Architecture and secondary structure of an entire HIV-1 RNA genome

Single-stranded RNA viruses encompass broad classes of infectious agents and cause the common cold, cancer, AIDS, and other serious health threats. Viral replication is regulated at many levels, including using conserved genomic RNA structures. Most potential regulatory elements within viral RNA genomes are uncharacterized. Here we report the structure of an entire HIV-1 genome at single nucleotide resolution using SHAPE, a high-throughput RNA analysis technology. The genome encodes protein structure at two levels. In addition to the correspondence between RNA and protein primary sequences, a correlation exists between high levels of RNA structure and sequences that encode inter-domain loops in HIV proteins. This correlation suggests RNA structure modulates ribosome elongation to promote native protein folding. Some simple genome elements previously shown to be important, including the ribosomal gag-pol frameshift stem-loop, are components of larger RNA motifs. We also identify organizational principles for unstructured RNA regions. Highly used splice acceptors lie in unstructured motifs and hypervariable regions are sequestered from flanking genome regions by stable insulator helices. These results emphasize that the HIV-1 genome and, potentially, many coding RNAs are punctuated by numerous previously unrecognized regulatory motifs and that extensive RNA structure may constitute an additional level of the genetic code

Molecular Architectures of Trimeric SIV and HIV-1 Envelope Glycoproteins on Intact Viruses: Strain-Dependent Variation in Quaternary Structure

The initial step in target cell infection by human, and the closely related simian immunodeficiency viruses (HIV and SIV, respectively) occurs with the binding of trimeric envelope glycoproteins (Env), composed of heterodimers of the viral transmembrane glycoprotein (gp41) and surface glycoprotein (gp120) to target T-cells. Knowledge of the molecular structure of trimeric Env on intact viruses is important both for understanding the molecular mechanisms underlying virus-cell interactions and for the design of effective immunogen-based vaccines to combat HIV/AIDS. Previous analyses of intact HIV-1 BaL virions have already resulted in structures of trimeric Env in unliganded and CD4-liganded states at ∼20 Å resolution. Here, we show that the molecular architectures of trimeric Env from SIVmneE11S, SIVmac239 and HIV-1 R3A strains are closely comparable to that previously determined for HIV-1 BaL, with the V1 and V2 variable loops located at the apex of the spike, close to the contact zone between virus and cell. The location of the V1/V2 loops in trimeric Env was definitively confirmed by structural analysis of HIV-1 R3A virions engineered to express Env with deletion of these loops. Strikingly, in SIV CP-MAC, a CD4-independent strain, trimeric Env is in a constitutively “open” conformation with gp120 trimers splayed out in a conformation similar to that seen for HIV-1 BaL Env when it is complexed with sCD4 and the CD4i antibody 17b. Our findings suggest a structural explanation for the molecular mechanism of CD4-independent viral entry and further establish that cryo-electron tomography can be used to discover distinct, functionally relevant quaternary structures of Env displayed on intact viruses

Tonsillar application of AT-2 SIV affords partial protection against rectal challenge with SIVmac239

Background: Although mucosal responses are important for preventing infections with HIV, the optimal strategies for inducing them remain unclear. To evaluate vaccine strategies targeting the oral mucosal lymphoid tissue inductive sites as an approach to provide immunity at distal sites, we vaccinated healthy macaques via the palatine/lingual tonsils with aldrithiol 2 (AT-2) inactivated Simian immunodeficiency virus (SIV)mac239, combined with CpG-C immunostimulatory oligonucleotide (CpG-C ISS-ODN, C274) as the adjuvant. Methods: Macaques received 5 doses of C274 or control ODN C661 and AT-2 SIV on the tonsillar tissues every 6 weeks before being challenged rectally with SIVmac239, 8 weeks after the last immunization. Results: Although no T-cell or B-cell responses were detected in the blood before challenge, antibody (Ab) responses were detected in the rectum. Immunization with AT-2 SIV significantly reduced the frequency of infection compared with nonimmunized controls, irrespective of adjuvant. In the vaccinated animals that became infected, peak viremias were somewhat reduced. SIV-specific responses were detected in the blood once animals became infected with no detectable differences between the differently immunized groups and the controls. Conclusion: This work provides evidence that vaccine immunogens applied to the oral mucosal associated lymphoid tissues can provide benefit against rectal challenge, a finding with important implications for mucosal vaccination strategies

