Comparative study of adaptive molecular evolution in different human immunodeficiency virus groups and subtypes

Molecular adaptation, as characterized by the detection of positive selection, was quantified in a number of genes from different human immunodeficiency virus type 1 (HIV-1) group M subtypes, group O, and an HIV-2 subtype using the codon-based maximum-likelihood method of Yang and coworkers (Z. H. Yang, R. Nielsen, N. Goldman, and A. M. K. Pedersen, Genetics 155:431-449, 2000). The env gene was investigated further since it exhibited the strongest signal for positive selection compared to those of the other two major HIV genes (gag and pol). In order to investigate the pattern of adaptive evolution across env, the location and strength of positive selection in different HIV-1 sequence alignments was compared. The number of sites having a significant probability of being positively selected varied among these different alignment data sets, ranging from 25 in HIV-1 group M subtype A to 40 in HIV-1 group O. Strikingly, there was a significant tendency for positively selected sites to be located at the same position in different HIV-1 alignments, ranging from 10 to 16 shared sites for the group M intersubtype comparisons and from 6 to 8 for the group O to M comparisons, suggesting that all HIV-1 variants are subject to similar selective forces. As the host immune response is believed to be the dominant driving force of adaptive evolution in HIV, this result would suggest that the same sites are contributing to viral persistence in diverse HIV infections. Thus, the positions of the positively selected sites were investigated in reference to the inferred locations of different epitope types (antibody, T helper, and cytotoxic T lymphocytes) and the positions of N and O glycosylation sites. We found a significant tendency for positively selected sites to fall outside T-helper epitopes and for positively selected sites to be strongly associated with N glycosylation sites.     

PHOSPHOLIPID METABOLISM IN EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS: ACTIVITY OF BRAIN PHOSPHOLIPASE A1, TOWARDS SPECIFICALLY LABELLED GLYCEROPHOSPHOLIPIDS

—The activity of phospholipase A₁ (phosphatide acylhydrolase, EC 3.1.1.4) was measured against phosphatidylcholine, -ethanolamine and -serine, specifically labelled with different fatty acids at either the 1 or 2 position, during experimental allergic encephalomyelitis in rats. In the acute stage of the disease there was a significant increase in enzyme activity in comparison to that from the control animals. The enhanced phospholipase A₁ activity was of the-same order of magnitude for all 1,2-diacyl-glycerophospholipids investigated. The phospholipase A₁-catalysed release of labelled fatty acids from molecular species of phosphatidylcholine decreased slightly with increasing degree of unsaturation of the fatty acids at the 1-position. The K_m-values of the highly purified enzyme were not altered by the demyelinating disorder. The phospholipase A₁ activity returned to the control level in the recovery stage.     

Molecular mechanisms of coupled monoubiquitination

Many proteins contain ubiquitin-binding domains or motifs (UBDs), such as the UIM (ubiquitin-interacting motif) and are referred to as ubiquitin receptors. Ubiquitin receptors themselves are frequently monoubiquitinated by a process that requires the presence of a UBD and is referred to as coupled monoubiquitination. Using a UIM-containing protein, eps15, as a model, we show here that coupled monoubiquitination strictly depends on the ability of the UIM to bind to monoubiquitin (mUb). We found that the underlying molecular mechanism is based on interaction between the UIM and a ubiquitin ligase (E3), which has itself been modified by ubiquitination. Furthermore, we demonstrate that the in vivo ubiquitination of members of the Nedd4 family of E3 ligases correlates with their ability to monoubiquitinate eps15. Thus, our results clarify the mechanism of coupled monoubiquitination and identify the ubiquitination of E3 ligases as a critical determinant in this process.     

Statistical deconvolution of enthalpic energetic contributions to MHC-peptide binding affinity

Background  

MHC Class I molecules present antigenic peptides to cytotoxic T cells, which forms an integral part of the adaptive immune response. Peptides are bound within a groove formed by the MHC heavy chain. Previous approaches to MHC Class I-peptide binding prediction have largely concentrated on the peptide anchor residues located at the P2 and C-terminus positions.     

