Deep sequencing of HIV-1 reverse transcripts reveals the multifaceted antiviral functions of APOBEC3G

Following cell entry, the RNA genome of HIV-1 is reverse transcribed into double-stranded DNA that ultimately integrates into the host-cell genome to establish the provirus. These early phases of infection are notably vulnerable to suppression by a collection of cellular antiviral effectors, called restriction or resistance factors. The host antiviral protein APOBEC3G (A3G) antagonizes the early steps of HIV-1 infection through the combined effects of inhibiting viral cDNA production and cytidine-to-uridine-driven hypermutation of this cDNA. In seeking to address the underlying molecular mechanism for inhibited cDNA synthesis, we developed a deep sequencing strategy to characterize nascent reverse transcription products and their precise 3′-termini in HIV-1 infected T cells. Our results demonstrate site- and sequence-independent interference with reverse transcription, which requires the specific interaction of A3G with reverse transcriptase itself. This approach also established, contrary to current ideas, that cellular uracil base excision repair (UBER) enzymes target and cleave A3G-edited uridine-containing viral cDNA. Together, these findings yield further insights into the regulatory interplay between reverse transcriptase, A3G and cellular DNA repair machinery, and identify the suppression of HIV-1 reverse transcriptase by a directly interacting host protein as a new cell-mediated antiviral mechanism.</p

Impaired Cellular Responses to Cytosolic DNA or Infection with Listeria monocytogenes and Vaccinia Virus in the Absence of the Murine LGP2 Protein

Innate immune signaling is crucial for detection of and the initial response to microbial pathogens. Evidence is provided indicating that LGP2, a DEXH box domain protein related to the RNA recognition receptors RIG-I and MDA5, participates in the cellular response to cytosolic double-stranded DNA (dsDNA). Analysis of embryonic fibroblasts and macrophages from mice harboring targeted disruption in the LGP2 gene reveals that LGP2 can act as a positive regulator of type I IFN and anti-microbial gene expression in response to transfected dsDNA. Results indicate that infection of LGP2-deficient mice with an intracellular bacterial pathogen, Listeria monocytogenes, leads to reduced levels of type I IFN and IL12, and allows increased bacterial growth in infected animals, resulting in greater colonization of both spleen and liver. Responses to infection with vaccinia virus, a dsDNA virus, are also suppressed in cells lacking LGP2, reinforcing the ability of LGP2 to act as a positive regulator of antiviral signaling. In vitro mechanistic studies indicate that purified LGP2 protein does not bind DNA but instead mediates these responses indirectly. Data suggest that LGP2 may be acting downstream of the intracellular RNA polymerase III pathway to activate anti-microbial signaling. Together, these findings demonstrate a regulatory role for LGP2 in the response to cytosolic DNA, an intracellular bacterial pathogen, and a DNA virus, and provide a plausible mechanistic hypothesis as the basis for this activity

APOBEC restriction goes nuclear

APOBEC3 restriction, known to inhibit retroviruses by interfering with genome replication and hypermutating viral DNA, targets the γ-herpesvirus Epstein–Barr virus and is antagonized by the viral BORF2 protein.</p

Nuclear import of SAMHD1 is mediated by a classical karyopherin α/β1 dependent pathway and confers sensitivity to VpxMAC induced ubiquitination and proteasomal degradation

