Author: Camille Jessica Cunanan
Myxovirus resistance protein 2, MxB, displays structural interfaces critical for anti-HIV activity
In an article published by Science in September 2017, Alvarez et al show that the Myxovirus resistance protein 2, MxB, displays an oligomerization interface that is known to be essential for the restriction of HIV. In “CryoEM structure of MxB reveals a novel oligomerization interface critical for HIV restriction,” Alvarez et al use cryogenic electron microscopy to determine the structure of MxB and show that MxB purifies as oligomers and self-assembles into helical arrays in physiological conditions (1). Alvarez et al also discover that guanosine triphosphate (GTP) binding dissembles the helical arrays, while GTP hydrolysis initiates its reformation. In addition, they apply mutational analysis to show that newly discovered structural interfaces play a critical role for HIV-1 restriction.
The Human Immunodeficiency Virus, more popularly known as HIV, is a disease that targets the immune system and weakens bodily defense systems against infections and some types of cancer (3). It has been previously shown that myxovirus resistance (Mx) proteins are restriction factors that have roles in interferon response against viruses, particularly HIV (2). While interferons are proteins that prevent viral reproduction, Mx proteins work by inhibiting the early steps in the viral replication cycle (4). Although the roles of MxA have already been known restrict a wide range of viruses, only recently have studies shown that MxB displays antiviral activity as well. MxB targets the viral core of HIV proceeding cell entry and reverse transcription. Additionally, MxB also inhibits nuclear import of preintegration complexes and proviral integration of the virus into host genomes.
The typical structure of Mx proteins consists of a GTPase, a bundle signaling element (BSE), and a stalk domain (2). In addition, members of the family exhibit GTP dependent self-assembly into helical arrays. Previous work have shown that the antiviral activity of MxB is dependent upon the capsid binding NTR and oligomerization; however, the structural information of the NTR and oligomerization of MxB remains unknown. Through GTPase assays and cryogenic electron microscopy, Alvarez et al obtained important structural information about MxB oligomers and its role in inhibiting HIV activity.
To gain a primary understanding of the structure of MxB, full-length wild-type MxB were fused with an N-terminal maltose binding protein and were then expressed and purified as oligomers. Electron microscopy showed that MxB proteins assembled into long helical tubes and immunogold labeling showed that the NTR of MxB were positioned on the outer circumference of the helices.
In addition to the discoveries made about the NTR, Alvarez et al conducted GTP binding assays to understand the effect of GTP binding on the helical assembly of MxB. Upon addition of GTP or non-hydrolysable analogs of GTP, disruption of the helices of MxB was disrupted entirely. Overnight treatment of GTP resulted in the reassembly of the MxB helices. The results suggested that GTP binding, but not hydrolysis, causes conformational changes that can disrupt helices assembly. On the other hand, hydrolysis reverses assembly disruption and causes native conformation of the helices.
To obtain further details about the helical tube assembly, cryogenic electron microcopy of the MxB samples was conducted. CryoEM allowed the authors to determine a 3D density map of the MxB helical assembly at a 4.6 Å resolution (Figure 3B). Microscopy showed that the MxB tubules are highly ordered with an inner and outer diameter of 55 and 275 Å, respectively (Figure 3A). The alpha helical turns and side chains, which make up the stalk and the BSE, are sequestered in the inner core of the protein. Additionally, the GTPase domain shows slight dynamic behavior, while the NTR was unable to be resolved suggesting its flexibility. CryoEM also shows that assembly is made of 6 MxB dimer units. Six interlock through the stalk and BSE domains to form a rung, while the sixth dimer forms a right-handed helix (Figure 3F and 3G). The surface of the tube shows a shallow groove, presumably representing the GTPase domain and the NTR (Figure 3F and 3G). Conclusively, the CryoEM data obtained by Alvarez et al develops a new assembly of interfaces that were previously ambiguous.
Further CryoEm experiments were conducted to unveil three novel interfaces on MxB – the dimer interface (interface 2; previously known), oligomer interfaces (interfaces 1 and 3), and the helical assembly interface (interface 4). Interface 1 involves the symmetric interaction of the stalk domain of one dimer and the BSE domain of another dimer, connected by a salt bridge and hydrophobic contacts. Interface 3 is composed of a connecting alpha helices and is composed of hydrophobic interactions between two adjacent dimers. Interface 4 involves the GTPase domain of dimer 1 and the stalk region of dimer 6, forming a rung.
After determining the structurally important domains of MxB, Alvarez et al performed mutational analyses to determine the functional importance of the interfaces in assembly and antiviral activity by exposure to HIV-1 cultured cells. Since the importance of the dimer interface (interface 2) on antiviral activity has been previously discovered, the authors expressed and purified MxB mutants with alterations on interfaces 1, 3, and 4. Residue substitutions at these interfaces involved F20D in interface 1, F495D and R449D in interface 3, and E285K in interface 4. Such mutations were discovered to decrease MxB tube formation. While interface 1 and 4 mutants retained the ability to form oligomers, the interface 3 mutants (F495D and R449D) could not form oligomers. In addition, only the interface 3 mutants displayed aberrant anti-HIV activity when exposed to HIV-1 cell cultures. Disrupted anti-viral activity and abnormal oligomerization suggests that interface 3 plays a critical role in the canonical function of MxB.
Conclusively, the high resolution CryoEm structure of wild-type MxB modeled by Alvarez et al initiates the ability to make further discoveries about the a MxB-capsid interaction in HIV progression and activity. Considering there still remains 70 million individuals suffering from HIV and that there are no known cures for HIV, only treatments, the discoveries made by Alvarez et al allows for cutting-edge pharmacological targets for HIV therapeutics (3).
- J. D. Alvarez, Frances et al, CryoEM structure of MxB reveals a novel oligomerization interface critical for HIV restriction
- Haller, P. Staeheli, M. Schwemmle, G. Kochs, Mx GTPases: Dynamin-like antiviral machines of innate immunity
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