A single polymorphic residue in humans underlies species-specific restriction of HSV-1 by the antiviral protein MxB.

IMPORTANCE: Herpesviruses present a major global disease burden. Understanding the host cell mechanisms that block viral infections, as well as how viruses can evolve to counteract these host defenses, is critically important for understanding viral disease pathogenesis. This study reveals that the major human variant of the antiviral protein myxovirus resistance protein B (MxB) inhibits the human pathogen herpes simplex virus (HSV-1), whereas a minor human variant and orthologous MxB genes from even closely related primates do not. Thus, in contrast to the many antagonistic virus-host interactions in which the virus is successful in thwarting the host's defense systems, here the human gene appears to be at least temporarily winning at this interface of the primate-herpesvirus evolutionary arms race. Our findings further show that a polymorphism at amino acid 83 in a small fraction of the human population is sufficient to abrogate MxB's ability to inhibit HSV-1, which could have important implications for human susceptibility to HSV-1 pathogenesis.

Antagonism of the Protein Kinase R Pathway in Human Cells by Rhesus Cytomegalovirus.

While cytomegalovirus (CMV) infections are often limited in host range by lengthy coevolution with a single host species, a few CMVs are known to deviate from this rule. For example, rhesus macaque CMV (RhCMV), a model for human CMV (HCMV) pathogenesis and vaccine development, can replicate in human cells, as well as in rhesus cells. Both HCMV and RhCMV encode species-specific antagonists of the broadly acting host cell restriction factor protein kinase R (PKR). Although the RhCMV antagonist of PKR, rTRS1, has very limited activity against human PKR, here, we show it is essential for RhCMV replication in human cells because it prevents human PKR from phosphorylating the translation initiation factor eIF2α, thereby allowing continued translation and viral replication. Although rTRS1 is necessary for RhCMV replication, it is not sufficient to rescue replication of HCMV lacking its own PKR antagonists in human fibroblasts. However, overexpression of rTRS1 in human fibroblasts enabled HCMV expressing rTRS1 to replicate, indicating that elevated levels or early expression of a weak antagonist can counteract a resistant restriction factor like human PKR. Exploring potential mechanisms that might allow RhCMV to replicate in human cells revealed that RhCMV makes no less double-stranded RNA than HCMV. Rather, in human cells, RhCMV expresses rTRS1 at levels 2 to 3 times higher than those of the HCMV-encoded PKR antagonists during HCMV infection. These data suggest that even a modest increase in expression of this weak PKR antagonist is sufficient to enable RhCMV replication in human cells.IMPORTANCE Rhesus macaque cytomegalovirus (RhCMV) offers a valuable model for studying congenital human cytomegalovirus (HCMV) pathogenesis and vaccine development. Therefore, it is critical to understand variations in how each virus infects and affects its host species to be able to apply insights gained from the RhCMV model to HCMV. While HCMV is capable only of infecting cells from humans and very closely related species, RhCMV displays a wider host range, including human as well as rhesus cells. RhCMV expresses an antagonist of a broadly acting antiviral factor present in all mammalian cells, and its ability to counter both the rhesus and human versions of this host factor is a key component of RhCMV's ability to cross species barriers. Here, we examine the molecular mechanisms that allow this RhCMV antagonist to function against a human restriction factor.

Human placental trophoblasts confer viral resistance to recipient cells.

Placental trophoblasts form the interface between the fetal and maternal environments and serve to limit the maternal-fetal spread of viruses. Here we show that cultured primary human placental trophoblasts are highly resistant to infection by a number of viruses and, importantly, confer this resistance to nonplacental recipient cells by exosome-mediated delivery of specific microRNAs (miRNAs). We show that miRNA members of the chromosome 19 miRNA cluster, which are almost exclusively expressed in the human placenta, are packaged within trophoblast-derived exosomes and attenuate viral replication in recipient cells by the induction of autophagy. Together, our findings identify an unprecedented paracrine and/or systemic function of placental trophoblasts that uses exosome-mediated transfer of a unique set of placental-specific effector miRNAs to directly communicate with placental or maternal target cells and regulate their immunity to viral infections.

