integration site remains unclear. Here, using fluorescently labeled HIV-1 cores and NS markers, we show that uncoating predominantly occurs within NSs. Treatment of infected cells with capsid inhibitors PF-3450074 (PF74) or lenacapavir (LEN) after nuclear entry induced rapid disruption of interactions between capsid and cleavage and polyadenylation specificity factor 6 (CPSF6) followed by exit of HIV-1 cores from NSs, indicating that CPSF6 binding is required to retain the viral cores in the NSs. Treatment with PF74 or LEN led to core disruption and appearance of transcriptionally active proviruses further from the NSs compared to viral cores that uncoated in the NSs in untreated cells. This spatial shift correlated with reduction in integration into gene-rich, transcriptionally active speckle-associated chromatin domains, the preferred sites of HIV-1 integration, and increased integration into gene-sparse lamina-associated domains located away from the nuclear envelope. These findings demonstrate that the HIV-1 uncoating site is a key determinant of integration targeting, and that capsid inhibitors can misdirect integration by relocalizing uncoating to outside of NSs. HIV-1 releases its DNA when its protective capsid shell disassembles (uncoats) inside membraneless subnuclear organelles called nuclear speckles. Drugs that force uncoating to occur elsewhere promote viral DNA insertion into different genomic sites.

The structure of the Cas1–Cas2 complex bound to a protospacer sequence illustrates how foreign DNA is captured and measured by bacterial proteins in preparation for integration into CRISPR loci. DNA recognition by the Cas1–Cas2 complex The CRISPR–Cas system widely used for genome editing in a variety of organisms derives from a bacterial immune system. In bacteria, DNA sequences from invading phage are incorporated into the host genome in loci known as CRISPR. How these 'spacer' sequences of 30–40 base pairs are selected and generated was unclear. Jennifer Doudna and colleagues now describe the structure of the Cas1–Cas2 complex bound to a protospacer sequence. The structure illustrates how the foreign DNA is captured and cleaved by the host proteins in preparation for integration into CRISPR loci. Bacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30–40-base-pair lengths into clustered regularly interspaced short palindromic repeat (CRISPR) loci as spacer segments 1 , 2 , 3 , 4 , 5 , 6 . The universally conserved Cas1–Cas2 integrase complex catalyses spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases 7 , 8 , 9 , 10 , 11 , 12 , 13 . How the Cas1–Cas2 complex selects foreign DNA substrates for integration remains unknown. Here we present X-ray crystal structures of the Escherichia coli Cas1–Cas2 complex bound to cognate 33-nucleotide protospacer DNA substrates. The protein complex creates a curved binding surface spanning the length of the DNA and splays the ends of the protospacer to allow each terminal nucleophilic 3′-OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo 2 , 3 , 4 . Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1–Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci.

Clonal expansion of HIV infected cells plays an important role in the formation and persistence of the reservoir that allows the virus to persist, in DNA form, despite effective antiretroviral therapy. We used integration site analysis to ask if there is a similar clonal expansion of SIV infected cells in macaques. We show that the distribution of HIV and SIV integration sites in vitro is similar and that both viruses preferentially integrate in many of the same genes. We obtained approximately 8000 integration sites from blood samples taken from SIV-infected macaques prior to the initiation of ART, and from blood, spleen, and lymph node samples taken at necropsy. Seven clones were identified in the pre-ART samples; one persisted for a year on ART. An additional 100 clones were found only in on-ART samples; a number of these clones were found in more than one tissue. The timing and extent of clonal expansion of SIV-infected cells in macaques and HIV-infected cells in humans is quite similar. This suggests that SIV-infected macaques represent a useful model of the clonal expansion of HIV infected cells in humans that can be used to evaluate strategies intended to control or eradicate the viral reservoir.