Biochemical Characterization of Novel Retroviral Integrase Proteins

Integrase is an essential retroviral enzyme, catalyzing the stable integration of reverse transcribed DNA into cellular DNA. Several aspects of the integration mechanism, including the length of host DNA sequence duplication flanking the integrated provirus, which can be from 4 to 6 bp, and the nucleotide preferences at the site of integration, are thought to cluster among the different retroviral genera. To date only the spumavirus prototype foamy virus integrase has provided diffractable crystals of integrase-DNA complexes, revealing unprecedented details on the molecular mechanisms of DNA integration. Here, we characterize five previously unstudied integrase proteins, including those derived from the alpharetrovirus lymphoproliferative disease virus (LPDV), betaretroviruses Jaagsiekte sheep retrovirus (JSRV), and mouse mammary tumor virus (MMTV), epsilonretrovirus walleye dermal sarcoma virus (WDSV), and gammaretrovirus reticuloendotheliosis virus strain A (Rev-A) to identify potential novel structural biology candidates. Integrase expressed in bacterial cells was analyzed for solubility, stability during purification, and, once purified, 3′ processing and DNA strand transfer activities in vitro. We show that while we were unable to extract or purify accountable amounts of WDSV, JRSV, or LPDV integrase, purified MMTV and Rev-A integrase each preferentially support the concerted integration of two viral DNA ends into target DNA. The sequencing of concerted Rev-A integration products indicates high fidelity cleavage of target DNA strands separated by 5 bp during integration, which contrasts with the 4 bp duplication generated by a separate gammaretrovirus, the Moloney murine leukemia virus (MLV). By comparing Rev-A in vitro integration sites to those generated by MLV in cells, we concordantly conclude that the spacing of target DNA cleavage is more evolutionarily flexible than are the target DNA base contacts made by integrase during integration. Given their desirable concerted DNA integration profiles, Rev-A and MMTV integrase proteins have been earmarked for structural biology studies

A supramolecular assembly mediates lentiviral DNA integration

Retroviral integrase (IN) functions within the intasome nucleoprotein complex to catalyze insertion of viral DNA into cellular chromatin. Using cryo–electron microscopy, we now visualize the functional maedi-visna lentivirus intasome at 4.9 angstrom resolution. The intasome comprises a homo-hexadecamer of IN with a tetramer-of-tetramers architecture featuring eight structurally distinct types of IN protomers supporting two catalytically competent subunits. The conserved intasomal core, previously observed in simpler retroviral systems, is formed between two IN tetramers, with a pair of C-terminal domains from flanking tetramers completing the synaptic interface. Our results explain how HIV-1 IN, which self-associates into higher-order multimers, can form a functional intasome, reconcile the bulk of early HIV-1 IN biochemical and structural data, and provide a lentiviral platform for design of HIV-1 IN inhibitors

A bipartite structural organization defines the SERINC family of HIV-1 restriction factors

The human integral membrane protein SERINC5 potently restricts HIV-1 infectivity and sensitizes the virus to antibody-mediated neutralization. Here, using cryo-EM, we determine the structures of human SERINC5 and its orthologue from Drosophila melanogaster at subnanometer and near-atomic resolution, respectively. The structures reveal a novel fold comprised of ten transmembrane helices organized into two subdomains and bisected by a long diagonal helix. A lipid binding groove and clusters of conserved residues highlight potential functional sites. A structure-based mutagenesis scan identified surface-exposed regions and the interface between the subdomains of SERINC5 as critical for HIV-1-restriction activity. The same regions are also important for viral sensitization to neutralizing antibodies, directly linking the antiviral activity of SERINC5 with remodeling of the HIV-1 envelope glycoprotein

Antiviral activity of intracellular nanobodies targeting the influenza virus RNA-polymerase core

Abstract Influenza viruses transcribe and replicate their genome in the nucleus of the infected cells, two functions that are supported by the viral RNA-dependent RNA-polymerase (FluPol). FluPol displays structural flexibility related to distinct functional states, from an inactive form to conformations competent for replication and transcription. FluPol machinery is constituted by a structurally-invariant core comprising the PB1 subunit stabilized with PA and PB2 domains, whereas the PA endonuclease and PB2 C-domains can pack in different configurations around the core. To get insights into the functioning of FluPol, we selected single-domain nanobodies (VHHs) specific of the influenza A FluPol core. When expressed intracellularly, several of them exhibited inhibitory activity on type A FluPol, but not on the type B one. The most potent VHH (VHH16) targets PA, but preferentially bind the PA-PB1 dimer with an affinity below the nanomolar range. Ectopic intracellular expression of VHH16 in virus permissive cells blocks multiplication of different influenza A subtypes, even when induced at late times post-infection. VHH16 was found to impair the transport of the PA-PB1 dimer to the nucleus, without affecting its handling by the importin β RanBP5 and subsequent steps in FluPol assembly. These data suggest that the VHH16 neutralization activity is likely due to an alteration of the import of the PA-PB1 dimer into the nucleus, resulting to an inhibition of FluPol functioning. VHH16 binding site represent a potential target for antiviral development. Author Summary The influenza virus RNA-polymerase (FluPol) ensures genome transcription and replication in the nucleus of the infected cells. To select ligands able to block FluPol activities, we screened a library of phages encoding nanobodies and resulting from the immunization of a llama with FluPol subunits. When expressed intracellularly, one of the nanobodies displays highly efficient FluPol blocking and virus neutralizing activities. This nanobody binds FluPol with high affinity and recognizes preferentially the PA-PB1 assembled subunits. Furthermore, it was found to interfere with the transport of the PA-PB1 dimer into the nucleus, suggesting that targeting FluPol trafficking between the cytoplasm and the nucleus may constitute a powerful strategy to develop new antivirals

