The Antiviral Factor SERINC5 Impairs the Expression of Non-Self-DNA.

SERINC5 is a restriction factor that becomes incorporated into nascent retroviral particles, impairing their ability to infect target cells. In turn, retroviruses have evolved countermeasures against SERINC5. For instance, the primate lentiviruses (HIV and SIV) use Nef, Moloney Murine Leukemia Virus (MLV) uses GlycoGag, and Equine Infectious Anemia Virus (EIAV) uses S2 to remove SERINC5 from the plasma membrane, preventing its incorporation into progeny virions. Recent studies have shown that SERINC5 also restricts other viruses, such as Hepatitis B Virus (HBV) and Classical Swine Fever Virus (CSFV), although through a different mechanism, suggesting that SERINC5 can interfere with multiple stages of the virus life cycle. To investigate whether SERINC5 can also impact other steps of the replication cycle of HIV, the effects of SERINC5 on viral transcripts, proteins, and virus progeny size were studied. Here, we report that SERINC5 causes significant defects in HIV gene expression, which impacts virion production. While the underlying mechanism is still unknown, we found that the restriction occurs at the transcriptional level and similarly affects plasmid and non-integrated proviral DNA (ectopic or non-self-DNA). However, SERINC5 causes no defects in the expression of viral RNA, host genes, or proviral DNA that is integrated in the cellular genome. Hence, our findings reveal that SERINC5's actions in host defense extend beyond blocking virus entry.

Exosomes mediate LTB4 release during neutrophil chemotaxis.

Leukotriene B4 (LTB4) is secreted by chemotactic neutrophils, forming a secondary gradient that amplifies the reach of primary chemoattractants. This strategy increases the recruitment range for neutrophils and is important during inflammation. Here, we show that LTB4 and its synthesizing enzymes localize to intracellular multivesicular bodies, which, upon stimulation, release their content as exosomes. Purified exosomes can activate resting neutrophils and elicit chemotactic activity in an LTB4 receptor-dependent manner. Inhibition of exosome release leads to loss of directional motility with concomitant loss of LTB4 release. Our findings establish that the exosomal pool of LTB4 acts in an autocrine fashion to sensitize neutrophils towards the primary chemoattractant, and in a paracrine fashion to mediate the recruitment of neighboring neutrophils in trans. We envision that this mechanism is used by other signals to foster communication between cells in harsh extracellular environments.

Selective Disruption of SERINC5 Antagonism by Nef Impairs SIV Replication in Primary CD4+ T Cells.

The Nef proteins of HIV-1 and SIV enhance viral infectivity by preventing the incorporation of the multipass transmembrane protein serine incorporator 5 (SERINC5), and to a lesser extent SERINC3, into virions. In addition to counteracting SERINCs, SIV Nef also downmodulates several transmembrane proteins from the surface of virus-infected cells, including simian tetherin, CD4 and MHC class I (MHC I) molecules. From a systematic analysis of alanine substitutions throughout the SIVmac239 Nef protein, we identified residues that are required to counteract SERINC5. This information was used to engineer an infectious molecular clone of SIV (SIVmac239nef AV), which differs by two amino acids in the N-terminal domain of Nef that make the virus sensitive to SERINC5 while retaining other activities of Nef. SIVmac239nef AV downmodulates CD3, CD4, MHC I and simian tetherin, but cannot counteract SERINC5. In primary rhesus macaque CD4+ T cells, SIVmac239nef AV exhibits impaired infectivity and replication compared to wild-type SIVmac239. These results demonstrate that SERINC5 antagonism can be separated from other Nef functions and reveal the impact of SERINC5 on lentiviral replication.Importance: SERINC5, a multipass transmembrane protein, is incorporated into retroviral particles during assembly. This leads to a reduction of particle infectivity by inhibiting virus fusion with the target cell membrane. The Nef proteins of HIV-1 and SIV enhance viral infectivity by preventing the incorporation of SERINC5 into virions. However, the relevance of this restriction factor in viral replication has not been elucidated. Here we report a systematic mapping of Nef residues required for SERINC5 antagonism. Counter screens for three other functions of Nef helped identify two residues in the N-terminal domain of Nef, which when mutated make Nef selectively susceptible to SERINC5. Since Nef is multi-functional, genetic separation of SERINC5 antagonism from its other functions affords comparison of the replication of isogenic viruses that are or are not sensitive to SERINC5. Such a strategy revealed the impact of SERINC5 on SIV replication in primary rhesus macaque CD4+ T-cells.

Loss of tetherin antagonism by Nef impairs SIV replication during acute infection of rhesus macaques.

