A directed evolution approach to select for novel Adeno-associated virus capsids on an HIV-1 producer T cell line.

A directed evolution approach was used to select for Adeno-associated virus (AAV) capsids that would exhibit more tropism toward an HIV-1 producer T cell line with the long-term goal of developing improved gene transfer vectors. A library of AAV variants was used to infect H9 T cells previously infected or uninfected by HIV-1 followed by AAV amplification with wild-type adenovirus. Six rounds of biological selection were performed, including negative selection and diversification after round three. The H9 T cells were successfully infected with all three wild-type viruses (AAV, adenovirus, and HIV-1). Four AAV cap mutants best representing the small number of variants emerging after six rounds of selection were chosen for further study. These mutant capsids were used to package an AAV vector and subsequently used to infect H9 cells that were previously infected or uninfected by HIV-1. A quantitative polymerase chain reaction assay was performed to measure cell-associated AAV genomes. Two of the four cap mutants showed a significant increase in the amount of cell-associated genomes as compared to wild-type AAV2. This study shows that directed evolution can be performed successfully to select for mutants with improved tropism for a T cell line in the presence of HIV-1.

Silver nanoparticles inhibit vaccinia virus infection by preventing viral entry through a macropinocytosis-dependent mechanism.

Silver nanoparticles have been shown to inhibit viruses. However, very little is known about the mechanism of antiviral activity. This study tested the hypothesis that 25-nm silver nanoparticles inhibited Vaccinia virus replication by preventing viral entry. Plaque reduction, confocal microscopy, and beta-galactosidase reporter gene assays were used to examine viral attachment and entry in the presence and absence of silver nanoparticles. To explore the mechanism of inhibition, viral entry experiments were conducted with silver nanoparticles and small interfering RNAs designed to silence the gene coding for p21-activated kinase 1, a key mediator of macropinocytosis. The silver nanoparticles caused a 4- to 5-log reduction in viral titer at concentrations that were not toxic to cells. Virus was capable of adsorbing to cells but could not enter cells in the presence of silver nanoparticles. Virus particles that had adsorbed to cells in the presence of silver nanoparticles were found to be infectious upon removal from the cells, indicating lack of direct virucidal effect. The half maximal inhibitory concentration for viral entry in the presence of silver nanoparticles was 27.4+/-3.3 microg/ml. When macropinocytosis was blocked, this inhibition was significantly reduced. Thus, macropinocytosis was required for the full antiviral effect. For the first time, this study points to the novel result that a cellular process involved in viral entry is responsible for the antiviral effects of silver nanoparticles.

Tangential flow ultrafiltration: a "green" method for the size selection and concentration of colloidal silver nanoparticles.

Nowadays, AgNPs are extensively used in the manufacture of consumer products,(1) water disinfectants,(2) therapeutics,(1, 3) and biomedical devices(4) due to their powerful antimicrobial properties.(3-6) These nanoparticle applications are strongly influenced by the AgNP size and aggregation state. Many challenges exist in the controlled fabrication(7) and size-based isolation(4,8) of unfunctionalized, homogenous AgNPs that are free from chemically aggressive capping/stabilizing agents or organic solvents.(7-13) Limitations emerge from the toxicity of reagents, high costs or reduced efficiency of the AgNP synthesis or isolation methods (e.g., centrifugation, size-dependent solubility, size-exclusion chromatography, etc.).(10,14-18) To overcome this, we recently showed that TFU permits greater control over the size, concentration and aggregation state of Creighton AgNPs (300 ml of 15.3 µg ml(-1) down to 10 ml of 198.7 µg ml(-1)) than conventional methods of isolation such as ultracentrifugation.(19) TFU is a recirculation method commonly used for the weight-based isolation of proteins, viruses and cells.(20,21) Briefly, the liquid sample is passed through a series of hollow fiber membranes with pore size ranging from 1,000 kD to 10 kD. Smaller suspended or dissolved constituents in the sample will pass through the porous barrier together with the solvent (filtrate), while the larger constituents are retained (retentate). TFU may be considered a "green" method as it neither damages the sample nor requires additional solvent to eliminate toxic excess reagents and byproducts. Furthermore, TFU may be applied to a large variety of nanoparticles as both hydrophobic and hydrophilic filters are available. The two main objectives of this study were: 1) to illustrate the experimental aspects of the TFU approach through an invited video experience and 2) to demonstrate the feasibility of the TFU method for larger volumes of colloidal nanoparticles and smaller volumes of retentate. First, unfuctionalized AgNPs (4 L, 15.2 µg ml(-1)) were synthesized using the well-established Creighton method(22,23) by the reduction of AgNO3 with NaBH4. AgNP polydispersity was then minimized via a 3-step TFU using a 50-nm filter (460 cm(2)) to remove AgNPs and AgNP-aggregates larger than 50 nm, followed by two 100-kD (200 cm(2) and 20 cm(2)) filters to concentrate the AgNPs. Representative samples were characterized using transmission electron microscopy, UV-Vis absorption spectrophotometry, Raman spectroscopy, and inductively coupled plasma optical emission spectroscopy. The final retentate consisted of highly concentrated (4 ml, 8,539.9 µg ml(-1)) yet lowly aggregated and homogeneous AgNPs of 1-20 nm in diameter. This corresponds to a silver concentration yield of about 62%.

