Dataset Information

Mutation in the loop C-terminal to the cyclophilin A binding site of HIV-1 capsid protein disrupts proper virus assembly and infectivity.

ABSTRACT: We have studied the effects associated with two single amino acid substitution mutations in HIV-1 capsid (CA), the E98A and E187G. Both amino acids are well conserved among all major HIV-1 subtypes. HIV-1 infectivity is critically dependent on proper CA cone formation and mutations in CA are lethal when they inhibit CA assembly by destabilizing the intra and/or inter molecular CA contacts, which ultimately abrogate viral replication. Glu98, which is located on a surface of a flexible cyclophilin A binding loop is not involved in any intra-molecular contacts with other CA residues. In contrast, Glu187 has extensive intra-molecular contacts with eight other CA residues. Additionally, Glu187 has been shown to form a salt-bridge with Arg18 of another N-terminal CA monomer in a N-C dimer. However, despite proper virus release, glycoprotein incorporation and Gag processing, electron microscopy analysis revealed that, in contrast to the E187G mutant, only the E98A particles had aberrant core morphology that resulted in loss of infectivity.

SUBMITTER: Abdurahman S

PROVIDER: S-EPMC1832212 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Mutation in the loop C-terminal to the cyclophilin A binding site of HIV-1 capsid protein disrupts proper virus assembly and infectivity.

Abdurahman Samir S Höglund Stefan S Höglund Anders A Vahlne Anders A

Retrovirology 20070319

We have studied the effects associated with two single amino acid substitution mutations in HIV-1 capsid (CA), the E98A and E187G. Both amino acids are well conserved among all major HIV-1 subtypes. HIV-1 infectivity is critically dependent on proper CA cone formation and mutations in CA are lethal when they inhibit CA assembly by destabilizing the intra and/or inter molecular CA contacts, which ultimately abrogate viral replication. Glu98, which is located on a surface of a flexible cyclophilin ...[more]

PMID: 17371591

Similar Datasets

Project description:The HI loop is a prominent domain on the adeno-associated virus (AAV) capsid surface that extends from each viral protein (VP) subunit overlapping the neighboring fivefold VP. Despite the highly conserved nature of the residues at the fivefold pore, the HI loops surrounding this critical region vary significantly in amino acid sequence between the AAV serotypes. In order to understand the role of this unique capsid domain, we ablated side chain interactions between the HI loop and the underlying EF loop in the neighboring VP subunit by generating a collection of deletion, insertion, and substitution mutants. A mutant lacking the HI loop was unable to assemble particles, while a substitution mutant (10 glycine residues) assembled particles but was unable to package viral genomes. Substitution mutants carrying corresponding regions from AAV1, AAV4, AAV5, and AAV8 yielded (i) particles with titers and infectivity identical to those of AAV2 (AAV2 HI1 and HI8), (ii) particles with a decreased virus titer (1 log) but normal infectivity (HI4), and (iii) particles that synthesized VPs but were unable to assemble into intact capsids (HI5). AAV5 HI is shorter than all other HI loops by one amino acid. Replacing the missing residue (threonine) in AAV2 HI5 resulted in a moderate particle assembly rescue. In addition, we replaced the HI loop with peptides varying in length and amino acid sequence. This region tolerated seven-amino-acid peptide substitutions unless they spanned a conserved phenylalanine at amino acid position 661. Mutation of this highly conserved phenylalanine to a glycine resulted in a modest decrease in virus titer but a substantial decrease (1 log order) in infectivity. Subsequently, confocal studies revealed that AAV2 F661G is incapable of efficiently completing a key step in the infectious pathway nuclear entry, hinting at a possible perturbation of VP1 phospholipase activity. Molecular modeling studies with the F661G mutant suggest that disruption of interactions between F661 and an underlying P373 residue in the EF loop of the neighboring subunit might adversely affect incorporation of the VP1 subunit at the fivefold axis. Western blot analysis confirmed inefficient incorporation of VP1, as well as a proteolytically processed VP1 subunit that could account for the markedly reduced infectivity. In summary, our studies show that the HI loop, while flexible in amino acid sequence, is critical for AAV capsid assembly, proper VP1 subunit incorporation, and viral genome packaging, all of which implies a potential role for this unique surface domain in viral infectivity.

