Susan Carpenter

PHOTOSENSITIZATION IS REQUIRED FOR INACTIVATION OF EQUINE INFECTIOUS ANEMIA VIRUS BY HYPERICIN

Abstract— Hypericin, a photoreactive polycyclic quinone, was found to dramatically reduce infectivity of cell‐free stocks of equine infectious anemia virus. However, the antiviral activity of hypericin was completely dependent on the presence of light. Short periods of photosensitization resulted in a partial loss of reverse transcriptase activity and complete inhibition of viral infectivity. These results suggest that the photodynamic effect of hypericin interferes with more than one stage in the virus replication cycle.

Effect of bovine immunodeficiency-like virus infection on immune function in experimentally infected cattle

Bovine immunodeficiency-like virus (BIV) is a bovine lentivirus that has antigenic and genetic homology with the human immunodeficiency virus. Little work has been reported on the effect of BIV infection on bovine immune function. This study was designed to evaluate lymphocyte blastogenesis, mononuclear cell subset numbers, neutrophil function, hematology, and clinical signs in three groups of cattle. These groups were evaluated at 0-2 months post inoculation (PI, Group 1), 4-5 months PI (Group 2), or 19-27 months PI (Group 3). BIV infected animals were inoculated with the R-29 isolate of BIV in tissue culture cells, peripheral blood mononuclear cells from a R-29 infected calf, or a molecular clone of the R-29 isolate. Most inoculated animals seroconverted to BIV by Western immunoblot. BIV was reisolated from most of the animals inoculated. BIV infection was associated with an increase in the lymphocyte blastogenic response to the mitogen phytohemagglutinin in Groups 2 and 3. Neutrophil antibody dependent cell mediated cytotoxicity and neutrophil iodination were decreased (P < 0.05) in BIV infected cattle (Groups 2 and 3 and Group 3, respectively). All animals were clinically normal during the evaluation periods. Notable differences were not observed in the other assessments performed. Work with additional BIV isolates and over longer time frames is warranted.

Antiretroviral activity of synthetic hypericin and related analogs

Hypericin and pseudohypericin are naturally occurring polycyclic quinones which have recently been shown to inhibit the infectivity of several retroviruses, including human immunodeficiency virus. To better understand the antiviral mechanisms of these compounds, hypericin and a series of analogous quinones were synthesized and tested for anti-retroviral activity against equine infectious anemia virus (EIAV). Treatment of EIAV-infected cells with hypericin reduced the production of infectious virus by 99.99%. None of the analogs were found to inhibit virus replication. These results suggest that the complete ring structure of hypericin is required, but not sufficient, for antiviral activity.

The Role of Oxygen in the Antiviral Activity of Hypericin and Hypocrellin

The light‐induced antiviral activity of hypericin and hypocrellin in the presence and absence of oxygen was examined under experimental conditions where the effect of oxygen depletion could be quantified. There was a significant reduction of light‐induced antiviral activity of hypericin and hypocrellin under hypoxic conditions. Interestingly, antiviral activity of hypocrellin was not observed at low oxygen levels at which hypericin retained measurable virucidal activity. This suggests that additional pathways, such as the generation of protons from excited states of hypericin, may enhance the biological activity of activated oxygen species.

Equine Infectious Anemia Virus Resists the Antiretroviral Activity of Equine APOBEC3 Proteins through a Packaging-Independent Mechanism

