A stable immature lattice packages IP6 for HIV capsid maturation

HIV virion assembly begins with the construction of an immature lattice consisting of Gag hexamers. Upon virion release, protease-mediated Gag cleavage leads to a maturation event in which the immature lattice disassembles and the mature capsid assembles. The cellular metabolite inositiol hexakisphosphate (IP6) and maturation inhibitors (MIs) both bind and stabilize immature Gag hexamers, but whereas IP6 promotes virus maturation, MIs inhibit it. Here we show that HIV is evolutionarily constrained to maintain an immature lattice stability that ensures IP6 packaging without preventing maturation. Replication-deficient mutant viruses with reduced IP6 recruitment display increased infectivity upon treatment with the MI PF46396 (PF96) or the acquisition of second-site compensatory mutations. Both PF96 and second-site mutations stabilise the immature lattice and restore IP6 incorporation, suggesting that immature lattice stability and IP6 binding are interdependent. This IP6 dependence suggests that modifying MIs to compete with IP6 for Gag hexamer binding could substantially improve MI antiviral potency.

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Functional Surfaces of the Hepatitis B Virus Capsid

Journal of Virology ◽

10.1128/jvi.01178-09 ◽

2009 ◽

Vol 83 (22) ◽

pp. 11616-11623 ◽

Cited By ~ 29

Author(s):

Alexander Pairan ◽

Volker Bruss

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Core Protein ◽

Point Mutations ◽

Amino Acid Residues ◽

Virion Release ◽

P Protein ◽

B Virus ◽

Capsid Maturation ◽

Almost All

ABSTRACT The hepatitis B virus (HBV) core protein (CP) forms the shell of an icosahedral nucleocapsid. In a former work, we identified 11 amino acid residues of CP exposed on the capsid surface by an alanine mutation scan as being important for capsid envelopment. We now introduced several other amino acids at six of these positions and found that almost all 27 tested point mutations at S17, K96, and I126 reproduced the phenotype of the alanine mutation (with only two exceptions): the formation of nucleocapsids and of the viral DNA genome was wild type, but capsid envelopment and virion release were strongly inhibited. This indicates that these side chains have a very specific function during nucleocapsid envelopment. We also identified several CP point mutations (e.g., F122V/S/Y and R127D/G) allowing the formation of capsids but preventing the packaging of pregenomic RNA. The envelopment of such mutant capsids was blocked. Apparently, these CP mutations hampered the recognition/packaging of the pregenome-P-protein complex by CP, a process which is still barely understood, and the mutant capsids devoid of HBV-specific nucleic acid did not express the capsid maturation signal required for envelopment.

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The C-Terminal End of HIV-1 Vpu Has a Clade-Specific Determinant That Antagonizes BST-2 and Facilitates Virion Release

Journal of Virology ◽

10.1128/jvi.02315-18 ◽

2019 ◽

Vol 93 (11) ◽

Cited By ~ 2

Author(s):

Shilpi Sharma ◽

Moein Jafari ◽

Amandip Bangar ◽

Karen William ◽

John Guatelli ◽

...

Keyword(s):

Plasma Membrane ◽

Chronic Infection ◽

High Surface ◽

Human Populations ◽

Accessory Proteins ◽

Virion Assembly ◽

Virion Release ◽

Clade B ◽

Clade C ◽

Hiv 1

ABSTRACTThe cellular protein bone marrow stromal antigen-2 (BST-2)/tetherin acts against a variety of enveloped viruses by restricting their release from the plasma membrane. The HIV-1 accessory protein Vpu counteracts BST-2 by downregulating it from the cell surface and displacing it from virion assembly sites. Previous comparisons of Vpus from transmitted/founder viruses and between viruses isolated during acute and chronic infection led to the identification of a tryptophan at position 76 in Vpu (W76) as a key determinant for the displacement of BST-2 from virion assembly sites. Although present in Vpus from clades B, D, and G, W76 is absent from Vpus from clades A, C, and H. Mutagenesis of the C-terminal region of Vpu from two clade C viruses led to the identification of a conserved LL sequence that is functionally analogous to W76 of clade B. Alanine substitution of these leucines partially impaired virion release. This impairment was even greater when the mutations were combined with mutations of the Vpu β-TrCP binding site, resulting in Vpu proteins that induced high surface levels of BST-2 and reduced the efficiency of virion release to less than that of virus lackingvpu. Microscopy confirmed that these C-terminal leucines in clade C Vpu, like W76 in clade B, contribute to virion release by supporting the displacement of BST-2 from virion assembly sites. These results suggest that although encoded differently, the ability of Vpu to displace BST-2 from sites of virion assembly on the plasma membrane is evolutionarily conserved among clade B and C HIV-1 isolates.IMPORTANCEAlthough targeted by a variety of restriction mechanisms, HIV-1 establishes chronic infection in most cases, in part due to the counteraction of these host defenses by viral accessory proteins. Using conserved motifs, the accessory proteins exploit the cellular machinery to degrade or mistraffic host restriction factors, thereby counteracting them. The Vpu protein counteracts the virion-tethering factor BST-2 in part by displacing it from virion assembly sites along the plasma membrane, but a previously identified determinant of that activity is clade specific at the level of protein sequence and not found in the clade C viruses that dominate the pandemic. Here, we show that clade C Vpu provides this activity via a leucine-containing sequence rather than the tryptophan-containing sequence found in clade B Vpu. This difference seems likely to reflect the different evolutionary paths taken by clade B and clade C HIV-1 in human populations.