Project description:The ability of human immunodeficiency virus type 1 (HIV-1) to transduce nondividing cells is key to infecting terminally differentiated macrophages, which can serve as a long-term reservoir of HIV-1 infection. The mutation N57A in the viral CA protein renders HIV-1 cell cycle dependent, allowing examination of HIV-1 infection of nondividing cells. Here, we show that the N57A mutation confers a postentry infectivity defect that significantly differs in magnitude between the common lab-adapted molecular clones HIV-1NL4-3 (>10-fold) and HIV-1LAI (2- to 5-fold) in multiple human cell lines and primary CD4+ T cells. Capsid permeabilization and reverse transcription are altered when N57A is incorporated into HIV-1NL4-3 but not HIV-1LAI The N57A infectivity defect is significantly exacerbated in both virus strains in the presence of cyclosporine (CsA), indicating that N57A infectivity is dependent upon CA interacting with host factor cyclophilin A (CypA). Adaptation of N57A HIV-1LAI selected for a second CA mutation, G94D, which rescued the N57A infectivity defect in HIV-1LAI but not HIV-1NL4-3 The rescue of N57A by G94D in HIV-1LAI is abrogated by CsA treatment in some cell types, demonstrating that this rescue is CypA dependent. An examination of over 40,000 HIV-1 CA sequences revealed that the four amino acids that differ between HIV-1NL4-3 and HIV-1LAI CA are polymorphic, and the residues at these positions in the two strains are widely prevalent in clinical isolates. Overall, a few polymorphic amino acid differences between two closely related HIV-1 molecular clones affect the phenotype of capsid mutants in different cell types.IMPORTANCE The specific mechanisms by which HIV-1 infects nondividing cells are unclear. A mutation in the HIV-1 capsid protein abolishes the ability of the virus to infect nondividing cells, serving as a tool to examine cell cycle dependence of HIV-1 infection. We have shown that two widely used HIV-1 molecular clones exhibit significantly different N57A infectivity phenotypes due to fewer than a handful of CA amino acid differences and that these clones are both represented in HIV-infected individuals. As such minor differences in closely related HIV-1 strains may impart significant infectivity differences, careful consideration should be given to drawing conclusions from one particular HIV-1 clone. This study highlights the potential for significant variation in results with the use of multiple strains and possible unanticipated effects of natural polymorphisms.

Project description:Enteric viruses exploit bacterial components, including lipopolysaccharides (LPS) and peptidoglycan (PG), to facilitate infection in humans. Because of their origin in the bat enteric system, we wondered if severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle East respiratory syndrome CoV (MERS-CoV) also use bacterial components to modulate infectivity. To test this question, we incubated CoVs with LPS and PG and evaluated infectivity, finding no change following LPS treatment. However, PG from Bacillus subtilis reduced infection >10,000-fold, while PG from other bacterial species failed to recapitulate this. Treatment with an alcohol solvent transferred inhibitory activity to the wash, and mass spectrometry revealed surfactin, a cyclic lipopeptide antibiotic, as the inhibitory compound. This antibiotic had robust dose- and temperature-dependent inhibition of CoV infectivity. Mechanistic studies indicated that surfactin disrupts CoV virion integrity, and surfactin treatment of the virus inoculum ablated infection in vivo Finally, similar cyclic lipopeptides had no effect on CoV infectivity, and the inhibitory effect of surfactin extended broadly to enveloped viruses, including influenza, Ebola, Zika, Nipah, chikungunya, Una, Mayaro, Dugbe, and Crimean-Congo hemorrhagic fever viruses. Overall, our results indicate that peptidoglycan-associated surfactin has broad viricidal activity and suggest that bacteria by-products may negatively modulate virus infection.IMPORTANCE In this article, we consider a role for bacteria in shaping coronavirus infection. Taking cues from studies of enteric viruses, we initially investigated how bacterial surface components might improve CoV infection. Instead, we found that peptidoglycan-associated surfactin is a potent viricidal compound that disrupts virion integrity with broad activity against enveloped viruses. Our results indicate that interactions with commensal bacterial may improve or disrupt viral infections, highlighting the importance of understanding these microbial interactions and their implications for viral pathogenesis and treatment.

Dataset Information

Mutation in the loop C-terminal to the cyclophilin A binding site of HIV-1 capsid protein disrupts proper virus assembly and infectivity.

Publications

Mutation in the loop C-terminal to the cyclophilin A binding site of HIV-1 capsid protein disrupts proper virus assembly and infectivity.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets