The role of the rhesus macaque (macaca mulatta) apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) in lentiviral replication and persistence

SHIV infections in rhesus macaques have been used to extensively study accessory proteins of HIV-1 involved in pathogenesis as well as in vaccine development. All primate lentiviruses encode for a Vif (virion infectivity factor) protein, which is required for HIV-1 replication in primary CD4+ T cells and macrophages. The Vif protein interacts with APOBEC3 (A3) proteins promoting their accelerated degradation by the 26S proteosome. Sequence analysis of Vif proteins from different lentiviruses revealed that there are two highly conserved domains in the carboxyl terminus that are required for recruitment of the of the Vif-CBF-β-Cul5/Elongin B/C/Rbx-1 E3 ubiquitin ligase complex. These domains are the viral BC box, SLQ(Y/F)LAL, and Zn2+ binding (H-X5-C-X17-18-C-X3-5-H; HCCH) motifs. Previous cell culture studies have shown that the introduction of amino acid substitutions into the SLQ(Y/F)LA motif resulted in decreased binding of Vif to Elongin C, while substitutions into the HCCH motif prevented the interaction of Vif with Cullin 5. In this first study, we introduced two amino acid changes in the highly conserved SLQYLA domain (S147A, L148A; AAQYLA) of the SIV Vif protein in SHIV. In vitro, the resulting virus, SHIVVifAAQYLA, replicated in A3G negative CEM-SS cells but failed to replicate in A3G positive CEM cells. We also showed that hA3G was incorporated into the virion. Following these in vitro studies, SHIVVifAAQYLA was inoculated into three rhesus macaques, which were followed for over six months to assess various viral and immunological factors throughout the duration of infection. All three macaques did not develop significant CD4+ T cell loss over the course of infection, had plasma viral loads that were over 100-fold lower than macaques inoculated with parental SHIVKU-1bMC33, and developed no histological lesions in various lymphoid tissues, and developed immunoprecipitating antibodies. DNA and RT-PCR analysis revealed that only a select number of tissues were infected with virus, while sequence analysis of PBMC and select tissue DNA (at necropsy) showed that the site-directed changes were stable during the first three weeks after inoculation but thereafter the S147A amino acid substitution changed to a threonine in two of the three macaques. However, the L148A substitution remained stale in the vif amplified from the PBMC and select tissues at necropsy in all three macaques. Extensive sequence analysis of the vif, vpu, nef, and env genes revealed a increased number of G-to-A mutations in the genes amplified from macaques inoculated with SHIVVifAAQYLA. The majority of these mutations (>85%) were in the context of 5'-T85%) were in the context of 5'-TC (minus strand) and not 3'-CC, suggesting that one or more of the rhesus A3 proteins may be responsible for the observed mutational patterns. To determine if infectious virus was present in the plasma at necropsy, plasma from the three macaques inoculated with SHIVVifAAQYLA were pooled and intravenously inoculated into a naïve macaque. This macaque maintained its levels of circulating CD4+ T cells throughout the duration of infection, maintained viral loads below the limits of detection, and did not produce immunoprecipitating antibodies. However, gag was present in the DNA and RNA isolated from PBMC throughout infection and in select tissues at necropsy. The results from this first study showed for the first time the importance of the SLYQLA domain in vivo in viral pathogenesis. It also showed that mutations in vif could lead to a persistent infection in rhesus macaques resulting in the accumulation of G-to-A substitutions in the viral genome. In the second study, we used the SHIV/macaque model of infection to compare the replication and pathogenicity of SHIVs that express a Vif protein in which the entire SLQYLA (SHIVVif5A) or HCCH (SHIVVifHCCH(-)) domains were substituted with alanine residues. Each virus was inoculated into three rhesus macaques where various viral and immunological parameters were followed for six months. Our in vitro results indicate that in the presence of these mutant Vif viruses, rhA3G is incorporated into the virion, stably