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UL25 capsid binding facilitates mechanical maturation of the Herpesvirus capsid and allows retention of pressurized DNA

Journal of Virology ◽

10.1128/jvi.00755-21 ◽

2021 ◽

Author(s):

Krista G. Freeman ◽

Jamie B. Huffman ◽

Fred L. Homa ◽

Alex Evilevitch

Keyword(s):

Dna Packaging ◽

Maturation Process ◽

Virion Assembly ◽

Force Microscopy ◽

Atomic Force ◽

Auxiliary Protein ◽

Capsid Maturation ◽

Simplex Virus ◽

Hsv 1

The maturation process that occurs in most viruses is evolutionarily driven as it resolves several conflicting virion assembly requirements. During herpesvirus assembly in a host cell nucleus, micron-long double-stranded herpes DNA is packaged into a nanometer-sized procapsid. This leads to strong confinement of the viral genome with resulting tens of atmospheres of intra-capsid DNA pressure. Yet, the procapsid is unstable due to weak, reversible interactions between its protein subunits, which ensures free energy minimization and reduces assembly errors. In this work we show that herpesviruses resolve these contradictory capsid requirements through a mechanical capsid maturation process facilitated by multi-functional auxiliary protein UL25. Through mechanical interrogation of herpes simplex virus type 1 (HSV-1) capsid with atomic force microscopy nano-indentation, we show that UL25 binding at capsid vertices post-assembly provides the critical capsid reinforcement required for stable DNA encapsidation; the absence of UL25 binding leads to capsid rupture. Furthermore, we demonstrate that gradual capsid reinforcement is a feasible maturation mechanism facilitated by progressive UL25 capsid binding, which is likely correlated with DNA packaging progression. This work provides insight into elegantly programmed viral assembly machinery where targeting of capsid assembly mechanics presents a new antiviral strategy that is resilient to development of drug resistance. Importance: Most viruses undergo a maturation process from a weakly assembled particle to a stable virion. Herpesvirus capsid undergoes mechanical maturation to withstand tens of atmospheres of DNA pressure. We demonstrate that this mechanical capsid maturation is mainly facilitated through binding of auxiliary protein UL25 in HSV-1 capsid vertices. We show that UL25 binding provides the critical capsid reinforcement required for stable DNA encapsidation. Our data also suggests that gradual capsid reinforcement by progressive UL25 binding is a feasible capsid maturation mechanism, correlated with DNA packaging progression.

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Derivation and characterization of an HIV-1 mutant that rescues IP6 binding deficiency

Retrovirology ◽

10.1186/s12977-021-00571-3 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Daniel Poston ◽

Trinity Zang ◽

Paul Bieniasz

Keyword(s):

Primary Amines ◽

Virion Assembly ◽

Gag Proteins ◽

Maturation Inhibitor ◽

Live Cell Microscopy ◽

Compensatory Mutations ◽

Virion Formation ◽

Cell Microscopy ◽

Hiv 1

Abstract Background A critical step in the HIV-1 replication cycle is the assembly of Gag proteins to form virions at the plasma membrane. Virion assembly and maturation are facilitated by the cellular polyanion inositol hexaphosphate (IP6), which is proposed to stabilize both the immature Gag lattice and the mature capsid lattice by binding to rings of primary amines at the center of Gag or capsid protein (CA) hexamers. The amino acids comprising these rings are critical for proper virion formation and their substitution results in assembly deficits or impaired infectiousness. To better understand the nature of the deficits that accompany IP6 binding deficiency, we passaged HIV-1 mutants that had substitutions in IP6 coordinating residues to select for compensatory mutations. Results We found a mutation, a threonine to isoleucine substitution at position 371 (T371I) in Gag, that restored replication competence to an IP6-binding-deficient HIV-1 mutant. Notably, unlike wild-type HIV-1, the assembly and infectiousness of resulting virus was not impaired when IP6 biosynthetic enzymes were genetically ablated. Surprisingly, we also found that the maturation inhibitor Bevirimat (BVM) could restore the assembly and replication of an IP6-binding deficient mutant. Moreover, using BVM-dependent mutants we were able to image BVM-induced assembly of individual HIV-1 particles assembly in living cells. Conclusions Overall these results suggest that IP6-Gag and Gag-Gag contacts are finely tuned to generate a Gag lattice of optimal stability, and that under certain conditions BVM can rescue IP6 deficiency. Additionally, our work identifies an inducible virion assembly system that can be utilized to visualize HIV-1 assembly events using live cell microscopy.

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