Rapid assessment of antiviral activity and cytotoxicity of silver nanoparticles using a novel application of the tetrazolium-based colorimetric assay.

This study centers on the development of a new screening tool for simultaneously evaluating the antiviral and cytotoxic properties of antiviral agents against an HIV-1-based, pseudotyped virus particle engineered to encode antibiotic resistance. The traditional colony-forming-unit assay for quantifying this type of virus was impractical as a screening tool due to the cumbersome nature of the setup and high costs in labor and supplies. Therefore, a smaller-scale and higher-throughput means of scoring antiviral activity was successfully developed and used to evaluate a specific batch of 25-nm silver nanoparticles (AgNPs). The new assay employed a unique application of the traditional cell proliferation/cytotoxicity test that is based on the chemical 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, which produces a colorimetric readout. The AgNPs showed a half maximal inhibitory concentration against the virus of 11.2±0.6 µg/ml (p<0.0001) with no significant toxicity against the cells. Because the virus was engineered to undergo only the first half of its replication cycle, the observed AgNP inhibition must have occurred at one of the early stages of infection. Overall, the new assay was very efficient and will be useful for testing different viral pseudotypes, screening different types of nanomaterials, and investigating other antiviral agents.

Size selection and concentration of silver nanoparticles by tangential flow ultrafiltration for SERS-based biosensors.

A proposed tangential flow ultrafiltration method was compared to the widely used ultracentrifugation method for efficiency and efficacy in concentrating, size selecting, and minimizing the aggregation state of a silver nanoparticle (AgNP) colloid while probing the AgNPs' SERS-based sensing capabilities. The ultrafiltration method proved to be more efficient and more effective and was found to tremendously boost the SERS-based sensing capabilities of these AgNPs through the increased number of homogeneous SERS hot spots available for a biotarget molecule within a minimal focal volume. Future research studies and applications addressing the physiochemical properties or biological impact of AgNPs would greatly benefit from ultrafiltration for its ability to generate monodisperse colloidal nanoparticles, to eliminate excess toxic chemicals from nanoparticle synthesis, and to obtain minimum levels of aggregation during nanoparticle concentration.

Is gene therapy a good therapeutic approach for HIV-positive patients?

Despite advances and options available in gene therapy for HIV-1 infection, its application in the clinical setting has been challenging. Although published data from HIV-1 clinical trials show safety and proof of principle for gene therapy, positive clinical outcomes for infected patients have yet to be demonstrated. The cause for this slow progress may arise from the fact that HIV is a complex multi-organ system infection. There is uncertainty regarding the types of cells to target by gene therapy and there are issues regarding insufficient transduction of cells and long-term expression. This paper discusses state-of-the-art molecular approaches against HIV-1 and the application of these treatments in current and ongoing clinical trials.

The reverse transcriptase sequence of human immunodeficiency virus type 1 is under positive evolutionary selection within the central nervous system.

The human immunodeficiency virus type 1 (HIV-1) enters the central nervous system (CNS) during the acute phase of infection and causes AIDS-related encephalitis and dementia in 30% of individuals. Previous studies show that HIV-1 sequences derived from the CNS of infected patients, including the sequence encoding reverse transcriptase (RT), are genetically distinct from sequences in other tissues. The hypothesis of the current study is that the RT sequence of HIV-1 is under positive selection within the CNS. Multiple alignments of non-CNS-derived and CNS-derived HIV-1 RT sequences were constructed using the ClustalW 1.8 program. The multiple alignments were analyzed with the Synonymous/Nonsynonymous Analysis Program. Codon positions 122-125, 135-149, and 166-212 of the CNS-derived RT sequences underwent a greater accumulation of nonsynonymous than synonymous substitutions, which was markedly different from the analysis results of the non-CNS-derived RT sequences. These residues are located in the finger and palm subdomains of the RT protein structure, which encodes the polymerase active site. The analysis of CNS-derived partial-length RT sequences that encompass these regions yielded similar results. A comparison of CNS-derived RT sequences to a non-CNS-derived RT consensus sequence revealed that a majority of the nonsynonymous substitutions resulted in a specific amino acid replacement. These results indicate that reverse transcriptase is under positive selection within the CNS. The amino acid replacements were visualized on a three-dimensional structure of HIV-1 RT using the Sybyl software suite. The protein structure analysis revealed that the amino acid replacements observed among the CNS-derived sequences occurred in areas of known structural and functional significance.