The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) occlusion-derived virus (ODV) envelope protein ODV-E56 is essential for oral infection of larvae of Heliothis virescens. Bioassays with recombinant clones of AcMNPV lacking a functional odv-e56 gene showed that ODV-E56 was required for infectivity of both polyhedra and to a lesser extent, purified ODV. However, binding and fusion assays showed that ODV lacking ODV-E56 bound and fused to midgut cells at levels similar to ODV of wild-type virus. Fluorescence microscopy of midguts from larvae inoculated with ODV-E56-positive and -negative viruses that express GFP indicated that ODV-E56 was required for infection of the midgut epithelium. Purified ODV-E56 bound to several proteins in midgut-derived brush border membrane vesicles, but failed to rescue infectivity of ODV-E56-negative viruses in trans. These results indicate that ODV-E56 is a per os infectivity factor (pif-5) required for primary midgut infection at a point before or after virion binding and fusion.

Equine infectious anemia virus (EIAV)Rev is an essential regulatory protein that facilitates expression of viral mRNAs encoding structural proteins and genomic RNA and regulates alternative splicing of the bicistronic tat/rev mRNA. EIAV Rev is characterized by a high rate of genetic variation in vivo, and changes in Rev genotype and phenotype have been shown to coincide with changes in clinical disease. To better understand how genetic variation alters Rev phenotype, we undertook deletion and mutational analyses to map functional domains and to identify specific motifs that are essential for EIAV Rev activity. All functional domains are contained within the second exon of EIAV Rev. The overall organization of domains within Rev exon 2 includes a nuclear export signal, a large central region required for RNA binding, a nonessential region, and a C-terminal region required for both nuclear localization and RNA binding. Subcellular localization of green fluorescent protein-Rev mutants indicated that basic residues within the KRRRK motif in the C-terminal region of Rev are necessary for targeting of Rev to the nucleus. Two separate regions of Rev were necessary for RNA binding: a central region encompassing residues 57 to 130 and a C-terminal region spanning residues 144 to 165. Within these regions were two distinct, short arginine-rich motifs essential for RNA binding, including an RRDRW motif in the central region and the KRRRK motif near the C terminus. These findings suggest that EIAV Rev utilizes a bipartite RNA-binding domain. Equine infectious anemia virus (EIAV) infection of horsescan result in a rapid, variable, and dynamic disease course. Moreover, horses that survive the early clinical episodes of disease are generally able to control virus replication and remain clinically normal, inapparent carriers of EIAV. The unique features of clinical disease, and the ability of some infected horses to eventually control virus replication, provide an excellent system for longitudinal analyses of virus and host factors important in lentivirus persistence and pathogenesis. Genetic diversity is a hallmark of lentiviruses and is considered an important mechanism of virus persistence and pathogenesis. Previous studies have identified a high rate of genetic variation in EIAV in the region overlapped by the transmembrane protein gp45 (TM) and the major exon of Rev (2, 30). Genetic variation in rev/tm can significantly alter Rev activity (7), and in vivo studies suggest that changes in Rev phenotype correlate with changes in the clinical stage of disease (4, 6). In particular, Rev is significantly less active during the inapparent compared to the chronic stage of disease, suggesting that the Rev phenotype contributes to selection of virus variants in vivo. Insight into the genetic changes and factors that contribute to Rev selection in vivo requires identification of the functional domains and motifs that mediate EIAV Rev activity.The Rev/Rex proteins of complex retroviruses differentially regulate expression of incomplet...

Development of ways to block virus transmission by aphids could lead to novel and broad-spectrum means of controlling plant viruses. Viruses in the Luteoviridae enhanced are obligately transmitted by aphids in a persistent manner that requires virion accumulation in the aphid hemocoel. To enter the hemocoel, the virion must bind and traverse the aphid gut epithelium. By screening a phage display library, we identified a 12-residue gut binding peptide (GBP3.1) that binds to the midgut and hindgut of the pea aphid Acyrthosiphon pisum. Binding was confirmed by labeling the aphid gut with a GBP3.1-green fluorescent protein fusion. GBP3.1 reduced uptake of Pea enation mosaic virus (Luteoviridae) from the pea aphid gut into the hemocoel. GBP3.1 also bound to the gut epithelia of the green peach aphid and the soybean aphid. These results suggest a novel strategy for inhibiting plant virus transmission by at least three major aphid pest species.

The principal neutralizing domain (PND) of equine infectious anemia virus (EIAV) is located in the V3 region of SU. Genetic variation in the PND is considered to play an important role in immune escape and EIAV persistence; however, few studies have characterized genetic variation in SU during the inapparent stage of disease. To better understand the mechanisms of virus persistence, we undertook a longitudinal study of SU variation in a pony experimentally inoculated with the virulent EIAV(Wyo). Viral RNA isolated from the inoculum and from sequential sera samples was amplified by RT-PCR, cloned, and individual clones were sequenced. Of the 147 SU clones obtained, we identified 71 distinct V3 variants that partitioned into five major non-overlapping groups, designated PND-1 to PND-5, which segregated with specific stages of clinical disease. Genotypes representative of each group were inserted into an infectious molecular clone, and chimeric viruses were tested for susceptibility to neutralization by autologous sera from successive times post-infection. Overall, there was a trend for increasing resistance to neutralizing antibody during disease progression. The PND genotype associated with recrudescence late in infection was resistant to both type-specific and broadly neutralizing antibody, and displayed a reduced replication phenotype in vitro. These findings indicate that neutralizing antibody exerts selective pressure throughout infection and suggest that viral strategies of immune evasion and persistence change in the face of an evolving and maturing host immune response.

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.Lentiviruses exhibit high mutation rates and exist in vivo as a population of closely related viral genotypes that are commonly referred to as a quasispecies (11,13,19). The population structure of a quasispecies consists of a master sequence, which is the dominant genotype, and the mutant spectrum, which includes reservoirs of genotypic and phenotypic variants with the potential to become dominant in the face of environmental change. Studies have demonstrated that the evolution of viral quasispecies adheres to basic principles of population genetics; however, quasispecies theory diverges from population genetics in that the entire deme of related sequences, rather than the individual replicon, is the main target of selection (14). The quasispecies occupies a region on a fitness landscape where adaptive mutations move the quasispecies toward a fitness peak, thereby increasing the mean fitness of the quasispecies. Understanding the principles that shape the evolution of viral quasispecies is becoming increasingly important as molecular information is used to model disease progression and to predict the emergence of antigenic and drug-resistant variants.Experimental infection of ponies with equine infectious anemia virus (EIAV) provides an excellent system for longitudinal studies of lentivirus evolution during disease progression.