A prominent characteristic of human immunodeficiency virus type 1 (HIV-1) is its high genetic variability, which generates diversity of the virus and often causes a serious problem of the emergence of drug-resistant mutants. Subtype B HIV-1 is dominant in advanced countries, and the mortality rate due to subtype B HIV-1 has been decreased during the past decade. In contrast, the number of patients with non-subtype B viruses is still increasing in developing countries. One of the reasons for the prevalence of non-subtype B viruses is lack of information about the biological and therapeutic differences between subtype B and non-subtype B viruses. M36I is the most frequently observed polymorphism in non-subtype B HIV-1 proteases. However, since the 36th residue is located at a non-active site of the protease and has no direct interaction with any ligands, the structural role of M36I remains unclear. Here, we performed molecular dynamics (MD) simulations of M36I protease in complex with nelfinavir and revealed the influence of the M36I mutation. The results show that M36I regulates the size of the binding cavity of the protease. The reason for the rare emergence of D30N variants in non-subtype B HIV-1 proteases was also clarified from our computational analysis.

Eight serotypes of human astroviruses (HAstV-1 to HAstV-8) have been described. To date, the entire genomes of HAstV-1 and HAstV-2 as well as the ORF2 sequences of HAstV-1-6 and 8 have been reported. In this study, the ORF2 sequences of seventeen strains of HAstVs originating from different countries were determined, as well as the sequence ORF2 of one porcine astrovirus (PAstV) strain. Afterwards, comparison of the capsid protein precursors encoded by ORF2 of 46 strains of HAstVs, PAstV, and feline astrovirus (FAstV) was carried out. A phylogenetic tree showed eight genogroups of HAstVs that corresponded exactly to the serotypes. HAstV-3 and 7 were the most closely related, whereas HAstVs, FAstV, and PAstV segregated from each other. Compared to a PAstV, a FAstV is closer to HAstVs. Furthermore, the capsid protein precursors were divided into four regions (after amino acid residues 424, 688, and 776, respectively) based on sequence identity. Region I was the most conserved, and FAstV was very close in identity to HAstVs. Two amino acid motifs in region I were predicted to contain the common antigenic epitopes. Region II was relatively variable. Deletions and insertions were characteristic of region III, and region IV was relatively conserved. To our knowledge, this is the first comparative sequence analysis of the capsid protein precursors of eight serotypes of HAstVs as well as two animal astroviruses (FAstV and PAstV).

Nelfinavir (NFV) is a currently available HIV-1 protease (PR) inhibitor. Patients in whom NFV treatment has failed predominantly carry D30N mutants of HIV-1 PRs if they have been infected with the subtype B virus. In contrast, N88S mutants of HIV-1 PRs predominantly emerge in patients in whom NFV treatment has failed and who carry the CRF01_AE virus. Both D30N and N88S confer resistance against NFV. However, it remains unclear why the nonactive site mutation N88S confers resistance against NFV. In this study, we examined the resistance mechanism through computational simulations. The simulations suggested that despite the nonactive site mutation, N88S causes NFV resistance by reducing interactions between PR and NFV. We also investigated why the emergence rates of D30N and N88S differ between subtype B and CRF01_AE HIV-1. The simulations suggested that polymorphisms of CRF01_AE PR are involved in the emergence rate of the drug-resistant mutants.

The majority of HIV-1 infections around the world result from non-B clade HIV-1 strains. The CRF01_AE (AE) strain is seen principally in Southeast Asia. AE protease differs by ϳ10% in amino acid sequence from clade B protease and carries several naturally occurring polymorphisms that are associated with drug resistance in clade B. AE protease has been observed to develop resistance through a nonactive-site N88S mutation in response to nelfinavir (NFV) therapy, whereas clade B protease develops both the active-site mutation D30N and the nonactive-site mutation N88D. Structural and biochemical studies were carried out with wild-type and NFV-resistant clade B and AE protease variants. The relationship between clade-specific sequence variations and pathways to inhibitor resistance was also assessed. AE protease has a lower catalytic turnover rate than clade B protease, and it also has weaker affinity for both NFV and darunavir (DRV). This weaker affinity may lead to the nonactive-site N88S variant in AE, which exhibits significantly decreased affinity for both NFV and DRV. The D30N/N88D mutations in clade B resulted in a significant loss of affinity for NFV and, to a lesser extent, for DRV. A comparison of crystal structures of AE protease shows significant structural rearrangement in the flap hinge region compared with those of clade B protease and suggests insights into the alternative pathways to NFV resistance. In combination, our studies show that sequence polymorphisms within clades can alter protease activity and inhibitor binding and are capable of altering the pathway to inhibitor resistance.