SummaryGram-negative bacterial peptidoglycan is specifically recognized by the host intracellular sensor NOD1, resulting in the generation of innate immune responses. Although epithelial cells are normally refractory to external stimulation with peptidoglycan, these cells have been shown to respond in a NOD1-dependent manner to Gramnegative pathogens that can either invade or secrete factors into host cells. In the present work, we report that Gram-negative bacteria can deliver peptidoglycan to cytosolic NOD1 in host cells via a novel mechanism involving outer membrane vesicles (OMVs). We purified OMVs from the Gramnegative mucosal pathogens: Helicobacter pylori, Pseudomonas aeruginosa and Neisseria gonorrhoea and demonstrated that these peptidoglycan containing OMVs upregulated NF-kB and NOD1-dependent responses in vitro. These OMVs entered epithelial cells through lipid rafts thereby inducing NOD1-dependent responses in vitro. Moreover, OMVs delivered intragastrically to miceinduced innate and adaptive immune responses via a NOD1-dependent but TLR-independent mechanism. Collectively, our findings identify OMVs as a generalized mechanism whereby Gramnegative bacteria deliver peptidoglycan to cytosolic NOD1. We propose that OMVs released by bacteria in vivo may promote inflammation and pathology in infected hosts.

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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.

Production of the human immunodeficiency virus type 1 (HIV-1) Gag-Pol precursor protein results from a ؊1 ribosomal frameshifting event. In infected cells, this generates Gag and Gag-Pol in a ratio that is estimated to be 20:1, a ratio that is conserved among retroviruses. To examine the impact of this ratio on HIV-1 replication and viral assembly, we altered the Gag/Gag-Pol ratio in virus

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This study provides the first direct evidence that HIV-1-associated cholesterol is important for the maintenance of virion structure and infectivity. While the buoyant density of cholesterol-defective HIV-1 can be restored by a cholesterol structural analogue, cholestenone, the requirement for cholesterol is essential for HIV-1 infectivity.

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) target HIV-1 reverse transcriptase (RT) by binding to a pocket in RT that is close to, but distinct, from the DNA polymerase active site and prevent the synthesis of viral cDNA. NNRTIs, in particular, those that are potent inhibitors of RT polymerase activity, can also act as chemical enhancers of the enzyme's inter-subunit interactions. However, the consequences of this chemical enhancement effect on HIV-1 replication are not understood. Here, we show that the potent NNRTIs efavirenz, TMC120, and TMC125, but not nevirapine or delavirdine, inhibit the late stages of HIV-1 replication. These potent NNRTIs enhanced the intracellular processing of Gag and Gag-Pol polyproteins, and this was associated with a decrease in viral particle production from HIV-1-transfected cells. The increased polyprotein processing is consistent with premature activation of the HIV-1 protease by NNRTI-enhanced Gag-Pol multimerization through the embedded RT sequence. These findings support the view that Gag-Pol multimerization is an important step in viral assembly and demonstrate that regulation of Gag-Pol/Gag-Pol interactions is a novel target for small molecule inhibitors of HIV-1 production. Furthermore, these drugs can serve as useful probes to further understand processes involved in HIV-1 particle assembly and maturation.