X4 and R5 HIV-1 Have Distinct Post-entry Requirements for Uracil DNA Glycosylase during Infection of Primary Cells

It has been assumed that R5 and X4 HIV utilize similar strategies to support viral cDNA synthesis post viral entry. In this study, we provide evidence to show that R5 and X4 HIV have distinct requirements for host cell uracil DNA glycosylase (UNG2) during the early stage of infection. UNG2 has been previously implicated in HIV infection, but its precise role remains controversial. In this study we show that, although UNG2 is highly expressed in different cell lines, UNG2 levels are low in the natural host cells of HIV. Short interfering RNA knockdown of endogenous UNG2 in primary cells showed that UNG2 is required for R5 but not X4 HIV infection and that this requirement is bypassed when HIV enters the target cell via vesicular stomatitis virus envelope-glycoprotein-mediated endocytosis. We also show that short interfering RNA knockdown of UNG2 in virus-producing primary cells leads to defective R5 HIV virions that are unable to complete viral cDNA synthesis. Quantitative PCR analysis revealed that endogenous UNG2 levels are transiently up-regulated post HIV infection, and this increase in UNG2 mRNA is ∼10–20 times higher in R5 versus X4 HIV-infected cells. Our data show that both virion-associated UNG2 and HIV infection-induced UNG2 expression are critical for reverse transcription during R5 but not X4 HIV infection. More importantly, we have made the novel observation that R5 and X4 HIV have distinct host cell factor requirements and differential capacities to induce gene expression during the early stages of infection. These differences may result from activation of distinct signaling cascades and/or infection of divergent T-lymphocyte subpopulations

Pharmacological and Anatomical Evidence for an Interaction Between mGluR5- and GABAA α1-Containing Receptors in the Discriminative Stimulus Effects of Ethanol

The discriminative stimulus properties of ethanol are mediated in part by positive modulation of GABA(A) receptors. Recent evidence indicates that metabotropic glutamate receptor subtype 5 (mGluR5) activity can influence GABA(A) receptor function. Therefore, the purpose of this work was to examine the potential involvement of mGluR5 in the discriminative stimulus effects of ethanol. In rats trained to discriminate ethanol (1 g/kg, intragastric gavage (i.g.)) from water, 2-methyl-6-(phenylethyl)-pyridine (MPEP) (1–50 mg/kg, i.p.) a selective noncompetitive antagonist of the mGlu5 receptor did not produce ethanol-like stimulus properties. However, pretreatment with MPEP (30 mg/kg) reduced the stimulus properties of ethanol as indicated by significant reductions in ethanol-appropriate responding, specifically at 0.5 and 1 g/kg ethanol, and a failure of ethanol test doses (1 and 2 g/kg) to fully substitute for the ethanol training dose. To test whether mGluR5 antagonism altered the GABA(A) receptor component of the ethanol stimulus, the ability of MPEP to modulate pentobarbital and diazepam substitution for ethanol was assessed. Pentobarbital substitution (1–10 mg/kg, i.p.) for ethanol was not altered by MPEP pretreatment. However, MPEP pretreatment inhibited the ethanol-like stimulus properties of diazepam (5 mg/kg, i.p.). To examine a potential anatomical basis for these pharmacological findings, expression patterns of mGluR5- and benzodiazepine-sensitive GABA(A) α1-containing receptors were examined by dual-label fluorescent immunohistochemistry with visualization by confocal microscopy. Results indicated that mGluR5- and GABA(A) α1-containing receptors were both coexpressed in limbic brain regions and colocalized on the same cells in specific brain regions including the amygdala, hippocampus, globus pallidus, and ventral pallidum. Together, these findings suggest an interaction between mGluR5- and benzodiazepine-sensitive GABA(A) receptors in mediating ethanol discrimination