ABSTRACT Equine infectious anemia virus (EIAV), uniquely among lentiviruses, does not encode a vif gene product. Other lentiviruses, including human immunodeficiency virus type 1 (HIV-1), use Vif to neutralize members of the APOBEC3 (A3) family of intrinsic immunity factors that would otherwise inhibit viral infectivity. This suggests either that equine cells infected by EIAV in vivo do not express active A3 proteins or that EIAV has developed a novel mechanism to avoid inhibition by equine A3 (eA3). Here, we demonstrate that horses encode six distinct A3 proteins, four of which contain a single copy of the cytidine deaminase (CDA) consensus active site and two of which contain two CDA motifs. This represents a level of complexity previously seen only in primates. Phylogenetic analysis of equine single-CDA A3 proteins revealed two proteins related to human A3A (hA3A), one related to hA3C, and one related to hA3H. Both equine double-CDA proteins are similar to hA3F and were named eA3F1 and eA3F2. Analysis of eA3F1 and eA3F2 expression in vivo shows that the mRNAs encoding these proteins are widely expressed, including in cells that are natural EIAV targets. Both eA3F1 and eA3F2 inhibit retrotransposon mobility, while eA3F1 is a potent inhibitor of a Vif-deficient HIV-1 mutant and induces extensive editing of HIV-1 reverse transcripts. However, both eA3F1 and eA3F2 are weak inhibitors of EIAV. Surprisingly, eA3F1 and eA3F2 were packaged into EIAV and HIV-1 virions as effectively as hA3G, although only the latter inhibited EIAV infectivity. Moreover, all three proteins bound both the HIV-1 and EIAV nucleocapsid protein specifically in vitro. It therefore appears that EIAV has evolved a novel mechanism to specifically neutralize the biological activities of the cognate eA3F1 and eA3F2 proteins at a step subsequent to virion incorporation.

Binding of Equine Infectious Anemia Virus Rev to an Exon Splicing Enhancer Mediates Alternative Splicing and Nuclear Export of Viral mRNAs

ABSTRACT In addition to facilitating the nuclear export of incompletely spliced viral mRNAs, equine infectious anemia virus (EIAV) Rev regulates alternative splicing of the third exon of thetat/rev mRNA. In the presence of Rev, this exon of the bicistronic RNA is skipped in a fraction of the spliced mRNAs. In this report, the cis-acting requirements for exon 3 usage were correlated with sequences necessary for Rev binding and transport of incompletely spliced RNA. The presence of a purine-rich exon splicing enhancer (ESE) was required for exon 3 recognition, and the addition of Rev inhibited exon 3 splicing. Glutathione-S-transferase (GST)-Rev bound to probes containing the ESE, and mutation of GAA repeats to GCA within the ESE inhibited both exon 3 recognition in RNA splicing experiments and GST-Rev binding in vitro. These results suggest that Rev regulates alternative splicing by binding at or near the ESE to block SR protein-ESE interactions. A 57-nucleotide sequence containing the ESE was sufficient to mediate Rev-dependent nuclear export of incompletely spliced RNAs. Rev export activity was significantly inhibited by mutation of the ESE or bytrans-complementation with SF2/ASF. These results indicate that the ESE functions as a Rev-responsive element and demonstrate that EIAV Rev mediates exon 3 exclusion through protein-RNA interactions required for efficient export of incompletely spliced viral RNAs.

Longitudinal studies of immune function in cattle experimentally infected with bovine immunodeficiency-like virus and/or bovine leukemia virus

The effects of single or dual infection with bovine immunodeficiency-like virus (BIV) and/or, bovine leukemia virus (BLV) on bovine immune function were examined over a 4 year period. Holstein calves were infected with BIV (four calves), BLV (five calves), BIV and BLV (five calves), or sham inoculated (three calves). Lymphocyte blastogenesis to mitogens, seven tests of neutrophil function, and mononuclear cell subset analysis by flow cytometry (BoCD4, BoCD8, BoCD2, BoWC1, sIgM+, and monocytes) were performed at regular intervals to 49 months post-infection. These data were analyzed for main effects of each virus and interaction as a 2 x 2 factorial. BIV infected cattle had lower neutrophil antibody-dependent cell-mediated cytotoxicity and iodination responses during 2 of the 4 years post-infection (P < 0.05). BIV infection was not associated with any long-term significant changes in lymphocyte blastogenesis to mitogens or changes in mononuclear cell subset numbers in blood. There was a tendency for animals infected with BIV alone to have decreased lymphocyte blastogenic responses to mitogens, but this was not statistically significant. BLV infection caused an increase in total mononuclear cells with no dramatic shift in the relative proportions of the various subsets. Co-infection with BIV and BLV did not consistently cause a different response than either virus did individually. One BIV infected animal died of non-BLV lymphosarcoma 7 months after infection. All other animals had no unusual clinical signs. In summary, infection with BIV caused a significant, temporary decrease in neutrophil function with no consistent statistically significant alteration in lymphocyte blastogenesis or mononuclear cell numbers during the first 4 years after infection. BLV infection caused an increase in lymphocyte numbers, and there appeared to be no synergism between the viruses.