IN expression, extraction, and purification.

<p>(A) Fractions of bacterially expressed His₆-tagged WDSV, LPDV, JSRV, MMTV, and Rev-A INs were visualized through western blotting. Lanes 1 and 2 represent the pellet (P1) and supernatant (S1) fractions obtained following centrifugation of cells lysed in 200 mM NaCl-containing buffer A. Pellet 2 (P2) and supernatant 2 (S2) were obtained following centrifugation (lanes 3 and 4) of fraction P1 homogenized in buffer B containing 1 M NaCl and 5 mM CHAPS. During the final extraction step, the pellet from step 2 was homogenized in buffer C containing 0.5 M NaCl and 2 M urea (lanes 5 and 6). (B) Schematic of the protocols utilized for JSRV, LPDV, MMTV, and Rev-A IN purification. All columns were run on an ÄKTA purifier system. (C) The purities of MMTV (lane 2) and Rev-A (lane 4) INs were assessed at 93% and 97%, respectively, following silver staining of SDS-polyacrylamide gels. Lanes 1 and 3 contain the indicated molecular mass standards.</p

Rev-A integration products obtained by DNA sequence analysis.

<p>Rev-A integration products obtained by DNA sequence analysis.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Sequence analysis of Rev-A integration sites and comparison to MLV.

<p>(A) Palindromic consensus sequence from sites of Rev-A integration <i>in vitro</i>. Observed frequencies of nucleotides at the insertion sites were compared to expected frequencies at each position based on the sequence of the pGEM-3 target DNA. The sequence of the target site duplication following DNA gap repair is indicated in the black box and underlined below the consensus sequence, which employs IUPAC-IUB nucleotide codes; positions of DNA strand transfer are labeled by vertical arrows. Green and red boxes highlight nucleotide positions that are >140% and <60% of the expected base, respectively. Yellow boxes and bold values indicate <i>P</i> values of <0.05 and 0.001, respectively. (B) Comparison of consensus Rev-A (from panel A) and MLV <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076638#pone.0076638-Wu1" target="_blank">[36]</a> integration site sequences.</p

Concerted integration assay design and IN activities.

<p>(A) Schematic showing precleaved U5 substrate (vDNA), circular plasmid target DNA (pGEM-3), and products of single-end versus concerted vDNA integration. Positions of ³²P label are shown by *. (B) EtBr stained image (upper panel) and phosphorimage (lower panel) of integration reactions, comparing MMTV and Rev-A INs to control PFV and HIV-1 IN proteins. Reactions fractionated through two separate gels delimitated by a white border were performed under the exact same conditions. Half-site products of Rev-A and PFV vDNA integration were evident upon long exposure of the phosphorImager screen. Migration positions of standards (in kb) are shown to the left, whereas positions of half-site and concerted vDNA integration products are to the right. Note the half-site products co-migrate with the open circular (o.c.) form of pGEM-3, whereas the concerted products migrate in between the o.c. and supercoiled (s.c.) forms of the plasmid. (C) Half-site and concerted integration quantification of panel B phosphorimage. Results (percent of vDNA substrate converted into half-site and concerted integration reaction products) are means ± SEM for three independent experiments.</p

IN 3′ processing activities.

<p>(A) Schematic of blunt-ended vDNA substrate processed by IN adjacent to the conserved CA 3′ dinucleotide (vertical arrowhead). Positions of ³²P label are shown by *. (B) Polyacrylamide sequencing gel of products of HIV-1, MMTV, and Rev-A IN 3′ processing reactions; Mn²⁺, Mg²⁺, and glycerol were included as indicated. The positions of the starting substrates (30 bp for HIV-1 IN; 32 bp for MMTV and Rev-A), the simple dinucleotide cleavage products (pGpT_OH for HIV-1 and pTpT_OH for MMTV and Rev-A), and form I and form II cleavage products are indicated. IN proteins were omitted from the initial reaction in each set of five reactions. (C) Mn²⁺ and Mg²⁺-dependent 3′ processing activities expressed as percentage of product formation ± standard error of the mean (SEM) for three independent experiments. Asterisks indicate <i>P</i> values <0.05 by paired t-test.</p