GARD: a genetic algorithm for recombination detection

MOTIVATION: Phylogenetic and evolutionary inference can be severely misled if recombination is not accounted for, hence screening for it should be an essential component of nearly every comparative study. The evolution of recombinant sequences can not be properly explained by a single phylogenetic tree, but several phylogenies may be used to correctly model the evolution of non-recombinant fragments. RESULTS: We developed a likelihood-based model selection procedure that uses a genetic algorithm to search multiple sequence alignments for evidence of recombination breakpoints and identify putative recombinant sequences. GARD is an extensible and intuitive method that can be run efficiently in parallel. Extensive simulation studies show that the method nearly always outperforms other available tools, both in terms of power and accuracy and that the use of GARD to screen sequences for recombination ensures good statistical properties for methods aimed at detecting positive selection. AVAILABILITY: Freely available http://www.datamonkey.org/GARD/     

Genetic Analyses of HIV-1 env Sequences Demonstrate Limited Compartmentalization in Breast Milk and Suggest Viral Replication within the Breast That Increases with Mastitis

The concentration of human immunodeficiency virus type 1 (HIV-1) is generally lower in breast milk than in blood. Mastitis, or inflammation of the breast, is associated with increased levels of milk HIV-1 and risk of mother-to-child transmission through breastfeeding. We hypothesized that mastitis facilitates the passage of HIV-1 from blood into milk or stimulates virus production within the breast. HIV-1 env sequences were generated from single amplicons obtained from breast milk and blood samples in a cross-sectional study. Viral compartmentalization was evaluated using several statistical methods, including the Slatkin and Maddison (SM) test. Mastitis was defined as an elevated milk sodium (Na⁺) concentration. The association between milk Na⁺ and the pairwise genetic distance between milk and blood viral sequences was modeled using linear regression. HIV-1 was compartmentalized within milk by SM testing in 6/17 (35%) specimens obtained from 9 women, but all phylogenetic clades included viral sequences from milk and blood samples. Monotypic sequences were more prevalent in milk samples than in blood samples (22% versus 13%; P = 0.012), which accounted for half of the compartmentalization observed. Mastitis was not associated with compartmentalization by SM testing (P = 0.621), but Na⁺ was correlated with greater genetic distance between milk and blood HIV-1 populations (P = 0.041). In conclusion, local production of HIV-1 within the breast is suggested by compartmentalization of virus and a higher prevalence of monotypic viruses in milk specimens. However, phylogenetic trees demonstrate extensive mixing of viruses between milk and blood specimens. HIV-1 replication in breast milk appears to increase with inflammation, contributing to higher milk viral loads during mastitis.Breastfeeding accounts for 30 to 50% of mother-to-child-transmission (MTCT) of human immunodeficiency virus type 1 (HIV-1) (38). MTCT through breastfeeding occurs primarily in sub-Saharan Africa, where the use of artificial infant formula is often not feasible because of cost and the associated infant mortality from infections due to the use of unsafe water and the lack of the protective effects of breast milk (19, 38, 51). Numerous strategies to reduce postnatal HIV-1 infection of infants while preserving the advantages of breastfeeding have been evaluated, including maternal use of combination antiretroviral therapy or infant antiretroviral prophylaxis during the period of breastfeeding (5, 25, 26, 30, 40). Understanding the biologic events that increase the concentration of HIV-1 in breast milk is critical to the development and evaluation of interventions to reduce postnatal MTCT.The risk of MTCT is strongly associated with the concentration of HIV-1 in breast milk (28, 46, 47). Although breast milk HIV-1 RNA concentrations correlate with those in plasma, levels in milk are typically 2 log₁₀ lower (15, 24, 43). This suggests that HIV-1 in blood and milk may not mix freely, likely because of the closure of tight junctions between mammary alveolar cells that occurs once milk production is established and before weaning (16). Thus, HIV-1 may evolve in the breast without substantial mixing with blood, i.e., evolving viral variants would become compartmentalized—a phenomenon that has been observed in the central nervous system (50) and in some studies of the genital tract (10, 44, 57). Compartmentalization of HIV-1 variants has been detected in the breast milk of a small number of women (3, 4), but other data suggest that compartmentalization in breast milk may be uncommon (22).Breast inflammation (mastitis) occurs frequently during lactation, most commonly without symptoms. Mastitis is associated with elevations in HIV-1 RNA levels in milk (15, 31, 47, 55), an increase in the number of inflammatory cells in milk, and opening of tight junctions in the mammary epithelium that allows passage of subcellular blood components, of which sodium (Na⁺) serves as a marker (15, 16, 36, 47, 55). Greater permeability of mammary epithelia may allow the passage of free virus from the blood into breast milk, which would result in the mixing of HIV-1 subpopulations from blood and milk. Alternatively, inflammation in the breast may induce replication of virus by HIV-1-infected cells within the breast, which would result in divergence between milk and blood HIV-1 subpopulations. Here we describe detailed genetic analyses of HIV-1 subpopulations in the blood and breast milk to determine whether mastitis affects the structure of these populations and to gain understanding of the processes that may lead to increased concentrations of HIV-1 in milk.     

