Late domain-dependent inhibition of equine infectious anemia virus budding

The Gag proteins of a number of different retroviruses contain late or L domains that promote the release of virions from the plasma membrane. Three types of L domains have been identified to date: Pro-Thr-Ala-Pro (PTAP), Pro-Pro-X-Tyr, and Tyr-Pro-Asp-Leu. It has previously been demonstrated that overexpression of the N-terminal, E2-like domain of the endosomal sorting factor TSG101 (TSG-5') inhibits human immunodeficiency virus type 1 (HIV-1) release but does not affect the release of the PPPY-containing retrovirus murine leukemia virus (MLV), whereas overexpression of the C-terminal portion of TSG101 (TSG-3') potently disrupts both HIV-1 and MLV budding. In addition, it has been reported that, while the release of a number of retroviruses is disrupted by proteasome inhibitors, equine infectious anemia virus (EIAV) budding is not affected by these agents. In this study, we tested the ability of TSG-5', TSG-3', and full-length TSG101 (TSG-F) overexpression, a dominant negative form of the AAA ATPase Vps4, and proteasome inhibitors to disrupt the budding of EIAV particles bearing each of the three types of L domain. The results indicate that (i) inhibition by TSG-5' correlates with dependence on PTAP; (ii) the release of wild-type EIAV (EIAV/WT) is insensitive to TSG-3', whereas this C-terminal TSG101 fragment potently impairs the budding of EIAV when it is rendered PTAP or PPPY dependent; (iii) budding of all EIAV clones is blocked by dominant negative Vps4; and (iv) EIAV/WT release is not impaired by proteasome inhibitors, while EIAV/PTAP and EIAV/PPPY release is strongly disrupted by these compounds. These findings highlight intriguing similarities and differences in host factor utilization by retroviral L domains and suggest that the insensitivity of EIAV to proteasome inhibitors is conferred by the L domain itself and not by determinants in Gag outside the L domain.     

The S2 gene of equine infectious anemia virus is a highly conserved determinant of viral replication and virulence properties in experimentally infected ponies

Equine infectious anemia virus (EIAV) is genetically one of the simplest lentiviruses in that the viral genome encodes only three accessory genes, tat, rev, and S2. Although serological analyses demonstrate the expression of the S2 protein in persistently infected horses, the role of this viral gene remains undefined. We recently reported that the S2 gene is not essential for EIAV replication in primary equine macrophages, as EIAV mutants lacking the S2 gene replicate to levels similar to those of the parental virus (F. Li, B. A. Puffer, and R. C. Montelaro, J. Virol. 72:8344-8348, 1998). We now describe in vivo studies that examine the evolution and role of the S2 gene in ponies experimentally infected with EIAV. The results of these studies reveal for the first time that the S2 gene is highly conserved during persistent infection and that deletion of the S2 gene reduces viral virulence and virus replication levels compared to those of the parental virus containing a functional S2 gene. These data indicate that the EIAV S2 gene is in fact an important determinant of viral replication and pathogenic properties in vivo, despite the evident lack of S2 influence on viral replication levels in vitro. Thus, these observations suggest in vivo functions of EIAV S2 that are not adequately reflected in simple infections of cultured cells, including natural target macrophages.     

Inferring the number of evolutionary events from DNA coding sequence differences

The estimation of the amount of evolutionary divergence that has taken place between two DNA coding sequences depends strongly on the degree of constraint on amino acid replacements. If amino acid replacements are relatively unconstrained, the individual nucleotide is the appropriate unit of analysis and the method of Tajima and Nei can be used. If amino acid replacements are constrained, however, this method is shown to be inapplicable. For sequences with strong amino acid constraints, a method is outlined analogous to the Tajima and Nei method using codons as the unit of analysis. Only synonymous substitutions are used. Codon usage data can be employed to estimate the necessary parameters of the calculation, or a priori models of substitution may be employed. Sequences with significant but intermediate constraints on amino acid replacements are, in principle, unanalyzable.      

