Dissociating Siv Env and Cd4: Consequenes for Virus and Host

CD4 tropism is conserved among all primate lentiviruses and likely contributes to viral pathogenesis by targeting cells that are critical for the adaptive anti-viral immune responses. Although CD4-independent variants of HIV and SIV have been described that can utilize coreceptors CCR5 or CXCR4 in the absence of CD4, these viruses typically retain their CD4 binding sites and can still interact with CD4. In this thesis, I present the characterization and evaluation, both in vitro and in vivo, of a novel CD4-independent variant of SIV lacking a CD4 binding site. I first describe the derivation of iMac239, a CD4-independent variant of SIVmac239. Like other CD4-independent variants, we found that a mutation in the V1/V2 loops of Env was required for CD4-independent entry, and that acquisition of CD4-independence resulted in an increase in neutralization sensitivity. While iMac239 was CD4-independent, its CD4-binding site was intact, thus we removed the Aspartic Acid residue at position 385 (analogous to D-368 in HIV-1) to ablate CD4 binding. We found that this novel variant, iMac239-ΔD385, exhibited replication kinetics similar to that of the parental iMac239 strain, and was insensitive to neutralization by soluble CD4. Both CD4-independent strains exhibited an expansion of cellular tropism in vitro with infection of CD4- CD8+ T cells in stimulated rhesus PBMCs. Next, I present our evaluation of iMac239-ΔD385 pathogenesis and immunogenicity in four rhesus macaques. iMac239-ΔD385 replicated to a high acute viral peak, but was quickly controlled to undetectable levels by the host immune response. iMac239-ΔD385 infection elicited high and sustained neutralizing antibody titers and polyfunctional T cell responses. Strikingly, we observed an alteration in the distribution of infected cells in the lymph node and expansion in the types of cells infected. We tested iMac239-ΔD385 as a live attenuated vaccine against a pathogenic SIVsmE660, and while the number of animals in the study is too small to determine significance we observed a trend toward improved outcomes post challenge, potentially due to a synergistic interaction between iMac239-ΔD385 vaccination and Trim5α alleles

Circadian Modulation of Gene Expression, but not Glutamate Uptake, in Mouse and Rat Cortical Astrocytes

Circadian clocks control daily rhythms including sleep-wake, hormone secretion, and metabolism. These clocks are based on intracellular transcription-translation feedback loops that sustain daily oscillations of gene expression in many cell types. Mammalian astrocytes display circadian rhythms in the expression of the clock genes Period1 (Per1) and Period2 (Per2). However, a functional role for circadian oscillations in astrocytes is unknown. Because uptake of extrasynaptic glutamate depends on the presence of Per2 in astrocytes, we asked whether glutamate uptake by glia is circadian.We measured glutamate uptake, transcript and protein levels of the astrocyte-specific glutamate transporter, Glast, and the expression of Per1 and Per2 from cultured cortical astrocytes and from explants of somatosensory cortex. We found that glutamate uptake and Glast mRNA and protein expression were significantly reduced in Clock/Clock, Per2- or NPAS2-deficient glia. Uptake was augmented when the medium was supplemented with dibutyryl-cAMP or B27. Critically, glutamate uptake was not circadian in cortical astrocytes cultured from rats or mice or in cortical slices from mice.We conclude that glutamate uptake levels are modulated by CLOCK, PER2, NPAS2, and the composition of the culture medium, and that uptake does not show circadian variations

Circadian Gene Regulation of Bmal1 in Astrocytes

Mentor: Erik Herzog From the Washington University Undergraduate Research Digest: WUURD, Volume 4, Issue 2, Spring 2009. Published by the Office of Undergraduate Research. Henry Biggs, Director of Undergraduate Research and Associate Dean in the College of Arts & Sciences; Joy Zalis Kiefer, Undergraduate Research Coordinator, Co-editor, and Assistant Dean in the College of Arts & Sciences; Kristin Sobotka, Editor

Glutamate uptake in mouse cortical slices is dose dependent, but not circadian.

