Dawn P. Wooley

The Envelope Glycoprotein of Human Endogenous Retrovirus HERV-W Induces Cellular Resistance to Spleen Necrosis Virus

Summary. Human endogenous retrovirus type W (HERV-W) envelope glycoprotein (Env) has recently been reported to induce fusion in cells expressing the RD-114 and type D retrovirus receptor (RDR) and to serve as a functional retroviral envelope protein. In this report, another biological function for HERV-W was demonstrated by testing its ability to protect cells against retroviral infection. Spleen necrosis virus (SNV), a gammaretrovirus was chosen for testing resistance because it uses RDR to enter cells. An HERV-W Env expression plasmid was transfected into canine osteosarcoma cells (D-17), which are permissive for SNV infection. Cell fusion assays were performed to demonstrate biological function of HERV-W Env in D-17 cells. The presence of HERV-W env sequences was confirmed in stably transfected cell clones by using polymerase chain reaction. Viral infectivity assays were performed with SNV and amphotropic Murine leukemia virus (MLV-A) pseudotyped vector viruses to measure titers in D-17 cells expressing HERV-W Env and in negative control cells. The HERV-W Env caused fusion of D-17 cells in culture and greatly reduced infection by SNV vector virus. A 1000- to 10,000-fold decrease in SNV infectivity was observed for D-17 cells expressing HERV-W Env as compared to D-17 cells that were not expressing HERV-W Env. In contrast, infection by MLV-A pseudotyped vector virus was not significantly reduced. Thus, HERV-W Env confers host cell resistance to infection by SNV. This is the first report of a human endogenous retrovirus gene product blocking infection by any exogenous retrovirus.

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.

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.

High fidelity of homologous retroviral recombination in cell culture.

Summary. Genetic variation continues to be a major obstacle in the development of therapies and vaccines against retroviral infections and contributes extensively to viral pathogenesis and persistence. Recombination is one mechanism that increases retroviral variation by shuffling mutations from different genomes. Recent studies suggest that recombination not only shuffles the mutations but also generates them at high rates during reverse transcription. In contrast to these recent studies, this investigation shows that recombination does not generate mutations during recombination. A spleen necrosis virus (SNV)-based homologous recombination system was used to test the hypothesis that retroviral recombination is a high-fidelity process during replication of the virus in cell culture. The system consisted of a pair of SNV vectors expressing two drug resistance genes. The vectors were constructed so that cells containing recombinant proviruses could be selected by a double drug-resistant phenotype. Restriction enzyme digestion and agarose gel electrophoresis were used to map the location of recombination within 182 proviruses. Sequencing and single-strand conformation polymorphism techniques were then used to check for mutations within the recombinant proviruses. Since no mutations were detected among the 182 recombinants that were analyzed, homologous recombination is a high-fidelity process for retroviruses in cell culture.

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.

Duplex Quantitative Polymerase Chain Reaction Assay for Detection of Adenoviral and Lentiviral Vectors

The ability to detect and quantify viral vector sequences from a variety of clinical sample types is crucial to performing biosafety risk assessments. Viral vector-mediated gene transfer studies are often performed in animals, and these animals must be placed under appropriate biocontainment conditions to protect the workers and environment. Data on the shedding of viral vectors from animals are limited, and the sample types are challenging for polymerase chain reaction (PCR). For this reason, we developed a quantitative PCR assay that could be used for such purpose. We designed a sequence-specific, probe-binding method for detecting adenoviral and lentiviral vector sequences. A duplex strategy was used that included a quality control sequence to be amplified in the same reaction as the viral vector. This sequence provided an internal control for normalization of noncellular sample types, such as animal excretions that inherently lack a natural control sequence. The new assay was used to establish the efficiency of reverse transcription and to detect viral genomes in stocks of whole virus particles. We identified sets of primers and probes for both adenoviral and lentiviral sequences that work well together with no interference. The average conversion rate of RNA into complementary DNA was 18.5%. The new quantitative PCR assay was efficient and specific, and it measured successfully the number of viral genomes in stocks of whole virus particles. This assay could be used to detect adenoviral and lentiviral vector sequences for biosafety and other research purposes.