Positively Charged Amino Acid Substitutions in the E2 Envelope Glycoprotein Are Associated with the Emergence of Venezuelan Equine Encephalitis Virus

Epidemic-epizootic Venezuelan equine encephalitis (VEE) viruses (VEEV) have emerged repeatedly via convergent evolution from enzootic predecessors. However, previous sequence analyses have failed to identify common sets of nucleotide or amino acid substitutions associated with all emergence events. During 1993 and 1996, VEEV subtype IE epizootics occurred on the Pacific Coast of the states of Chiapas and Oaxaca in southern Mexico. Like other epizootic VEEV strains, when inoculated into guinea pigs and mice, the Mexican isolates were no more virulent than closely related enzootic strains, complicating genetic studies of VEE emergence. Complete genomic sequences of 4 of the Mexican strains were determined and compared to those of closely related enzootic subtype IE isolates from Guatemala. The epizootic viruses were less than 2% different at the nucleotide sequence level, and phylogenetic relationships confirmed that the equine-virulent Mexican strains probably evolved from enzootic progenitors on the Pacific Coast of Mexico or Guatemala. Of 35 amino acids that varied among the Guatemalan and Mexican isolates, only 8 were predicted phylogenetically to have accompanied the phenotypic change. One mutation at position 117 of the E2 envelope glycoprotein, involving replacement of Glu by Lys, resulted in a small-plaque phenotype characteristic of epizootic VEEV strains. Analysis of additional E2 sequences from representative enzootic and epizootic VEEV isolates implicated similar surface charge changes in the emergence of previous South American epizootic phenotypes, indicating that E2 mutations are probably important determinants of the equine-virulent phenotype and of VEE emergence. Maximum-likelihood analysis indicated that one change at E2 position 213 has been influenced by positive selection and convergent evolution of the epizootic phenotype.     

Evidence for the non-quasispecies evolution of RNA viruses [corrected

The quasispecies model of RNA virus evolution differs from those formulated in conventional population genetics in that neutral mutations do not lead to genetic drift of the population, and natural selection acts on the mutant distribution as a whole rather than on individual variants. By computer simulation, we show that this model could be inappropriate for many RNA viruses because the neutral sequence space may be too large to allow the formation of a quasispecies distribution. This view is supported by our analysis of gene sequences from vesicular stomatitis virus, which is considered a prototype RNA virus quasispecies. Our results are relevant to the evolution of RNA systems in general.     

Variable Immune-Driven Natural Selection in the Attachment (G) Glycoprotein of Respiratory Syncytial Virus (RSV)

A maximum-likelihood analysis of selection pressures acting on the attachment (G) glycoprotein gene of respiratory syncytial virus (RSV) from humans (HRSV) and bovines (BRSV) is presented. Six positively selected sites were identified in both group A and group B of HRSV, although only one site was common between them, while no positively selected sites were detected in BRSV. All positively selected sites were located within the ectodomain of the G protein and showed some association with positions of immunoglobulin (Ig) epitopes and sites of O-glycosylation. These results suggest that immune (antibody)-driven natural selection is an important determinant of RSV evolution and that this selection pressure differs among strains. The passage histories of RSV strains were also shown to affect the distribution of positively selected sites, particularly in HRSV B, and should be considered whenever retrospective analysis of adaptive evolution is undertaken. Received: 15 August 2000 / Accepted: 2 November 2000