Cellular and viral specificities of human immunodeficiency virus type 1 vif protein

The vif gene of human immunodeficiency virus type 1 (HIV-1) greatly enhances the infectivity of HIV-1 virions that are released from cells classified as nonpermissive (e.g., lymphocytes, macrophages, and H9 leukemic T cells) but is irrelevant in permissive cells (e.g., HeLa or COS cells). Recently, it was reported that vif expression in nonpermissive cells dramatically increases infectivity not only of HIV-1 but also of other enveloped viruses, including murine leukemia viruses (MLVs). This was surprising in part because MLVs and other murine retroviruses lack vif genes yet replicate efficiently in T lymphocytes. To investigate these issues, we first developed improved methods for producing substantial quantities of HIV-1 virions with vif deletions from healthy H9 cells. These virions had approximately the same amounts of major core proteins and envelope glycoproteins as the control wild-type virions but were only approximately 1% as infectious. We then produced H9 cells that contained wild-type or vif deletion HIV-gpt proviruses, which lack a functional env gene. After superinfection with either xenotropic or amphotropic MLVs, these cells released HIV-gpt virions pseudotyped with an MLV envelope plus replication-competent MLV. Interestingly, the pseudotyped HIV-gpt (vif deletion) virions were noninfectious, whereas the MLV virions simultaneously released from the same H9 cells were fully infectious. These results strongly suggest that the Vif protein functions in a manner that is both cell specific and at least substantially specific for HIV-1 and related lentiviruses. In addition, these results confirm that vif deletion HIV-1 virions from nonpermissive cells are blocked at a postpenetration stage of the infection pathway.     

Substrate translocation kinetics of excitatory amino acid carrier 1 probed with laser-pulse photolysis of a new photolabile precursor of D-aspartic acid

Here we report the synthesis and photochemical and biological characterization of a new photolabile precursor of D-aspartic acid, alpha-carboxynitrobenzyl-caged D-aspartate (alpha-CNB-caged D-aspartate), and its application for studying the molecular mechanism of the neuronal excitatory amino acid carrier 1 (EAAC1). Investigation of the photochemical properties of alpha-CNB-caged D-aspartate by transient absorption spectroscopy of the aci-nitro intermediate revealed that it photolyzes with a quantum yield of 0. 19 at pH 7.0. The major component of the aci-nitro intermediate (77% of the total absorbance) decays with a time constant of 26 s. This decay is slowed by only a factor of 2 when increasing the pH to 10. A minor component (21%) decays with a time constant of 410 s and is pH insensitive. The compound was tested with respect to its biological activity with the glutamate transporter EAAC1 expressed in HEK293 cells. Whole-cell current recordings from these cells in the presence and absence of alpha-CNB-caged D-aspartate demonstrated that the compound neither activates nor inhibits EAAC1. Upon photolysis, D-aspartate-mediated whole-cell currents were generated. In contrast to laser-pulse photolysis experiments with alpha-CNB-caged L-glutamate, only a minor and much slower transient current component was observed. These results indicate that the substrate translocation step, which is not rate-limiting for the overall turnover of the transporter with L-glutamate, becomes rate-limiting when D-aspartate is translocated. The results demonstrate that the new caged D-aspartate derivative is a useful tool for the investigation of the molecular mechanism of glutamate transporters and probably other aspartate translocating systems using rapid chemical kinetic techniques.     

Functional mimicry of a human immunodeficiency virus type 1 coreceptor by a neutralizing monoclonal antibody

Interaction of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein with the primary receptor, CD4, promotes binding to a chemokine receptor, either CCR5 or CXCR4. The chemokine receptor-binding site on gp120 elicits CD4-induced (CD4i) antibodies in some HIV-1-infected individuals. Like CCR5 itself, the CD4i antibody 412d exhibits a preference for CCR5-using HIV-1 strains and utilizes sulfated tyrosines to achieve binding to gp120. Here, we show that 412d binding requires the gp120 beta19 strand and the base of the V3 loop, elements that are important for the binding of the CCR5 N terminus. Two gp120 residues in the V3 loop base determined 412d preference for CCR5-using HIV-1 strains. A chimeric molecule in which the 412d heavy-chain third complementarity-determining loop sequence replaces the CCR5 N terminus functioned as an efficient second receptor, selectively supporting the entry of CCR5-using HIV-1 strains. Sulfation of N-terminal tyrosines contributed to the function of this chimeric receptor. These results emphasize the close mimicry of the CCR5 N terminus by the gp120-interactive region of a naturally elicited CD4i antibody.     