Control of Viremia and Prevention of Simian-Human Immunodeficiency Virus-Induced Disease in Rhesus Macaques Immunized with Recombinant Vaccinia Viruses plus Inactivated Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 1 Particles

An effective vaccine against the human immunodeficiency virus type 1 (HIV-1) will very likely have to elicit both cellular and humoral immune responses to control HIV-1 strains of diverse geographic and genetic origins. We have utilized a pathogenic chimeric simian-human immunodeficiency virus (SHIV) rhesus macaque animal model system to evaluate the protective efficacy of a vaccine regimen that uses recombinant vaccinia viruses expressing simian immunodeficiency virus (SIV) and HIV-1 structural proteins in combination with intact inactivated SIV and HIV-1 particles. Following virus challenge, control animals experienced a rapid and complete loss of CD4(+) T cells, sustained high viral loads, and developed clinical disease by 17 to 21 weeks. Although all of the vaccinated monkeys became infected, they displayed reduced postpeak viremia, had no significant loss of CD4(+) T cells, and have remained healthy for more than 15 months postinfection. CD8(+) T-cell and neutralizing antibody responses in vaccinated animals following challenge were demonstrable. Despite the control of disease, virus was readily isolated from the circulating peripheral blood mononuclear cells of all vaccinees at 22 weeks postchallenge, indicating that immunologic control was incomplete. Virus recovered from the animal with the lowest postchallenge viremia generated high virus loads and an irreversible loss of CD4(+) T-cell loss following its inoculation into a naïve animal. These results indicate that despite the protection from SHIV-induced disease, the vaccinated animals still harbored replication-competent and pathogenic virus

Carraguard inhibits infection in immature DCs coincident with DC maturation.

<p>(A) Immature DCs were pre-incubated with graded doses of Carraguard, after which the DCs were challenged with Bal (down triangles) or HIV Δenv pseudotyped with the VSVg envelope (Δenv, filled squares). Cells were harvested 5 d later and stained for (A) HIV capsid p24 protein (mAb KC-57-RD1) or (B) the surface maturation markers CD83 and CD86. (A) Percent inhibition (mean±SD, triplicates) of infection and (B) the MFIs (mean±SD of triplicates) of CD83 (black bar) and CD86 (grey bar) expression (on the entire DC population) are shown for 1 of 4 replicate experiments. CD83 and CD86 up-regulation in response to increasing doses of Carraguard correlate closely (r = 0.99). (C) p24 expression is plotted against CD83, showing the correlation between lower CD83 levels and HIV infection. Comparable results were obtained when comparing CD86 and p24 expression (data not shown).</p

<i>In vivo</i> activity of MIV-150-containing MC gels.

<p>Depo-Provera-treated animals were treated with (A) 3 ml of MC containing 500 µM MIV-150 30 min prior to vaginal challenge with 10³ or 10⁴ TCID₅₀ of RT-SHIV or (B) 3 ml of MC containing 500 µM MIV-150 24 h before, 30 min before, and 24 h after vaginal challenge with 10⁴ TCID₅₀ of RT-SHIV. Plasma viral loads over time are shown for the indicated numbers of animals in each group. One year after challenge the 3 animals with the low-level initial infection (now with undetectable virus) and two of the normally infected animals were treated with the anti-CD8 mAb to deplete CD8 cells. (C) Effective depletion of CD8 cells was verified by flow cytometry and the CD8 cells per µl of blood are shown for each animal. (D) Analysis of the plasma virus loads before during and after CD8 depletion, revealed no rebound in virus levels in the 3 animals with the unusual acute low-level infection. Each symbol denotes a different animal that are detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003162#pone-0003162-t001" target="_blank">Table 1</a>.</p

Carraguard can inhibit or enhance cell-free infection.

<p>(A) TZM.bl cells were exposed to 0–10 µg/ml of Carraguard, before graded doses of HIV_Bal or RT-SHIV were added. 24 h later, the media was replaced and cells were cultured for 4 d. The numbers of β-gal expressing SFCs per well are shown (mean±SD, triplicate cultures). (B) Titrated amounts of Carraguard were tested against of HIV_MN (MN, up triangles), HIV_Bal (Bal) (down triangles), or RT-SHIV (circles) in the TZM.bl cell line as in (A). The data are shown as the percent inhibition (mean±SD, triplicate cultures) of infection in the test conditions relative to the no Carraguard control. Negative % inhibition values represent enhancement, with no inhibitor effect at 0%. (C) Titrated amounts of Carraguard were added to PHA activated PBMCs before the cells were cultured with Bal or RT-SHIV for 5 d. Infection was measured by Q-PCR. Data are shown for triplicate cultures (mean±SD). Data in (A–C) are representative of 3 independent experiments with different donors in each case.</p

Infection and immune status of RT-SHIV-challenged macaques.

ˆ<p>CD4 counts taken on day 84 for DE38, day 189 for FI76, FI78, FI70, FI71, FI73, and FI75, and day 196 for FI86 and FI87.</p>$<p>Co-cultures of PBMCs with 174×CEM cells were scored as positive by microscopic examination of syncytia formation and in some instances verified by PCR for SIV gag.</p>&<p>DE38 had two timepoints of positive IFNγ responses at weeks 8 and 10 post challenge.</p>*<p>GF14 - average 715 copies/ml on day 42 post challenge, but the animal remained below the level of detection at all other time points.</p>%<p>500 µM of MIV-150 in MC administered once 30 min prior to challenge (1X) or 24 h before, 30 min before, and 30 min after virus challenge (3X).</p>@<p>These animals exhibited low-level viremia but ultimately controlled infection to undetectable levels even after CD8 depletion.</p><p>Ab positivity was defined as having at least two positive OD values above baseline at 4 weeks post challenge and IFNγ positivity was defined as having at least 50 SIV-specific IFNγ SFCs per 10⁶ PBMCs on more than one time point post challenge.</p><p>ND, not determined.</p

Carraguard-based gels inhibit vaginal infection in macaques.

<p>Depo-Provera-treated animals were treated with 3 ml of MC, Carraguard (Carr), or PC-817 30 min prior to challenge with 1 ml of or 10³–10⁵ TCID₅₀ RT-SHIV. (A) Plasma viral loads were quantified by PCR and SIV gag RNA copies per ml of plasma are shown for each animal over time. The numbers of animals in the respective groups are indicated in each panel. Each symbol denotes a different animal (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003162#pone-0003162-t001" target="_blank">Table 1</a>). (B) The frequencies (percentage of infected animals, mean±SEM) of infection in the MC, Carraguard, and PC-817-treated groups challenged with 10³ and 10⁴ TCID₅₀ are plotted. Carraguard (p<0.02) and PC-817 (p<0.03) significantly reduced the frequency of immunodeficiency virus infection compared to the MC-treated placebo group.</p

Carraguard augments mature DC-mediated amplification of infection.

<p>(A) Mature moDCs were pre-incubated with 0, 10, or 200 µg/ml Carraguard and challenged with graded doses of Bal or RT-SHIV, washed, and co-cultured with TZM.bl cells. Mean SFCs (±SD, triplicate conditions) are shown from 1 of 4 experiments. (B) Carraguard-treated mature DCs were pulsed with Bal or RT-SHIV, washed, and co-cultured with TZM.bl cells as in (A). The percent inhibition of infection (mean±SD, triplicate conditions) is shown for 1 of 4 experiments. A statistically significant difference (p<0.05, two-tailed paired <i>t</i>-test) between the enhancement effects on Bal vs RT-SHIV infection is noted by the asterisk. (C) Mature DCs pre-treated with Carraguard (Pre, open squares) were challenged with 3000 TCID₅₀ of Bal or 4500 TCID₅₀ of RT-SHIV, washed, and co-cultured with TZM.bl cells. Alternatively, mature DCs were pulsed with virus, washed, added to TZM.bl cells and the graded doses of Carraguard added to the co-cultures (Post, filled squares). The percent inhibition (mean±SD, triplicates) are shown from 1 of 4 experiments. A statistically significant difference (p<0.01, two-tailed paired <i>t</i>-test) between the pre versus post Carraguard enhancement effects on Bal infection is noted by the asterisk.</p