<p>A–B, Two independent experiments showing that glutamate uptake is approximately 10 fold higher for a 10 µM glutamate concentration than for a 1 µM concentration. In addition, glutamate uptake did not show circadian variations at any concentration (mean±SEM). n = 3 for each time and concentration (A), n = 2 for each time and concentration (B).</p

Glutamate uptake in rat cortical astrocytes depends on sodium and a high affinity transporter.

<p>A, Glutamate uptake in sodium-free buffer (LiCl) was significantly lower than in culture medium with sodium (n = 3, mean±SEM). Uptake was initiated with 500 µM glutamate. B, <i>Trans</i>-2,4-PDC, a high-affinity transporter inhibitor (100 µM), reduced glutamate uptake (mean±SEM, n = 3) by 55–65%. Uptake was initiated with 1 µM glutamate. C, Glutamate uptake was 18 times higher in cultures immunopositive for GLAST (GLAST+). All cells which expressed GLAST also expressed GFAP. Separate cultures of cells immunonegative for GLAST (GLAST-) were also immunonegative for GFAP and did not uptake glutamate (n = 3 cultures per cell type and concentration, mean±SEM).</p

Glutamate uptake in slices of somatosensory cortex is not circadian.

<p>Explants were taken either 2 h before dusk (ZT 10, black squares) or 2 h before dawn (ZT 23, gray circles) and cultured for 36 h <i>in vitro</i> until 10-min uptake of 25 µM tritiated glutamate was measured. White and black bars on top of the X axis represent the projected subjective day and subjective night, respectively. (n = 3 slices per time point; mean±SEM).</p

Glutamate uptake depends on the <i>Clock</i> and <i>Per2</i> genes.

<p>Dose–response curves for glutamate uptake were generated comparing wild-type astrocyte cultures and either <i>Clock/Clock</i> or <i>Per2^m</i> mutant astrocytes. A, Glutamate uptake was significantly reduced in astrocytes derived from <i>Clock/Clock</i> mutants compared to wild-type (+/+) glia (n = 3 cultures per concentration, mean±SEM). B, Glutamate uptake was significantly reduced in astrocytes derived from <i>Per2^m</i> mutants compared to wild-type (+/+) glia (n = 3 per concentration, mean±SEM).</p

B27 or dB-cAMP as culture supplements do not induce a circadian rhythm in glutamate uptake.

<p>A, Uptake was measured in rat cortical astrocytes with (black line) or without B27 supplements (gray line) (500 µM tritiated glutamate, n = 3 cultures per time point; mean±SEM). B, A 10-day treatment of rat astrocytes with dB-cAMP (250 µM, black line) significantly increased overall glutamate uptake (500 µM tritiated glutamate), but did not induce circadian rhythmicity (n = 3 cultures per time point; mean±SEM).</p

Clonal expansion of SIV-infected cells in macaques on antiretroviral therapy is similar to that of HIV-infected cells in humans.

Clonal expansion of HIV infected cells plays an important role in the formation and persistence of the reservoir that allows the virus to persist, in DNA form, despite effective antiretroviral therapy. We used integration site analysis to ask if there is a similar clonal expansion of SIV infected cells in macaques. We show that the distribution of HIV and SIV integration sites in vitro is similar and that both viruses preferentially integrate in many of the same genes. We obtained approximately 8000 integration sites from blood samples taken from SIV-infected macaques prior to the initiation of ART, and from blood, spleen, and lymph node samples taken at necropsy. Seven clones were identified in the pre-ART samples; one persisted for a year on ART. An additional 100 clones were found only in on-ART samples; a number of these clones were found in more than one tissue. The timing and extent of clonal expansion of SIV-infected cells in macaques and HIV-infected cells in humans is quite similar. This suggests that SIV-infected macaques represent a useful model of the clonal expansion of HIV infected cells in humans that can be used to evaluate strategies intended to control or eradicate the viral reservoir