MX2 Viral Substrate Breadth and Inhibitory Activity Are Regulated by Protein Phosphorylation.

Human immunodeficiency virus type-1 (HIV-1) infection is potently inhibited by human myxovirus resistance 2 (MX2/MxB), which binds to the viral capsid and blocks the nuclear import of viral DNA. We have recently shown that phosphorylation is a key regulator of MX2 antiviral activity, with phosphorylation of serine residues at positions 14, 17, and 18 repressing MX2 function. Here, we extend the study of MX2 posttranslational modifications and identify serine and threonine phosphorylation in all domains of MX2. By substituting these residues with aspartic acid or alanine, hence mimicking the presence or absence of a phosphate group, respectively, we identified key positions that control MX2 antiviral activity. Aspartic acid substitutions of residues Ser306 or Thr334 and alanine substitutions of Thr343 yielded proteins with substantially reduced antiviral activity, whereas the presence of aspartic acid at positions Ser28, Thr151, or Thr343 resulted in enhanced activity: referred to as hypermorphic mutants. In some cases, these hypermorphic mutations, particularly when paired with other MX2 mutations (e.g., S28D/T151D or T151D/T343A) acquired the capacity to inhibit HIV-1 capsid mutants known to be insensitive to wild-type MX2, such as P90A or T210K, as well as MX2-resistant retroviruses such as equine infectious anemia virus (EIAV) and murine leukemia virus (MLV). This work highlights the complexity and importance of MX2 phosphorylation in the regulation of antiviral activity and in the selection of susceptible viral substrates. IMPORTANCE Productive infection by human immunodeficiency virus type-1 (HIV-1) requires the import of viral replication complexes into the nuclei of infected cells. Myxovirus resistance 2 (MX2/MxB) blocks this step, halting nuclear accumulation of viral DNA and virus replication. We recently demonstrated how phosphorylation of a stretch of three serines in the amino-terminal domain of MX2 inhibits the antiviral activity. Here, we identify additional positions in MX2 whose phosphorylation status reduces or enhances antiviral function (hypomorphic and hypermorphic variants, respectively). Importantly, hypermorphic mutant proteins not only increased inhibitory activity against wild-type HIV-1 but can also exhibit antiviral capabilities against HIV-1 capsid mutant viruses that are resistant to wild-type MX2. Furthermore, some of these proteins were also able to inhibit retroviruses that are insensitive to MX2. Therefore, we propose that phosphorylation comprises a major element of MX2 regulation and substrate determination.

Effects of HIV-1 reverse transcriptase connection subdomain mutations on polypurine tract removal and initiation of (+)-strand DNA synthesis.

HIV-1 reverse transcriptase (RT) connection subdomain mutations at positions 348, 369 and 376 have been associated with resistance to non-nucleoside RT inhibitors (NNRTIs). N348I may interfere with the initiation of (+)-strand DNA synthesis by reducing polypurine tract (PPT) removal in the presence of nevirapine. The effect of NNRTIs on the RNase H-mediated cleavage of PPT-containing template-primers has been studied with wild-type HIV-1 RT and mutants N348I, T369I, T369V, T376S and N348I/T369I. In the presence of NNRTIs, all RTs were able to stimulate PPT cleavage after primer elongation. The enhancing effects of nevirapine and efavirenz were reduced in RTs carrying mutation N348I, and specially N348I/T369I. However, those mutations had no effect on rilpivirine-mediated cleavage. Prior to elongation, the PPT remains resilient to cleavage, although efavirenz and rilpivirine facilitate RNase H-mediated trimming of its 3'-end. The integrity of the 3'-end is essential for the initiation of (+)-strand DNA synthesis. In the presence of dNTPs, rilpivirine was the most effective inhibitor of (+)-strand DNA synthesis blocking nucleotide incorporation and preventing usage of available PPT primers. The N348I/T369I RT showed reduced ability to generate short RNA products revealing a cleavage window defect. Its lower RNase H activity could be attributed to enhanced rigidity compared to the wild-type enzyme.