expressed, restricts, and accumulates G-to-A substitutions (plus strand) in the nef,Italics/> gene of the mutated viral genomes. gene of the mutated viral genomes. In vivo, all macaques maintained a stable level of circulating CD4+ T cells, developed low viral burdens, maintained engineered mutations, yielded no histological lesions, and developed immunoprecipitating antibodies by 12 weeks post-inoculation. However, the production of viral RNA only persisted in macaques inoculated with SHIVVifHCCH(-). The analysis of vif sequences amplified from PBMC DNA between weeks 0-16 during SHIVVifHCCH(-) infection revealed an increased number of G-to-A substitutions that increased with time in two of the three macaques. Sequence analysis of nef and vpu from the small intestine (ileum), thymus, and the spleen showed G-to-A substitutions in nef genes isolated from macaques inoculated with SHIVVifHCCH(-). Macaques inoculated with SHIVVif5A effectively controlled the virus three weeks post-inoculation and no viral sequences could be amplified from tissue DNA. These studies showed that the SLQYLA and HCCH domains are critical for viral pathogenesis in vivo and that there may exist APOBEC3 negative reservoirs in the rhesus macaque that allow for low levels of viral replication and persistence but not disease. Therefore, this study suggests that mutations targeted to one or more functional conserved domains within the Vif protein may limit viral replication and generate an effective immune response leading to the "self-inactivation" of the virus by the activities of various APOBEC3 proteins resulting in a possible live-attenuated vaccine candidate. The APOBEC3 family of restriction factors has been shown to inhibit certain retroviruses and retroelements. The APOBEC3 family in humans is comprised of seven cytidine deaminases (A3A, A3B, A3C, A3D, A3F, A3G, and A3H) that catalyze the deamination of cytidine to uracil on single-stranded DNA or RNA. While the human APOBEC3 repertoire has been extensively studied, the full complement of these proteins in the rhesus macaque remains unknown. Sequencing of the rhesus macaque genome has led to the identification of the rhesus homologues A3B, A3C, A3D, A3F, A3G, and A3H. Finally, we identified a human A3A (hA3A) homologue in the rhesus macaque (rhA3A) and presents evidence that both the human and rhesus Apobec3 genes are orthologous. We show that rhA3A is highly expressed in activated CD4+ T cells, widely expressed in both the visceral and central nervous system tissues of the rhesus macaque, and is degraded in the presence of the human immunodeficiency virus (HIV-1) and simian-human immunodeficiency virus (SHIV) genomes in a Vif-dependent manner. Our results also indicate that rhA3A reduced the level of infectious SHIVΔvif by approximately 20-fold and HIV-1Δvif by 3-fold. Human and monkey A3A amino acid sequences are 81% homologous and can be distinguished by a three amino acid indel located between residues 27-30. When these residues were deleted from rhA3A (rhA3AΔSVR), the antiviral activity of rhA3A was abolished suggesting that these residues are critical for lentivirus inhibition. Select APOBEC3 proteins are incorporated into the virion and can inhibit reverse transcription and/or induce G-to-A hypermutation in nascent reverse transcripts in the next target cell. Previous studies revealed that rhA3G is incorporated into SHIVΔvif virions and exerts its antiviral activity in target cells by an increase in cytidine deamination of newly synthesized minus-strand viral DNA from cytosines to uracils, leading to G-to-A substitutions (plus strand) in the viral genome. We were able to detect the incorporation of rhA3A into SHIVΔvif and to a lesser extent in SHIV virions; however, we were unable to detect the incorporation of rhA3A into either HIV-1 or HIV-1Δvif virions. Even though rhA3A is incorporated into SHIVΔvif virions and potently restricts SHIVΔvif similar to rhA3G, rhA3A produced an approximately 5-fold decrease in the number of G-to-A mutations compared to rhA3G. Unlike hA3A, rhA3A did not inhibit adeno-associated virus 2 (AAV-2) replication and L1 retrotransposition. This data suggests for the first time an evolutionary switch in primate A3A virus specificity and provides evidence that a primate A3A protein can inhibit lentiviral replication