Background: The deoxynucleotide-triphosphate (dNTP) hydrolase sterile alpha motif domain and HD domain 1 (SAMHD1) is a nuclear protein that inhibits HIV-1 infection in myeloid cells as well as quiescent CD4 T-cells, by decreasing the intracellular dNTP concentration below a level that is required for efficient reverse transcription. The Vpx proteins of the SIVSMM/HIV-2 lineage of lentiviruses bind SAMHD1 and recruit an ubiquitin ligase, leading to polyubiquitination and proteasomal degradation.Results: Here, we have investigated the importance of nuclear localization for SAMHD1's antiviral function as well as its sensitivity to the Vpx protein of SIVMAC. Using GST pull down assays, as well as RNA silencing approaches, we show that SAMHD1 preferentially uses karyopherin alpha 2 (KPNA2) and a classical N-terminal nuclear localization signal ((KRPR17)-K-14) to enter the nucleus. Reduction of karyopherin beta 1 (KPNB1) or KPNA2 by RNAi also led to cytoplasmic re-distribution of SAMHD1. Using primary human monocyte-derived macrophages (MDM), a cell type in which SAMHD1 is naturally expressed to high levels, we demonstrate that nuclear localization is not required for its antiviral activity. Cytoplasmic SAMHD1 still binds to Vpx(MAC), is efficiently polyubiquitinated, but is not degraded. We also find that Vpx(MAC)-induced SAMHD1 degradation was partially reversed by ubiquitin carrying the K48R or K11R substitution mutations, suggesting involvement of K48 and K11 linkages in SAMHD1 polyubiquitination. Using ubiquitin K-R mutants also revealed differences in the ubiquitin linkages between wild type and cytoplasmic forms of SAMHD1, suggesting a potential association with the resistance of cytoplasmic SAMHD1 to Vpx(MAC) induced degradation.Conclusions: Our work extends published observations on SAMHD1 nuclear localization to a natural cell type for HIV-1 infection, identifies KPNA2/KPNB1 as cellular proteins important for SAMHD1 nuclear import, and indicates that components of the nuclear proteasomal degradation machinery are required for SAMHD1 degradation.</p

LGP2 selectively interacts with dsRNA but not dsDNA.

<p>Electrophoretic mobility shift assay of LGP2 with RNA or DNA. a) Single stranded (ss) or double-stranded (ds) RNA (27-mer) or DNA (ISD,45-mer) or b) dsRNA (27-mer) or ds(dA-dT) (28-mer) were incubated with or without LGP2 and analyzed by native PAGE and autoradiography.</p

<i>Listeria monocytogenes-</i>induced IFN production in BMDM is dependent on RNA Polymerase III.

<p>BMDM from wild-type mice were treated with the indicated concentrations of RNA polymerase III inhibitor ML-60218 for 10 h and then infected with (a) LM (5 cfu/cell) or (b) Sendai Virus, Cantell Strain (5 pfu/cell) for indicated amounts of time and cell supernatants were analyzed for secreted IFNβ by ELISA. Student's t test was performed: *(p<0.05), **(p<0.01), and ***(p<0.005).</p

LGP2 increases <i>Listeria monocytogenes</i> infection-mediated cytokine and chemokine secretion and contributes to limitation of LM growth <i>in vivo</i>.

<p>Serum from wild-type (+/+) or LGP2-deficient (−/−) mice (n = 4–5 each) infected with LM were collected 24 h after infection. Serum IFNα (a) and IL-12p70 (b) levels were analyzed by ELISA and IL-6 (c), CCL2 (d) levels were analyzed by Milliplex assay (Millipore). Plotted dots indicate average cytokine concentration of duplicate readings from each mouse. Student's t test was performed: *(p<0.05), **(p<0.01), and ***(p<0.005). (e,f) Wild-type (+/+) and LGP2-deficient mice (−/−) (n = 4) were infected with 1.5×10⁶ wild-type LM and bacterial growth in the liver (e) and spleen (f) was measured 72 h after inoculation. The median of colony forming units (CFUs) from each group is indicated by bars. A representative experiment of two independent experiments is shown.</p

LGP2 deficiency decreases intracellular dsDNA signaling.

<p>(a) MEFs from wild-type mice (+/+) and LGP2-deficient mice (−/−) were transfected with 5 µg/ml of poly(dA-dT) for the indicated amount of time and total RNA was isolated from cells. The relative abundance of <i>IFNβ</i>, <i>CXCL10</i>, and <i>CCL5</i> mRNA was determined by quantitative RT-PCR. Error bars indicate standard deviation for duplicate PCR reactions. (b) MEFs or BMDM from wild-type mice (+/+) and LGP2-deficient mice (−/−) were transfected with 5 µg/ml poly(dA-dT) or (c) ISD. At indicated time points after transfection, media was collected and IFNβ levels were quantified by ELISA. Results are representative of at least 2 independent transfection experiments carried out in duplicate. Student’s t test was performed: *(p<0.05), **(p<0.01), and ***(p<0.005).</p