The cellular protein SERINC5 inhibits the infectivity of diverse retroviruses, and its activity is counteracted by the glycosylated Gag (glycoGag) protein of murine leukemia virus (MLV), the S2 protein of equine infectious anemia virus (EIAV), and the Nef protein of human immunodeficiency virus type 1 (HIV-1). Determining the regions within SERINC5 that provide restrictive activity or Nef sensitivity should inform mechanistic models of the SERINC5/HIV-1 relationship. Here, we report that deletion of the conserved sequence EDTEE, which is located within a cytoplasmic loop of SERINC5 and which is reminiscent of an acidic-cluster membrane trafficking signal, increases the sensitivity of SERINC5 to antagonism by Nef, while it has no effect on the intrinsic activity of the protein as an inhibitor of infectivity. These effects correlated with enhanced removal of the ΔEDTEE mutant relative to that of wild-type SERINC5 from the cell surface and with enhanced exclusion of the mutant protein from virions by Nef. Mutational analysis indicated that the acidic residues, but not the threonine, within the EDTEE motif are important for the relative resistance to Nef. Deletion of the EDTEE sequence did not increase the sensitivity of SERINC5 to antagonism by the glycoGag protein of MLV, suggesting that its virologic role is Nef specific. These results are consistent with the reported mapping of the cytoplasmic loop that contains the EDTEE sequence as a general determinant of Nef responsiveness, but they further indicate that sequences inhibitory to as well as supportive of Nef activity reside in this region. We speculate that the EDTEE motif might have evolved to mediate resistance against retroviruses that use Nef-like proteins to antagonize SERINC5. IMPORTANCE Cellular membrane proteins in the SERINC family, especially SERINC5, inhibit the infectivity of retroviral virions. This inhibition is counteracted by retroviral proteins, specifically, HIV-1 Nef, MLV glycoGag, and EIAV S2. One consequence of such a host-pathogen “arms race” is a compensatory change in the host antiviral protein as it evolves to escape the effects of viral antagonists. This is often reflected in a genetic signature, positive selection, which is conspicuously missing in SERINC5. Here we show that despite this lack of genetic evidence, a sequence in SERINC5 nonetheless provides relative resistance to antagonism by HIV-1 Nef.

Hypochilus is a relictual lineage of Nearctic spiders distributed disjunctly across the United States in three montane regions (California, southern Rocky Mountains, southern Appalachia). Phylogenetic resolution of species relationships in Hypochilus has been challenging, and conserved morphology coupled with extreme genetic divergence has led to uncertain species limits in some complexes. Here, Hypochilus interspecies relationships have been reconstructed and cryptic speciation more critically evaluated using a combination of ultraconserved elements, mitochondrial CO1 by-catch, and morphology. Phylogenomic data strongly support the monophyly of regional clades and support a ((California, Appalachia), southern Rocky Mountains) topology. In Appalachia, five species are resolved as four lineages (H. thorelli Marx, 1888 and H. coylei Platnick, 1987 are clearly sister taxa), but the interrelationships of these four lineages remain unresolved. The Appalachian species H. pococki Platnick, 1987 is recovered as monophyletic but is highly genetically structured at the nuclear level. While algorithmic analyses of nuclear data indicate many species (e.g., all H. pococki populations as species), male morphology instead reveals striking stasis. Within the California clade, nuclear and mitochondrial lineages of H. petrunkevitchi Gertsch, 1958 correspond directly to drainage basins of the southern Sierra Nevada, with H. bernardino Catley, 1994 nested within H. petrunkevitchi and sister to the southernmost basin populations. Combining nuclear, mitochondrial, geographical, and morphological evidence a new species from the Tule River and Cedar Creek drainages is described, Hypochilus xomotesp. nov. We also emphasize the conservation issues that face several microendemic, habitat-specialized species in this remarkable genus.

28The cellular protein SERINC5 inhibits the infectivity of diverse retroviruses and is 29 counteracted by the glycoGag protein of MLV, the S2 protein of EIAV, and the Nef 30 protein of HIV-1. Determining regions within SERINC5 that provide restrictive activity or 31Nef-sensitivity should inform mechanistic models of the SERINC5/HIV-1 relationship. 32Here, we report that deletion of the highly conserved sequence EDTEE, which is 33 located within a cytoplasmic loop of SERINC5 and is reminiscent of an acidic cluster 34 membrane trafficking signal, increases the sensitivity of SERINC5 to antagonism by Nef 35 while having no effect on the intrinsic activity of the protein as an inhibitor of infectivity. 36The effects on infectivity correlated with enhanced removal of the EDTEE mutant 37 relative to wild type SERINC5 from the cell surface and with enhanced exclusion of the 38 mutant protein from virions by Nef. Mutational analysis revealed that the acidic residues, 39 but not the threonine, within the EDTEE motif are important for the relative resistance to 40Nef. Deletion of the EDTEE sequence did not increase the sensitivity of SERINC5 to 41 antagonism by the glycoGag protein of MLV, suggesting that its virologic role is Nef-42 specific. These results are consistent with the reported mapping of the cytoplasmic loop 43 that contains the EDTEE sequence as a general determinant of Nef-responsiveness, 44 but they further indicate that sequences inhibitory to as well as supportive of Nef-activity 45 reside in this region. We speculate that the EDTEE motif might have evolved to mediate 46 resistance against retroviruses that use Nef-like proteins to antagonize SERINC5. 47 Importance 50Cellular membrane proteins in the SERINC family, especially SERINC5, inhibit the 51 infectivity of retroviral virions. This inhibition is counteracted by retroviral proteins, 52 specifically HIV-1 Nef, MLV glycoGag, and EIAV S2. One consequence of such a host-53 pathogen "arms race" is compensatory change in the host antiviral protein as it evolves 54 to escape the effects of the viral antagonist. This is often reflected in a genetic 55 signature, positive selection, which is conspicuously missing in SERINC5. Here we 56show that despite this lack of genetic evidence, a sequence in SERINC5 nonetheless 57 provides relative resistance to antagonism by HIV-1 Nef. 58 59 Introduction 60HIV-1 is a complex retrovirus, encoding "accessory" genes that evolved to 61 enhance viral fitness in response to host-selective pressures (1). The accessory gene 62 nef accelerates in vivo pathogenesis and progression to AIDS, despite being non-63 essential for viral propogation in cell-culture (2-4). Expression of the Nef protein occurs 64 early during the viral replication cycle, preceding the expression of structural proteins 65 such as the envelope glycoprotein (Env) and preceding virion assembly (5). Post-66 translational myristoylation on an N-terminal glycine residue enables Nef to associate 67 with lipid membranes (6), where it modulates the trafficking of ...