The use of nanotrap particles technology in capturing HIV-1 virions and viral proteins from infected cells

HIV-1 infection results in a chronic but incurable illness since long-term HAART can keep the virus to an undetectable level. However, discontinuation of therapy rapidly increases viral burden. Moreover, patients under HAART frequently develop various metabolic disorders and HIV-associated neuronal disease. Today, the main challenge of HIV-1 research is the elimination of the residual virus in infected individuals. The current HIV-1 diagnostics are largely comprised of serological and nucleic acid based technologies. Our goal is to integrate the nanotrap technology into a standard research tool that will allow sensitive detection of HIV-1 infection. This study demonstrates that majority of HIV-1 virions in culture supernatants and Tat/Nef proteins spiked in culture medium can be captured by nanotrap particles. To determine the binding affinities of different baits, we incubated target molecules with nanotrap particles at room temperature. After short sequestration, materials were either eluted or remained attached to nanotrap particles prior to analysis. The unique affinity baits of nanotrap particles preferentially bound HIV-1 materials while excluded albumin. A high level capture of Tat or Tat peptide by NT082 and NT084 particles was measured by western blot (WB). Intracellular Nef protein was captured by NT080, while membrane-associated Nef was captured by NT086 and also detected by WB. Selective capture of HIV-1 particles by NT073 and NT086 was measured by reverse transcriptase assay, while capture of infectious HIV-1 by these nanoparticles was demonstrated by functional transactivation in TZM-bl cells. We also demonstrated specific capture of HIV-1 particles and exosomes-containing TAR-RNA in patients\u27 serum by NT086 and NT082 particles, respectively, using specific qRT-PCR. Collectively, our data indicate that certain types of nanotrap particles selectively capture specific HIV-1 molecules, and we propose to use this technology as a platform to enhance HIV-1 detection by concentrating viral proteins and infectious virions from infected samples

p53 Activation following Rift Valley Fever Virus Infection Contributes to Cell Death and Viral Production

Rift Valley fever virus (RVFV) is an emerging viral zoonosis that is responsible for devastating outbreaks among livestock and is capable of causing potentially fatal disease in humans. Studies have shown that upon infection, certain viruses have the capability of utilizing particular cellular signaling pathways to propagate viral infection. Activation of p53 is important for the DNA damage signaling cascade, initiation of apoptosis, cell cycle arrest and transcriptional regulation of multiple genes. The current study focuses on the role of p53 signaling in RVFV infection and viral replication. These results show an up-regulation of p53 phosphorylation at several serine sites after RVFV MP-12 infection that is highly dependent on the viral protein NSs. qRT-PCR data showed a transcriptional up-regulation of several p53 targeted genes involved in cell cycle and apoptosis regulation following RVFV infection. Cell viability assays demonstrate that loss of p53 results in less RVFV induced cell death. Furthermore, decreased viral titers in p53 null cells indicate that RVFV utilizes p53 to enhance viral production. Collectively, these experiments indicate that the p53 signaling pathway is utilized during RVFV infection to induce cell death and increase viral production

Human metapneumovirus - what we know now [version 1; referees: 2 approved]

Human metapneumovirus (HMPV) is a leading cause of acute respiratory infection, particularly in children, immunocompromised patients, and the elderly. HMPV, which is closely related to avian metapneumovirus subtype C, has circulated for at least 65 years, and nearly every child will be infected with HMPV by the age of 5. However, immunity is incomplete, and re-infections occur throughout adult life. Symptoms are similar to those of other respiratory viral infections, ranging from mild (cough, rhinorrhea, and fever) to more severe (bronchiolitis and pneumonia). The preferred method for diagnosis is reverse transcription-polymerase chain reaction as HMPV is difficult to culture. Although there have been many advances made in the past 16 years since its discovery, there are still no US Food and Drug Administration-approved antivirals or vaccines available to treat HMPV. Both small animal and non-human primate models have been established for the study of HMPV. This review will focus on the epidemiology, transmission, and clinical manifestations in humans as well as the animal models of HMPV pathogenesis and host immune response

The use of Nanotrap particles in the enhanced detection of Rift Valley fever virus nucleoprotein.

