Oligomerization Requirements for MX2-Mediated Suppression of HIV-1 Infection

Abstract

Human myxovirus resistance 2 (MX2/MXB) is an interferon-stimulated gene (ISG) and was recently identified as a late postentry suppressor of human immunodeficiency virus type 1 (HIV-1) infection, inhibiting the nuclear accumulation of viral cDNAs. Although the HIV-1 capsid (CA) protein is believed to be the viral determinant of MX2-mediated inhibition, the precise mechanism of antiviral action remains unclear. The MX family of dynamin-like GTPases also includes MX1/MXA, a well-studied inhibitor of a range of RNA and DNA viruses, including influenza A virus (FLUAV) and hepatitis B virus but not retroviruses. MX1 and MX2 are closely related and share similar domain architectures and structures. However, MX2 possesses an extended N terminus that is essential for antiviral function and confers anti-HIV-1 activity on MX1 [MX1(NMX2)]. Higher-order oligomerization is required for the antiviral activity of MX1 against FLUAV, with current models proposing that MX1 forms ring structures that constrict around viral nucleoprotein complexes. Here, we performed structure-function studies to investigate the requirements for oligomerization of both MX2 and chimeric MX1(NMX2) for the inhibition of HIV-1 infection. The oligomerization state of mutated proteins with amino acid substitutions at multiple putative oligomerization interfaces was assessed using a combination of covalent cross-linking and coimmunoprecipitation. We show that while monomeric MX2 and MX1(NMX2) mutants are not antiviral, higher-order oligomerization does not appear to be required for full antiviral activity of either protein. We propose that lower-order oligomerization of MX2 is sufficient for the effective inhibition of HIV-1.IMPORTANCE Interferon plays an important role in the control of virus replication during acute infection in vivo. Recently, cultured cell experiments identified human MX2 as a key effector in the interferon-mediated postentry block to HIV-1 infection. MX2 is a member of a family of large dynamin-like GTPases that includes MX1/MXA, a closely related interferon-inducible inhibitor of several viruses, including FLUAV, but not HIV-1. MX GTPases form higher-order oligomeric structures, and the oligomerization of MX1 is required for inhibitory activity against many of its viral targets. Through structure-function studies, we report that monomeric mutants of MX2 do not inhibit HIV-1. However, in contrast to MX1, oligomerization beyond dimer assembly does not seem to be required for the antiviral activity of MX2, implying that fundamental differences exist between the antiviral mechanisms employed by these closely related proteins.

Oligomerization and GTP-binding Requirements of MxA for Viral Target Recognition and Antiviral Activity against Influenza A Virus

“…The fact that the dimeric forms MxA(R640A) and MxA(L617D) exert antiviral activity strongly suggests that the GTP-dependent formation of dimers is important for the function of MxA. In this context, it is interesting to note that human MxB (Mx2), shown recently to restrict human immunodeficiency virus, appears to act as a dimer, albeit in a GTP-independent manner (43)(44)(45).…”

Capsid-Dependent Host Factors in HIV-1 Infection

“…Oligomerization is another MX2 property critical for its antiviral activity, and MX2 forms an extended antiparallel dimer [94, 100]. Dimerization, but not higher-order oligomerization, which is mediated by the MX2 CTD, is essential for anti-HIV-1 activity [88, 89, 94, 98, 100–102]. Oligomerization is required for MX2 binding to capsid [94, 101].…”

Role of MxB in Alpha Interferon-Mediated Inhibition of HIV-1 Infection

“…The formation of oligomers is indispensable for both MxA and MxB to restrict their target viruses. While MxA depends on its GTPase activity for its antiviral function, the GTPase activity of MxB is required to inhibit human herpesviruses but not HIV-1 (6,8,(11)(12)(13)(14)(15)(16)(17)(18)(19). MxA is cytoplasmic, whereas MxB is seen more at the cytoplasmic face of the nuclear envelope.…”