IFITM proteins inhibit HIV-1 protein synthesis

Interferon induced transmembrane proteins (IFITMs) inhibit the cellular entry of a broad range of viruses, but it has been suspected that for HIV-1 IFITMs may also inhibit a post-integration replicative step. We show that IFITM expression reduces HIV-1 viral protein synthesis by preferentially excluding viral mRNA transcripts from translation and thereby restricts viral production. Codon-optimization of proviral DNA rescues viral translation, implying that IFITM-mediated restriction requires recognition of viral RNA elements. In addition, we find that expression of the viral accessory protein Nef can help overcome the IFITM-mediated inhibition of virus production. Our studies identify a novel role for IFITMs in inhibiting HIV replication at the level of translation, but show that the effects can be overcome by the lentiviral protein Nef.Wellcome Trust-University of Edinburgh Institutional Strategic Support Fun

A comparative analysis of host responses to avian influenza infection in ducks and chickens highlights a role for the interferon-induced transmembrane proteins in viral resistance

Chickens are susceptible to infection with a limited number of Influenza A viruses and are a potential source of a human influenza pandemic. In particular, H5 and H7 haemagglutinin subtypes can evolve from low to highly pathogenic strains in gallinaceous poultry. Ducks on the other hand are a natural reservoir for these viruses and are able to withstand most avian influenza strains. Results: Transcriptomic sequencing of lung and ileum tissue samples from birds infected with high (H5N1) and low (H5N2) pathogenic influenza viruses has allowed us to compare the early host response to these infections in both these species. Chickens (but not ducks) lack the intracellular receptor for viral ssRNA, RIG-I and the gene for an important RIG-I binding protein, RNF135. These differences in gene content partly explain the differences in host responses to low pathogenic and highly pathogenic avian influenza virus in chicken and ducks. We reveal very different patterns of expression of members of the interferon-induced transmembrane protein (IFITM) gene family in ducks and chickens. In ducks, IFITM1, 2 and 3 are strongly up regulated in response to highly pathogenic avian influenza, where little response is seen in chickens. Clustering of gene expression profiles suggests IFITM1 and 2 have an anti-viral response and IFITM3 may restrict avian influenza virus through cell membrane fusion. We also show, through molecular phylogenetic analyses, that avian IFITM1 and IFITM3 genes have been subject to both episodic and pervasive positive selection at specific codons. In particular, avian IFITM1 showed evidence of positive selection in the duck lineage at sites known to restrict influenza virus infection. Conclusions: Taken together these results support a model where the IFITM123 protein family and RIG-I all play a crucial role in the tolerance of ducks to highly pathogenic and low pathogenic strains of avian influenza viruses when compared to the chicken

Role of S-Palmitoylation on IFITM5 for the Interaction with FKBP11 in Osteoblast Cells

Recently, one of the interferon-induced transmembrane (IFITM) family proteins, IFITM3, has become an important target for the activity against influenza A (H1N1) virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane (TM1) domain and in the cytoplasmic (CP) loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 (FKBP11) to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid (17-ODYA), and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP), revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation

Derivative-mediated inhibition of viral replication in macrophages.

<p>Terminally differentiated primary macrophages (2×10⁵ cells/well) derived from four healthy donors were infected with HIV-1 (a total of 1 ng of p24) and then incubated with serial 10-fold dilutions of derivative (concentration range, 0 to 10 μM). The levels of virus in the culture supernatants were measured at 4, 8, and 12 days after inoculation in a p24 antigen ELISA. Data represent the mean p24 value from two wells.</p

Analysis of the conserved regions within Vpr using the Wu-Kabat method.