δ13C and δ15N in the endangered Kemp’s ridley sea turtle Lepidochelys kempii after the Deepwater Horizon oil spill

The Deepwater Horizon explosion in April 2010 and subsequent oil spill released 3.19 × 106 barrels (5.07 × 108 l) of MC252 crude oil into important foraging areas of the endangered Kemp’s ridley sea turtle Lepidochelys kempii (Lk) in the northern Gulf of Mexico (GoM). We measured δ13C and δ15N in scute biopsy samples from 33 Lk nesting in Texas during the period 2010 to 2012. Of these, 27 were equipped with satellite transmitters and were tracked to traditional foraging areas in the northern GoM after the spill. Differences in δ13C between the oldest and newest scute layers from 2010 nesters were not significant, but δ13C in the newest layers from 2011 and 2012 nesters was significantly lower compared to 2010. δ15N differences were not statis- tically significant. Collectively, the stable isotope and tracking data indicate that the lower δ13C values reflect the incorporation of oil rather than changes in diet or foraging area. Discriminant analysis indicated that 51.5% of the turtles sampled had isotope signatures indicating oil exposure. Growth of the Lk population slowed in the years following the spill. The involvement of oil exposure in recent population trends is unknown, but long-term effects may not be evident for many years. Our results indicate that C isotope signatures in scutes may be useful biomarkers of sea turtle exposure to oil

Cellular HIV-1 inhibition by truncated old world primate APOBEC3A proteins lacking a complete deaminase domain

AbstractThe APOBEC3 (A3) deaminases are retrovirus restriction factors that were proposed as inhibitory components of HIV-1 gene therapy vectors. However, A3 mutational activity may induce undesired genomic damage and enable HIV-1 to evade drugs and immune responses. Here, we show that A3A protein from Colobus guereza (colA3A) can restrict HIV-1 replication in producer cells in a deaminase-independent manner without inducing DNA damage. Neither HIV-1 reverse transcription nor integration were significantly affected by colA3A, but capsid protein synthesis was inhibited. The determinants for colA3A restriction mapped to the N-terminal region. These properties extend to A3A from mandrills and De Brazza׳s monkeys. Surprisingly, truncated colA3A proteins expressing only the N-terminal 100 amino acids effectively exclude critical catalytic regions but retained potent cellular restriction activity. These highlight a unique mechanism of cellular HIV-1 restriction by several Old World monkey A3A proteins that may be exploited for functional HIV-1 cure strategies

AKAP7 Regulates CaM Kinase Activation in MCF-7 Cells

Abstract Estrogen (E2) activates calcium/calmodulin-dependent protein kinases (CaM Kinases) in MCF-7 breast cancer cells. In particular E2 activates a CaM KK, CaM KI, and ERK pathway to promote proliferation. CaM Kinase activation of ERK may be blocked by PKA in certain cell types through direct phosphorylation and inhibition of CaM KK. The ability of PKA to phosphorylate its cellular targets may be dictated by protein kinase A anchoring proteins (AKAPs). Hormones that elevate cAMP and activate PKA may utilize AKAPs to regulate signal transduction. Our goal was to evaluate the role of AKAPs in regulating E2 activation of the CaM KK and CaM KI pathway in MCF-7 cells. We also examined the ability of vitamin D (VitD) working through cAMP and PKA to block CaM KK signaling in breast cancer cells. Our results suggest that E2 activates CaM KK and CaM KI within 5 minutes. VitD promoted PKA-dependent phosphorylation of CaM KK. VitD and epinephrine treatment of cells triggered a potent increase in cAMP levels. Interestingly, purified GST-RII pulled down both CaM KK and CaM KI. Similarly, purified AKAP7 but not AKAP5 bound CaM KK and CaM KI an effect that is enhanced with E2. Endogenous AKAP7 and CaM KK associated in E2-stimulated but not in VitD-treated cells and VitD also blocked CaM KK activation in MCF-7 cells. Our results suggest that VitD blocks E2 activation of CaM Kinases and their association with AKAP7 in MCF-7 cells

Differential virus restriction patterns of rhesus macaque and human APOBEC3A: Implications for lentivirus evolution

AbstractThe human apolipoprotein B mRNA editing enzyme catalytic peptide-like 3 (APOBEC3; A3) family of proteins (A3A-H) are known to restrict various retroviruses and retroelements, but the full complement of rhesus macaque A3 proteins remains unclear. We report the isolation and characterization of the hA3A homologue from rhesus macaques (rhA3A) and show that the rhesus macaque and human A3 genes are orthologous. RhA3A is expressed at high levels in activated CD4+ T cells, is widely expressed in macaque tissues, and is degraded in the presence of the human immunodeficiency virus (HIV-1) and simian–human immunodeficiency virus (SHIV) genomes. Our results indicate that rhA3A is a potent inhibitor of SHIVΔvif and to a lesser extent HIV-1Δvif. Unlike hA3A, rhA3A did not inhibit adeno-associated virus 2 (AAV-2) replication and L1 retrotransposition. These data suggest an evolutionary switch in primate A3A virus specificity and provide the first evidence that a primate A3A can inhibit lentivirus replication