A procedure for the preferential radioiodination of small amounts of protein in the presence of excess lipids.

The chloramine-T, iodine monochloride, and lactoperoxidase radioiodination procedures were evaluated for their ability to label proteins in the presence of large amounts of reactive lipid. Mouse serum very low density lipoproteins (VLDL) (10% protein, 90% lipid) and detergent-disrupted Friend murine leukemia virus (FLV) (60% protein, 33% lipid) were used as model systems. Based on the distribution of label between protein and lipid, as well as the total amount of label incorporated by each procedure, only a modification of the iodine monochloride procedure preferentially labeled proteins to a high specific activity in both VLDL and FLV. The technique, which is described in detail, is technically simple, rapid, and causes no detectable degradation of proteins.     

Radiolabeling of proteins and viruses in vitro by acetylation with radioactive acetic anhydride.

We describe a convenient, rapid, and reproducible method for labeling proteins in vitro by acetylation with [3H] or [14-C]acetic anhydride dissolved in small amounts of anhydrous dioxane. The reaction is carried out at neutral pH and does not require the use of detergents, water-immiscible organic solvents, oxidizing, or reducing agents. Thus undesirable solvent-induced alterations in protein structure and biological activity are minimized. A method for calculating the specific activity of the protein and the efficiency of acetylation at known concentrations of protein and acetic anhydride is presented. Radioacetylated proteins were shown to be suitable for use as molecular weight calibration standards and as protein markers in polyacrylamide gel electrophoresis, gel filtration, and enzyme studies. Acetic anhydride was used to label intact oncornaviruses, which consist of a complex ribonucleo-protein core within a lipid envelope. Some of the viral lipid and all of the viral proteins, including the internal ones, were labeled without detectable alterations in viral morphology or buoyant density. This result suggests that acetic anhydride, evidently by virtue of its small size and neutral charge, penetrates freely throughout the viral membrane and core structures. The reactivity of RNA with acetic anhydride was less than 1% that of protein under similar reaction conditions.     

Isolation and comparative biochemical properties of the major internal polypeptides of equine infectious anemia virus.

We describe procedures for the large-scale production of equine infectious anemia virus (EIAV) and for the isolation of the four major non-glycosylated virion proteins, designated p26, p15, p11, and p9. Comparisons of the purified proteins by peptide mapping procedures and by enzyme-linked immunosorbent assays demonstrated the unrelatedness of the four proteins. The characteristic properties of each purified protein were examined by determining isoelectric points and amino acid compositions. We found that EIAV p26 and p9 focus at pI values of 6.2 and 5.0, respectively, and that these proteins contain no unusual amino acids. In contrast, EIAV p15 reproducibly displayed a heterogeneous isoelectric focusing pattern, with major pI values ranging from 5.7 to 8.3. This charge variation evidently correlated with different levels of phosphorylated serine or threonine or both, which could be detected by an amino acid analysis of purified p15. EIAV p11 apparently focused at a pI of greater than 10, reflecting its high content of basic amino acids. Moreover, localization experiments indicated that all four nonglycosylated proteins constitute the internal components of the virus, with all of the virion p11 closely associated with the viral RNA genome. Thus, our results demonstrated that EIAV, a lentivirus, contains structural polypeptides which are analogous to the structural polypeptides described previously in prototype C oncoviruses.     

Effects of common radioiodination procedures on the binding of glycoproteins to immobilized lectins

Representative glycoproteins including fetuin, protein A, ovalbumin, α₁ acid glycoprotein, and the major glycoprotein of equine infectious anemia virus were labelled with ¹²⁵I by the chloramine-T or Bolton-Hunter procedure and their binding to immobilized Con A or lentil lectin compared to untreated samples of each glycoprotein. Glycoprotein modification was no greater than one substituted residue per protein molecule. Yet the radioiodinated glycoproteins typically displayed only 0–50% of the lectin binding observed with untreated samples. These results indicate that lectin glycoprotein binding can be markedly altered by minor modifications in protein structure.     