Subpopulations of Equine Infectious Anemia Virus Rev Coexist In Vivo and Differ in Phenotype

ABSTRACT Lentiviruses exist in vivo as a population of related, nonidentical genotypes, commonly referred to as quasispecies. The quasispecies structure is characteristic of complex adaptive systems and contributes to the high rate of evolution in lentiviruses that confounds efforts to develop effective vaccines and antiviral therapies. Here, we describe analyses of genetic data from longitudinal studies of genetic variation in a lentivirus regulatory protein, Rev, over the course of disease in ponies experimentally infected with equine infectious anemia virus. As observed with other lentivirus data, the Rev variants exhibited a quasispecies character. Phylogenetic and partition analyses suggested that the Rev quasispecies comprised two distinct subpopulations that coexisted during infection. One subpopulation appeared to accumulate changes in a linear, time-dependent manner, while the other evolved radially from a common variant. Over time, the two subpopulations cycled in predominance coincident with changes in the disease state, suggesting that the two groups differed in selective advantage. Transient expression assays indicated the two populations differed significantly in Rev nuclear export activity. Chimeric proviral clones containing Rev genotypes representative of each population differed in rate and overall level of virus replication in vitro. The coexistence of genetically distinct viral subpopulations that differ in phenotype provides great adaptability to environmental changes within the infected host. A quasispecies model with multiple subpopulations may provide additional insight into the nature of lentivirus reservoirs and the evolution of antigenic and drug-resistant variants.

PAQ: Partition Analysis of Quasispecies

MOTIVATION The complexities of genetic data may not be accurately described by any single analytical tool. Phylogenetic analysis is often used to study the genetic relationship among different sequences. Evolutionary models and assumptions are invoked to reconstruct trees that describe the phylogenetic relationship among sequences. Genetic databases are rapidly accumulating large amounts of sequences. Newly acquired sequences, which have not yet been characterized, may require preliminary genetic exploration in order to build models describing the evolutionary relationship among sequences. There are clustering techniques that rely less on models of evolution, and thus may provide nice exploratory tools for identifying genetic similarities. Some of the more commonly used clustering methods perform better when data can be grouped into mutually exclusive groups. Genetic data from viral quasispecies, which consist of closely related variants that differ by small changes, however, may best be partitioned by overlapping groups. RESULTS We have developed an intuitive exploratory program, Partition Analysis of Quasispecies (PAQ), which utilizes a non-hierarchical technique to partition sequences that are genetically similar. PAQ was used to analyze a data set of human immunodeficiency virus type 1 (HIV-1) envelope sequences isolated from different regions of the brain and another data set consisting of the equine infectious anemia virus (EIAV) regulatory gene rev. Analysis of the HIV-1 data set by PAQ was consistent with phylogenetic analysis of the same data, and the EIAV rev variants were partitioned into two overlapping groups. PAQ provides an additional tool which can be used to glean information from genetic data and can be used in conjunction with other tools to study genetic similarities and genetic evolution of viral quasispecies.

The role of oxygen in the photoinduced antiviral activity of hypericin

Abstract Hypericin displays photoinduced antiviral activity. We examine the photoinduced antiviral activity of hypericin under both oxygenated and hypoxic conditions and observe that hypericin is equally toxic under both conditions. These results indicate that while singlet oxygen may play a role in the antiviral activity of hypericin, it does not play a major role.