δ13C and δ15N in the endangered Kemp’s ridley sea turtle Lepidochelys kempii after the Deepwater Horizon oil spill

The Deepwater Horizon explosion in April 2010 and subsequent oil spill released 3.19 × 106 barrels (5.07 × 108 l) of MC252 crude oil into important foraging areas of the endangered Kemp’s ridley sea turtle Lepidochelys kempii (Lk) in the northern Gulf of Mexico (GoM). We measured δ13C and δ15N in scute biopsy samples from 33 Lk nesting in Texas during the period 2010 to 2012. Of these, 27 were equipped with satellite transmitters and were tracked to traditional foraging areas in the northern GoM after the spill. Differences in δ13C between the oldest and newest scute layers from 2010 nesters were not significant, but δ13C in the newest layers from 2011 and 2012 nesters was significantly lower compared to 2010. δ15N differences were not statis- tically significant. Collectively, the stable isotope and tracking data indicate that the lower δ13C values reflect the incorporation of oil rather than changes in diet or foraging area. Discriminant analysis indicated that 51.5% of the turtles sampled had isotope signatures indicating oil exposure. Growth of the Lk population slowed in the years following the spill. The involvement of oil exposure in recent population trends is unknown, but long-term effects may not be evident for many years. Our results indicate that C isotope signatures in scutes may be useful biomarkers of sea turtle exposure to oil

BST-2 mediated restriction of simian–human immunodeficiency virus

AbstractPathogenic simian–human immunodeficiency viruses (SHIV) contain HIV-1 Vpu and SIV Nef, both shown to counteract BST-2 (HM1.24; CD317; tetherin) inhibition of virus release in a species-specific manner. We show that human and pig-tailed BST-2 (ptBST-2) restrict SHIV. We found that sequential “humanization” of the transmembrane domain (TMD) of the pig-tailed BST-2 (ptBST-2) protein resulted in a fluctuation in sensitivity to HIV-1 Vpu. Our results also show that the length of the TMD in human and ptBST-2 proteins is important for BST-2 restriction and susceptibility to Vpu. Taken together, our results emphasize the importance of tertiary structure in BST-2 antagonism and suggests that the HIV-1 Vpu transmembrane domain may have additional functions in vivo unrelated to BST-2 antagonism

Cryovolcanic flooding in Viking Terra on Pluto

A prominent fossa trough (Uncama Fossa) and adjacent 28-km diameter impact crater (Hardie) in Pluto's Viking Terra, as seen in the high-resolution images from the New Horizons spacecraft, show morphological evidence of in-filling with a material of uniform texture and red-brown color. A linear fissure parallel to the trough may be the source of a fountaining event yielding a cryoclastic deposit having the same composition and color properties as is found in the trough and crater. Spectral maps of this region with the New Horizons LEISA instrument reveal the spectral signature of H₂O ice in these structures and in distributed patches in the adjacent terrain in Viking Terra. A detailed statistical analysis of the spectral maps shows that the colored H₂O ice filling material also carries the 2.2-μm signature of an ammoniated component that may be an ammonia hydrate (NH₃nH₂O) or an ammoniated salt. This paper advances the view that the crater and fossa trough have been flooded by a cryolava debouched from Pluto's interior along fault lines in the trough and in the floor of the impact crater. The now frozen cryolava consisted of liquid H₂O infused with the red-brown pigment presumed to be a tholin, and one or more ammoniated compounds. Although the abundances of the pigment and ammoniated compounds entrained in, or possibly covering, the H₂O ice are unknown, the strong spectral bands of the H₂O ice are clearly visible. In consideration of the factors in Pluto's space environment that are known to destroy ammonia and ammonia-water mixtures, the age of the exposure is of order ≤10⁹ years. Ammoniated salts may be more robust, and laboratory investigations of these compounds are needed