Most simian immunodeficiency viruses use Nef to counteract the tetherin proteins of their nonhuman primate hosts. Nef also downmodulates cell-surface CD4 and MHC class I (MHC I) molecules and enhances viral infectivity by counteracting SERINC5. We previously demonstrated that tetherin antagonism by SIV Nef is genetically separable from CD4- and MHC I-downmodulation. Here we show that disruption of tetherin antagonism by Nef impairs virus replication during acute SIV infection of rhesus macaques. A combination of mutations was introduced into the SIVmac239 genome resulting in three amino acid substitutions in Nef that impair tetherin antagonism, but not CD3-, CD4- or MHC I-downmodulation. Further characterization of this mutant (SIVmac239AAA) revealed that these changes also result in partial sensitivity to SERINC5. Separate groups of four rhesus macaques were infected with either wild-type SIVmac239 or SIVmac239AAA, and viral RNA loads in plasma and sequence changes in the viral genome were monitored. Viral loads were significantly lower during acute infection in animals infected with SIVmac239AAA than in animals infected with wild-type SIVmac239. Sequence analysis of the virus population in plasma confirmed that the substitutions in Nef were retained during acute infection; however, changes were observed by week 24 post-infection that fully restored anti-tetherin activity and partially restored anti-SERINC5 activity. These observations reveal overlap in the residues of SIV Nef required for counteracting tetherin and SERINC5 and selective pressure to overcome these restriction factors in vivo.

Polymorphisms in Rhesus Macaque Tetherin Are Associated with Differences in Acute Viremia in Simian Immunodeficiency Virus Δnef-Infected Animals.

Tetherin (BST-2 or CD317) is an interferon-inducible transmembrane protein that inhibits virus release from infected cells. To determine the extent of sequence variation and the impact of polymorphisms in rhesus macaque tetherin on simian immunodeficiency virus (SIV) infection, tetherin alleles were sequenced from 146 rhesus macaques, including 68 animals infected with wild-type SIVmac239 and 47 animals infected with SIVmac239Δnef Since Nef is the viral gene product of SIV that counteracts restriction by tetherin, these groups afford a comparison of the effects of tetherin polymorphisms on SIV strains that are, and are not, resistant to tetherin. We identified 15 alleles of rhesus macaque tetherin with dimorphic residues at 9 positions. The relationship between these alleles and plasma viral loads was compared during acute infection, prior to the onset of adaptive immunity. Acute viremia did not differ significantly among the wild-type SIV-infected animals; however, differences in acute viral loads were associated with polymorphisms in tetherin among the animals infected with SIVΔnef In particular, polymorphisms at positions 43 and 111 (P43 and H111) were associated with lower acute-phase viral loads for SIVΔnef infection. These observations reveal extensive polymorphism in rhesus macaque tetherin, maintained perhaps as a consequence of variability in the selective pressure of diverse viral pathogens, and identify tetherin alleles that may have an inherently greater capacity to restrict SIV replication in the absence of Nef.IMPORTANCE As a consequence of ongoing evolutionary conflict with viral pathogens, tetherin has accumulated numerous species-specific differences that represent important barriers to the transmission of viruses between species. This study reveals extensive polymorphism in rhesus macaque tetherin and identifies specific alleles that are associated with lower viral loads during the first few weeks after infection with nef-deleted SIV. These observations suggest that the variable selective pressure of viral pathogens, in addition to driving the diversification of tetherin among species, also operates within certain species to maintain sequence variation in tetherin.

The inflammatory chemokine Cxcl18b exerts neutrophil-specific chemotaxis via the promiscuous chemokine receptor Cxcr2 in zebrafish.

Cxcl18b is a chemokine found in zebrafish and in other piscine and amphibian species. Cxcl18b is a reliable inflammatory marker; however, its function is yet to be elucidated. Here, we found that Cxcl18b is chemotactic towards neutrophils, similarly to Cxcl8a/Interleukin-8, the best characterised neutrophil chemoattractant in humans and teleosts. Like Cxcl8a, Cxcl18b-dependent recruitment required the chemokine receptor Cxcr2, while it was unaffected by depletion of the other two neutrophil receptors cxcr1 and cxcr4b. To visualise cxcl18b induction, we generated a Tg(cxcl18b:eGFP) reporter line. The transgene is induced locally upon bacterial infection with the fish pathogen Mycobacterium marinum, but strikingly is not directly expressed by infected cells. Instead, cxcl18b is induced by non-phagocytic uninfected cells that compose the stroma of the granulomas, typical inflammatory lesions formed upon mycobacterial infections. Together, these results suggest that Cxcl18b might be an important contributor to neutrophil chemotaxis in the inflammatory microenvironment and indicate that the zebrafish model could be explored to further investigate in vivo the biological relevance of different Cxcl8-like chemokine lineages.

Exosomes Mediate LTB4 Release during Neutrophil Chemotaxis.

Leukotriene B4 (LTB4) is secreted by chemotactic neutrophils, forming a secondary gradient that amplifies the reach of primary chemoattractants. This strategy increases the recruitment range for neutrophils and is important during inflammation. Here, we show that LTB4 and its synthesizing enzymes localize to intracellular multivesicular bodies that, upon stimulation, release their content as exosomes. Purified exosomes can activate resting neutrophils and elicit chemotactic activity in a LTB4 receptor-dependent manner. Inhibition of exosome release leads to loss of directional motility with concomitant loss of LTB4 release. Our findings establish that the exosomal pool of LTB4 acts in an autocrine fashion to sensitize neutrophils towards the primary chemoattractant, and in a paracrine fashion to mediate the recruitment of neighboring neutrophils in trans. We envision that this mechanism is used by other signals to foster communication between cells in harsh extracellular environments.