Rift Valley fever virus (RVFV) is a highly pathogenic arthropod-borne virus that has a detrimental effect on both livestock and human populations. While there are several diagnostic methodologies available for RVFV detection, many are not sensitive enough to diagnose early infections. Furthermore, detection may be hindered by high abundant proteins such as albumin. Previous findings have shown that Nanotrap particles can be used to significantly enhance detection of various small analytes of low abundance. We have expanded upon this repertoire to show that this simple and efficient sample preparation technology can drastically improve the detection of the RVFV nucleoprotein (NP), the most abundant and widely used viral protein for RVFV diagnostics.After screening multiple Nanotrap particle architectures, we found that one particle, NT45, was optimal for RVFV NP capture, as demonstrated by western blotting. NT45 significantly enhanced detection of the NP at levels undetectable without the technology. Importantly, we demonstrated that Nanotrap particles are capable of concentrating NP in a number of matrices, including infected cell lysates, viral supernatants, and animal sera. Specifically, NT45 enhanced detection of NP at various viral titers, multiplicity of infections, and time points. Our most dramatic results were observed in spiked serum samples, where high abundance serum proteins hindered detection of NP without Nanotrap particles. Nanotrap particles allowed for sample cleanup and subsequent detection of RVFV NP. Finally, we demonstrated that incubation of our samples with Nanotrap particles protects the NP from degradation over extended periods of time (up to 120 hours) and at elevated temperatures (at 37ºC).This study demonstrates that Nanotrap particles are capable of drastically lowering the limit of detection for RVFV NP by capturing, concentrating, and preserving RVFV NP in clinically relevant matrices. These studies can be extended to a wide range of pathogens and their analytes of diagnostic interest

Protein Phosphatase-1 regulates Rift Valley fever virus replication

Rift Valley fever virus (RVFV), genus Phlebovirus family Bunyaviridae, is an arthropod-borne virus endemic throughout sub-Saharan Africa. Recent outbreaks have resulted in cyclic epidemics with an increasing geographic footprint, devastating both livestock and human populations. Despite being recognized as an emerging threat, relatively little is known about the virulence mechanisms and host interactions of RVFV. To date there are no FDA approved therapeutics or vaccines for RVF and there is an urgent need for their development. The Ser/Thr protein phosphatase 1 (PP1) has previously been shown to play a significant role in the replication of several viruses. Here we demonstrate for the first time that PP1 plays a prominent role in RVFV replication early on during the viral life cycle. Both siRNA knockdown of PP1α and a novel PP1-targeting small molecule compound 1E7-03, resulted in decreased viral titers across several cell lines. Deregulation of PP1 was found to inhibit viral RNA production, potentially through the disruption of viral RNA transcript/protein interactions, and indicates a potential link between PP1α and the viral L polymerase and nucleoprotein. These results indicate that PP1 activity is important for RVFV replication early on during the viral life cycle and may prove an attractive therapeutic target

Nanotrap particles can capture and enrich NP from virally infected cells.

<p>A) One ml of cytoplasmic extract (CE) at 2.6 μg/ml obtained from RVFV infected Vero lysates were incubated with 100 μl of NT45, NT53, and NT69. No Nanotrap particle sample (-NT) was processed in parallel. After 30 minutes, the (+)NT samples were centrifuged. The unbound material (S) from the spin was saved and 10 μl was processed in parallel. The bound material (P) was resuspended in blue lysis buffer and boiled for 10 minutes. The samples were centrifuged at maximum speed and the supernatants were then loaded onto a NuPage 4–12% Bis-Tris gel. Samples were subsequently analyzed by western blot for NP. B) One ml of CE was serially diluted in 50mM Tris-HCl from 15 μg/ml to 0.75 μg/ml and incubated with 100 μl of NT45 for 30 minutes. No Nanotrap particle samples (-NT) were processed in parallel. The control sample is CE at 770 μg/ml (10 μl volume). The samples were processed as in panel A.</p

Non-virion associated NP can be detected in viral supernatants.