An Endogenous Inhibitor of Human Immunodeficiency Virus in Human Lymphocytes Is Overcome by the Viral Vif Protein

The vif gene of human immunodeficiency virus type 1 (HIV-1) encodes a basic M_r 23,000 protein that is necessary for production of infectious virions by nonpermissive cells (human lymphocytes and macrophages) but not by permissive cells such as HeLa-CD4. It had been proposed that permissive cells may contain an unidentified factor that functions like the viral Vif protein. To test this hypothesis, we produced pseudotyped wild-type and vif-deleted HIV gpt virions (which contain the HIV-1 genome with the bacterial mycophenolic acid resistance gene gpt in place of the viral env gene) in permissive cells, and we used them to generate nonpermissive H9 leukemic T cells that express these proviruses. We then fused these H9 cells with permissive HeLa cells that express the HIV-1 envelope glycoprotein gp120-gp41, and we asked whether the heterokaryons would release infectious HIV gpt virions. The results clearly showed that the vif-deleted virions released by the heterokaryons were noninfectious whereas the wild-type virions were highly infectious. This strongly suggests that nonpermissive cells, the natural targets of HIV-1, contain a potent endogenous inhibitor of HIV-1 replication that is overcome by Vif.     

First External Quality Assessment of Molecular and Serological Detection of Rift Valley Fever in the Western Mediterranean Region

Rift Valley fever (RVF) is a mosquito-borne viral zoonosis which affects humans and a wide range of domestic and wild ruminants. The large spread of RVF in Africa and its potential to emerge beyond its geographic range requires the development of surveillance strategies to promptly detect the disease outbreaks in order to implement efficient control measures, which could prevent the widespread of the virus to humans. The Animal Health Mediterranean Network (REMESA) linking some Northern African countries as Algeria, Egypt, Libya, Mauritania, Morocco, Tunisia with Southern European ones as France, Italy, Portugal and Spain aims at improving the animal health in the Western Mediterranean Region since 2009. In this context, a first assessment of the diagnostic capacities of the laboratories involved in the RVF surveillance was performed. The first proficiency testing (external quality assessment—EQA) for the detection of the viral genome and antibodies of RVF virus (RVFV) was carried out from October 2013 to February 2014. Ten laboratories participated from 6 different countries (4 from North Africa and 2 from Europe). Six laboratories participated in the ring trial for both viral RNA and antibodies detection methods, while four laboratories participated exclusively in the antibodies detection ring trial. For the EQA targeting the viral RNA detection methods 5 out of 6 laboratories reported 100% of correct results. One laboratory misidentified 2 positive samples as negative and 3 positive samples as doubtful indicating a need for corrective actions. For the EQA targeting IgG and IgM antibodies methods 9 out of the 10 laboratories reported 100% of correct results, whilst one laboratory reported all correct results except one false-positive. These two ring trials provide evidence that most of the participating laboratories are capable to detect RVF antibodies and viral RNA thus recognizing RVF infection in affected ruminants with the diagnostic methods currently available.     

6-Dimethylaminopurine (6-DMAP), a reversible inhibitor of the transition to metaphase during the first meiotic cell division of the mouse oocyte

The first meiotic cell division (meiotic maturation) of dictyate stage mouse oocytes removed from the follicle resumes spontaneously in vitro. We used the puromycin analog 6-dimethylaminopurine (6-DMAP) to test the respective roles of protein synthesis and protein phosphorylation in driving this process. While protein synthesis inhibitors do not block meiosis resumption, 6-DMAP was found to inhibit germinal vesicle breakdown (GVBD), by inhibiting the burst of protein phosphorylation without changing the rate of incorporation of [35S]methionine into proteins. This effect is reversible; it depends both upon drug concentration and the particular female. When added after GVBD and before the emission of the first polar body, 6-DMAP decreases the level of protein phosphorylation and induces decondensation of the chromosomes and reformation of the nuclear envelope. In contrast, 6-DMAP did not trigger these processes in metaphase II oocytes which only produce resting nuclei when treated by protein synthesis inhibitors. From these data, we conclude that (1) the early appearance and stability of mouse MPF in Metaphase I oocytes depend on protein phosphorylation rather than on protein synthesis, and (2) protein synthesis is necessary to maintain the condensation of the chromosomes in metaphase II oocytes.     

Remote Sensing Derived Fire Frequency,Soil Moisture and Ecosystem Productivity Explain Regional Movements in Emu over Australia

Species distribution modeling has been widely used in studying habitat relationships and for conservation purposes. However, neglecting ecological knowledge about species, e.g. their seasonal movements, and ignoring the proper environmental factors that can explain key elements for species survival (shelter, food and water) increase model uncertainty. This study exemplifies how these ecological gaps in species distribution modeling can be addressed by modeling the distribution of the emu (Dromaius novaehollandiae) in Australia. Emus cover a large area during the austral winter. However, their habitat shrinks during the summer months. We show evidence of emu summer habitat shrinkage due to higher fire frequency, and low water and food availability in northern regions. Our findings indicate that emus prefer areas with higher vegetation productivity and low fire recurrence, while their distribution is linked to an optimal intermediate (~0.12 m³ m^-3) soil moisture range. We propose that the application of three geospatial data products derived from satellite remote sensing, namely fire frequency, ecosystem productivity, and soil water content, provides an effective representation of emu general habitat requirements, and substantially improves species distribution modeling and representation of the species’ ecological habitat niche across Australia.     

Ferritin synthesis on polyribosomes attached to the endoplasmic reticulum.

The evidence that ferritin is synthesized both on free polyribosomes and on polyribosomes attached to the endoplasmic reticulum is reviewed. Evidence that some ferritin is secreted from cells after synthesis on bound polyribosomes was found to be inconclusive.