Evolution of envelope-specific antibody responses in monkeys experimentally infected or immunized with simian immunodeficiency virus and its association with the development of protective immunity.

Previous studies of attenuated simian immunodeficiency virus (SIV) vaccines in rhesus macaques have demonstrated the development of broad protection against experimental challenge, indicating the potential for the production of highly effective immune responses to SIV antigens. However, the development of this protective immune status was found to be critically dependent on the length of time postvaccination with the attenuated virus strain, suggesting a necessary maturation of immune responses. In this study, the evolution of SIV envelope-specific antibodies in monkeys experimentally infected with various attenuated strains of SIV was characterized by using a comprehensive panel of serological assays to assess the progression of antibodies in longitudinal serum samples that indicate the development of protective immunity. In parallel studies, we also used the same panel of antibody assays to characterize the properties of SIV envelope-specific antibodies elicited by inactivated whole-virus and envelope subunit vaccines previously reported to be ineffective in producing protective immunity. The results of these studies demonstrate that the evolution of protective immunity in monkeys inoculated with attenuated strains of SIV is associated with a complex and lengthy maturation of antibody responses over the first 6 to 8 months postinoculation, as reflected in progressive changes in antibody conformational dependence and avidity properties. The establishment of long-term protective immunity at this time in general parallels the absence of further detectable changes in antibody responses and a maintenance of relatively constant antibody titer, avidity, conformational dependence, and the presence of neutralizing antibody for at least 2 years postinoculation. In contrast to the mature antibody responses elicited by the attenuated SIV vaccines, the whole-virus and envelope subunit vaccines in general elicited only immature antibody responses characterized by poor reactivity with native envelope proteins, low avidity, low conformational dependence, and the absence of neutralization activity against the challenge strain. Thus, these studies establish for the first time an association between the effectiveness of experimental vaccines and the capacity of the vaccine to produce a mature antibody response to SIV envelope proteins and further indicate that a combination of several antibody parameters (including titer, avidity, conformational dependence, and virus neutralization) are superior to any single antibody parameter as prognostic indicators to evaluate candidate AIDS vaccines.     

Positionally independent and exchangeable late budding functions of the Rous sarcoma virus and human immunodeficiency virus Gag proteins.

The Gag proteins of Rous sarcoma virus and human immunodeficiency virus (HIV) each contain a function involved in a late step in budding, defects in which result in the accumulation of these molecules at the plasma membrane. In the Rous sarcoma virus Gag protein (Pr76gag), this assembly domain is associated with a PPPY motif, which is located at an internal position between the MA and CA sequences. This motif is not contained anywhere within the HIV Gag protein (Pr55gag), and the MA sequence is linked directly to CA. Instead, a late assembly function of HIV has been associated with the p6 sequence situated at the C terminus of Gag. Here we demonstrate the remarkable finding that the late assembly domains from these two unrelated Gag proteins are exchangeable between retroviruses and can function in a positionally independent manner.     

Maturation of envelope-specific antibody responses to linear determinants in monkeys inoculated with attenuated SIV

We have previously used a panel of quantitative and qualitative serological assays to define a lengthy and complex maturation of envelope-specific antibody responses in monkeys experimentally infected with attenuated simian immunodeficiency virus (SIV) that is closely associated with the temporal development of enduring and protective immunity to experimental virus challenge. To elucidate in more detail the changes in antibody specificity associated with this maturation, we describe here 'domain-specific' serological studies to characterize the evolution of antibody responses to defined linear determinants of the SIV envelope protein. The results of these studies reveal for the first time distinguishing differences in the evolution of antibody populations to distinct envelope peptide segments, as determined by measurements of antibody titer and avidity, indicating different patterns of antibody maturation to distinct linear envelope antigenic determinants. Thus, these data demonstrate the potential for domain-specific serology to produce a high-resolution characterization of SIV-specific antibody responses that can be used to evaluate experimental vaccine responses and to identify potential immune correlates of protection.