<p>Vivaspin 20 centrifugal concentrators with a 300,000 Da MWCO were used to filter viral supernatants harvested from Vero cells infected at an MOI 1 with MP12. Five milliliters of viral supernatant was added to the concentrators and centrifuged at 1400 rpm for 7 minutes until approximately 500 μl remained on the top portion of the concentrator. A) The top (T) and bottom (B) fractions, as well as control sample (C) containing the original sample before processing were analyzed by western blot using antibodies against NP (EC22 antibody). C, T, and B lysates were undiluted (neat). Cytoplasmic extract (CE) control is 7.7 μg/ml of RVFV infected Vero cell lysates. B) Plaque assays were performed with both fractions and the control sample. C) Densitometry analysis was performed to determine the NP band densities in the T and B fractions. The percent of total NP was determined by dividing the top or bottom portion's band density by the total NP band density (top and bottom portions added together) and multiplying by 100. D) NP band density per pfu values were calculated by dividing the NP band densities by the total pfu values.</p

Nanotrap particles can capture RVFV NP.

<p>A) Recombinant histidine-tagged NP (His-NP) at a starting concentration of 0.4 mg/ml and a volume of 100 μl was incubated with 75 μl of NT45, NT46, NT53, NT55, NT69, or NT71 for 30 minutes at ambient temperature. After 30 minutes, the (+)NT samples were centrifuged. Both bound (P) and 10uL of unbound (S) material was resuspended in blue lysis buffer and boiled for 10 minutes. The samples were centrifuged at maximum speed and the supernatants were then analyzed for presence of NP protein by western blot using antibodies directed against the histidine tag. No Nanotrap particle samples (-NT) at a 10 μl volume of 0.4 mg/ml His-NP were processed in parallel. B) Purified NP (obtained from BEI Resources) at 2 μg in a volume of 100 μl was incubated with 75 μl of NT45, NT46, NT53, NT55, NT69, or NT71 for 30 minutes at an ambient temperature. A control—NT sample (10 μl volume) was processed in parallel. Samples were processed as describe in panel A. After electrophoresis, NP was visualized by Commassie blue staining. C) His-NP (obtained from Immune Technology) at 1 μg/ml and a volume of one milliliter was incubated with 100 μl of NT45, NT53, and NT69. A control—NT sample (10 μl volume) was processed in parallel. The control sample is 100 μg/ml NP (volume of 10 μl). The samples were processed as in panel A and analyzed by western blotting for NP by using antibodies against NP. D) Viral supernatants at 1E+06 pfu/ml and a volume of 1 ml were incubated with 100 μl NT45, NT46, NT53, NT55, and NT69 for 30 minutes at an ambient temperature. Control—NT samples (10 μl volumes) at 1E+07pfu/ml and 1E+06 pfu/ml were processed in parallel. The samples were processed as in panel A and analyzed by western blot for NP.</p

Varying modulation of HIV-1 LTR activity by BAF complexes

The human immunodeficiency virus type 1 (HIV-1) long terminal repeat is present on both ends of the integrated viral genome and contains regulatory elements needed for transcriptional initiation and elongation. Post-integration, a highly ordered chromatin structure consisting of at least five nucleosomes, is found at the 5′ long terminal repeat, the location and modification state of which control the state of active viral replication as well as silencing of the latent HIV-1 provirus. In this context, the chromatin remodeling field rapidly emerges as having a critical role in the control of viral gene expression. In the current study, we focused on unique Baf subunits that are common to the most highly recognized of chromatin remodeling proteins, the SWI/SNF (switching-defective-sucrose non-fermenting) complexes. We find that at least two Baf proteins, Baf53 and Baf170, are highly regulated in HIV-1-infected cells. Previously, studies have shown that the depletion of Baf53 in uninfected cells leads to the expansion of chromosomal territories and the decompaction of the chromatin. Baf53, in the presence of HIV-1 infection, co-elutes off of a chromatographic column as a different-sized complex when compared to uninfected cells and appears to be predominantly phosphorylated. The innate function of Baf53-containing complexes appears to be transcriptionally suppressive, in that knocking down Baf53 increases viral gene expression from cells both transiently and chronically infected with HIV-1. Additionally, cdk9/cyclin T in the presence of Tat is able to phosphorylate Baf53 in vitro, implying that this posttranslationally modified form relieves the suppressive effect and allows for viral transcription to proceed. © 2011 Elsevier Ltd. All rights reserved