Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses.

Nucleic acids are used in many therapeutic modalities, including gene therapy, but their ability to trigger host immune responses in vivo can lead to decreased safety and efficacy. In the case of adeno-associated viral (AAV) vectors, studies have shown that the genome of the vector activates Toll-like receptor 9 (TLR9), a pattern recognition receptor that senses foreign DNA. Here, we engineered AAV vectors to be intrinsically less immunogenic by incorporating short DNA oligonucleotides that antagonize TLR9 activation directly into the vector genome.

A broad-spectrum antiviral molecule, QL47, selectively inhibits eukaryotic translation.

Small-molecule inhibitors of translation are critical tools to study the molecular mechanisms of protein synthesis. In this study, we sought to characterize how QL47, a host-targeted, small-molecule antiviral agent, inhibits steady-state viral protein expression. We demonstrate that this small molecule broadly inhibits both viral and host protein synthesis and targets a translation step specific to eukaryotic cells.

Global analysis of polysome-associated mRNA in vesicular stomatitis virus infected cells.

Infection of mammalian cells with vesicular stomatitis virus (VSV) results in the inhibition of cellular translation while viral translation proceeds efficiently. VSV RNA synthesis occurs entirely within the cytoplasm, where during transcription the viral polymerase produces 5 mRNAs that are structurally indistinct to cellular mRNAs with respect to their 5' cap-structure and 3'-polyadenylate tail. Using the global approach of massively parallel sequencing of total cytoplasmic, monosome- and polysome-associated mRNA, we interrogate the impact of VSV infection of HeLa cells on translation.

Polymorphisms in Rhesus Macaque Tetherin Are Associated with Differences in Acute Viremia in Simian Immunodeficiency Virus Δ-Infected Animals.

Tetherin (BST-2 or CD317) is an interferon-inducible transmembrane protein that inhibits virus release from infected cells. To determine the extent of sequence variation and the impact of polymorphisms in rhesus macaque tetherin on simian immunodeficiency virus (SIV) infection, tetherin alleles were sequenced from 146 rhesus macaques, including 68 animals infected with wild-type SIV239 and 47 animals infected with SIV239Δ Since Nef is the viral gene product of SIV that counteracts restriction by tetherin, these groups afford a comparison of the effects of tetherin polymorphisms on SIV strains that are, and are not, resistant to tetherin. We identified 15 alleles of rhesus macaque tetherin with dimorphic residues at 9 positions.

STING-dependent translation inhibition restricts RNA virus replication.

In mammalian cells, IFN responses that occur during RNA and DNA virus infections are activated by distinct signaling pathways. The RIG-I-like-receptors (RLRs) bind viral RNA and engage the adaptor MAVS (mitochondrial antiviral signaling) to promote IFN expression, whereas cGAS (cGMP-AMP synthase) binds viral DNA and activates an analogous pathway via the protein STING (stimulator of IFN genes). In this study, we confirm that STING is not necessary to induce IFN expression during RNA virus infection but also find that STING is required to restrict the replication of diverse RNA viruses.

KIR3DL01 recognition of Bw4 ligands in the rhesus macaque: maintenance of Bw4 specificity since the divergence of apes and Old World monkeys.

The identification of MHC class I ligands for rhesus macaque killer cell Ig-like receptors (KIRs) is fundamental to our basic understanding of KIR and MHC class I coevolution and to the study of NK cell responses in this nonhuman primate model for AIDS and other viral diseases. In this study, we show that Mamu-KIR3DL01, which is expressed by ∼90% of rhesus macaques, recognizes MHC class I molecules with a Bw4 motif. Primary NK cells expressing Mamu-KIR3DL01 were identified by staining with a mAb which, in this study, was shown to bind Mamu-KIR3DL01 allotypes with an aspartic acid at position 233.

Tetherin upregulation in simian immunodeficiency virus-infected macaques.

Here we show that simian immunodeficiency virus (SIV) infection of rhesus macaques results in rapid upregulation of tetherin (BST-2 or CD317) on peripheral blood lymphocytes, including the CD4(+) CCR5(+) T cell targets of virus infection, with a peak of induction that coincides with peak alpha interferon (IFN-α) levels in plasma, and that tetherin remains above baseline levels throughout chronic infection. These observations are consistent with a role for tetherin in innate immunity to immunodeficiency virus infection.

KIR polymorphisms modulate peptide-dependent binding to an MHC class I ligand with a Bw6 motif.

Molecular interactions between killer immunoglobulin-like receptors (KIRs) and their MHC class I ligands play a central role in the regulation of natural killer (NK) cell responses to viral pathogens and tumors. Here we identify Mamu-A1*00201 (Mamu-A*02), a common MHC class I molecule in the rhesus macaque with a canonical Bw6 motif, as a ligand for Mamu-KIR3DL05. Mamu-A1*00201 tetramers folded with certain SIV peptides, but not others, directly stained primary NK cells and Jurkat cells expressing multiple allotypes of Mamu-KIR3DL05.

RB regulates pancreas development by stabilizing Pdx1.

RB is a key substrate of Cdks and an important regulator of the mammalian cell cycle. RB either represses E2Fs that promote cell proliferation or enhances the activity of cell-specific factors that promote differentiation, although the mechanism that facilitates this dual interaction is unclear. Here, we demonstrate that RB associates with and stabilizes pancreatic duodenal homeobox-1 (Pdx-1) that is essential for embryonic pancreas development and adult β-cell function.

Envelope-modified single-cycle simian immunodeficiency virus selectively enhances antibody responses and partially protects against repeated, low-dose vaginal challenge.

Immunization of rhesus macaques with strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection elicits T-cell responses to multiple viral gene products and antibodies capable of neutralizing lab-adapted SIV, but not neutralization-resistant primary isolates of SIV. In an effort to improve upon the antibody responses, we immunized rhesus macaques with three strains of single-cycle SIV (scSIV) that express envelope glycoproteins modified to lack structural features thought to interfere with the development of neutralizing antibodies. These envelope-modified strains of scSIV lacked either five potential N-linked glycosylation sites in gp120, three potential N-linked glycosylation sites in gp41, or 100 amino acids in the V1V2 region of gp120.

Species-specific activity of SIV Nef and HIV-1 Vpu in overcoming restriction by tetherin/BST2.

Tetherin, also known as BST2, CD317 or HM1.24, was recently identified as an interferon-inducible host-cell factor that interferes with the detachment of virus particles from infected cells. HIV-1 overcomes this restriction by expressing an accessory protein, Vpu, which counteracts tetherin.