<p>In total, 2,004 Vpr sequences were obtained from the HIV sequence database and used to calculate the Wu-Kabat index for each amino acid residue (a). The four Vpr regions were α Helix 1 domain (αH1, residues 17 to 33), α Helix 2 domain (αH2, residues 38 to 50), α Helix 3 domain (αH3, residues 54 to 74), and the non-Helix region (residues 1 to 16, residues 34 to 37, residues 51 to 53, and residues 75 to 96). These four regions plus the whole Vpr region (residues 1 to 96) were used to estimate the average Wu-Kabat index value (b).</p

Synthesis of a Vpr-Binding Derivative for Use as a Novel HIV-1 Inhibitor

<div><p>The emergence of multidrug-resistant viruses compromises the efficacy of anti-human immunodeficiency virus type 1 (HIV-1) therapy and limits treatment options. Therefore, new targets that can be used to develop novel antiviral agents need to be identified. We previously identified a potential parent compound, hematoxylin, which suppresses the nuclear import of HIV-1 via the Vpr-importin α interaction and inhibits HIV-1 replication in a Vpr-dependent manner by blocking nuclear import of the pre-integration complex. However, it was unstable. Here, we synthesized a stable derivative of hematoxylin that bound specifically and stably to Vpr and inhibited HIV-1 replication in macrophages. Furthermore, like hematoxylin, the derivative inhibited nuclear import of Vpr in an <i>in vitro</i> nuclear import assay, but had no effect on Vpr-induced G2/M phase cell cycle arrest or caspase activity. Interestingly, this derivative bound strongly to amino acid residues 54–74 within the C-terminal α-helical domain (αH3) of Vpr. These residues are highly conserved among different HIV strains, indicating that this region is a potential target for drug-resistant HIV-1 infection. Thus, we succeeded in developing a stable hematoxylin derivative that bound directly to Vpr, suggesting that specific inhibitors of the interaction between cells and viral accessory proteins may provide a new strategy for the treatment of HIV-1 infection.</p></div

Synthesis of a stable hematoxylin derivative.

<p>Treatment of hematoxylin with 2,2-dimethoxypropane in acetone in the presence of <i>p</i>-toluene sulphonic acid and phosphorus pentoxide under reflux conditions yielded acetonide-protected hematoxylin (yield, 28%). Next, the acetonide was reacted with allyl bromide in the presence of potassium carbonate in dimethylformamide at room temperature to yield stable hematoxylin (yield, 22%). ¹H-NMR(CDCl₃) δ 6.97 (1 H, d, <i>J</i> = 8.5 Hz, ArH), 6.67 (1 H, s, ArH), 6.66 (1 H, d, <i>J</i> = 8.5 Hz, ArH), 6.56 (1 H, s, ArH), 6.05 (1 H, dddd, <i>J</i> = 6.3, 6.3, 10, 17 Hz, OCH₂C<u>H</u>CH₂), 5.79 (1 H, br s, ArOH), 5.34 (1 H, br dd, <i>J</i> = 1.4, 17 Hz, OCH₂CHC<u>H</u>_<u>2</u>), 5.24 (1 H, br d, <i>J</i> = 10 Hz, OCH₂CHC<u>H</u>_<u>2</u>), 4.59 (1 H, dd, <i>J</i> = 6.3, 12 Hz, OC<u>H</u>_<u>2</u>CHCH₂), 4.52 (1 H, dd, <i>J</i> = 6.3, 12 Hz, OC<u>H</u>_<u>2</u>CHCH₂), 4.11 (1 H, d, <i>J</i> = 11 Hz, CH₂), 4.05 (1 H, s, CH), 3.85 (1 H, d, <i>J</i> = 11 Hz, CH₂), 3.17 (1 H, d, <i>J</i> = 15 Hz, CH₂), 2.83 (1H, d, <i>J</i> = 15 Hz, CH₂), 2.64 (1 H, br, OH), 1.65 (3 H, s, CH₃), 1.60 (3 H, s, CH₃); MS (ESI pos.) <i>m</i>/<i>z</i> 383 [M+H]⁺.</p

Purification of recombinant Vpr protein and binding analysis by SPR.

<p>(a) SDS-PAGE analysis of purified recombinant proteins. COS-7 cells were transfected with mammalian pCAGGS vectors encoding FLAG-mRFP (mRFP) or FLAG-mRFP-Vpr (mRFP-Vpr) and then purified on ANTI-FLAG M2 agarose beads. Proteins were separated on reducing 15% SDS-PAGE and stained with Coomassie brilliant blue. (b) SPR to determine the binding of the derivative to Vpr. The derivative was coupled to PGS and incubated with mRFP or mRFP-Vpr. DMSO was used as a negative control.</p