Human trophoblasts confer resistance to viruses implicated in perinatal infection.

OBJECTIVE: Primary human trophoblasts were previously shown to be resistant to viral infection, and able to confer this resistance to nontrophoblast cells. Can trophoblasts protect nontrophoblastic cells from infection by viruses or other intracellular pathogens that are implicated in perinatal infection? STUDY DESIGN: Isolated primary term human trophoblasts were cultured for 48-72 hours. Diverse nonplacental human cell lines (U2OS, human foreskin fibroblast, TZM-bl, MeWo, and Caco-2) were preexposed to either trophoblast conditioned medium, nonconditioned medium, or miR-517-3p for 24 hours. Cells were infected with several viral and nonviral pathogens known to be associated with perinatal infections. Cellular infection was defined and quantified by plaque assays, luciferase assays, microscopy, and/or colonization assays. Differences in infection were assessed by Student t test or analysis of variance with Bonferroni correction. RESULTS: Infection by rubella and other togaviruses, human immunodeficiency virus-1, and varicella zoster was attenuated in cells preexposed to trophoblast-conditioned medium (P < .05), and a partial effect by the chromosome 19 microRNA miR-517-3p on specific pathogens. The conditioned medium had no effect on infection by Toxoplasma gondii or Listeria monocytogenes. CONCLUSION: Our findings indicate that medium conditioned by primary human trophoblasts attenuates viral infection in nontrophoblastic cells. Our data point to a trophoblast-specific antiviral effect that may be exploited therapeutically.

Requirement of the N-terminal activation domain of herpes simplex virus ICP4 for viral gene expression.

ICP4 is the major activator of herpes simplex virus (HSV) transcription. Previous studies have defined several regions of ICP4 that are important for viral gene expression, including a DNA binding domain and transactivation domains that are contained in the C-terminal and N-terminal 520 and 274 amino acids, respectively. Here we show that the N-terminal 210 amino acids of ICP4 are required for interactions with components of TFIID and mediator and, as a consequence, are necessary for the activation of viral genes. A mutant of ICP4 deleted for amino acids 30 to 210, d3-10, was unable to complement an ICP4 null virus at the level of viral replication. This was the result of a severe deficiency in viral gene and protein expression. The absence of viral gene expression coincided with a defect in the recruitment of RNA polymerase II to a representative early promoter (thymidine kinase [TK]). Affinity purification experiments demonstrated that d3-10 ICP4 was not found in complexes with components of TFIID and mediator, suggesting that the defect in RNA polymerase II (Pol II) recruitment was the result of ablated interactions between d3-10 and TFIID and mediator. Complementation assays suggested that the N-terminal and C-terminal regions of ICP4 cooperate to mediate gene expression. The complementation was the result of the formation of more functional heterodimers, which restored the ability of the d3-10-containing molecules to interact with TFIID. Together, these studies suggest that the N terminus contains a true activation domain, mediating interactions with TFIID, mediator, and perhaps other transcription factors, and that the C terminus of the molecule contains activities that augment the functions of the activation domain.

Natural variation in the heparan sulfate binding domain of the eastern equine encephalitis virus E2 glycoprotein alters interactions with cell surfaces and virulence in mice.

Recently, we compared amino acid sequences of the E2 glycoprotein of natural North American eastern equine encephalitis virus (NA-EEEV) isolates and demonstrated that naturally circulating viruses interact with heparan sulfate (HS) and that this interaction contributes to the extreme neurovirulence of EEEV (C. L. Gardner, G. D. Ebel, K. D. Ryman, and W. B. Klimstra, Proc. Natl. Acad. Sci. U. S. A., 108:16026-16031, 2011). In the current study, we have examined the contribution to HS binding of each of three lysine residues in the E2 71-to-77 region that comprise the primary HS binding site of wild-type (WT) NA-EEEV viruses. We also report that the original sequence comparison identified five virus isolates, each with one of three amino acid differences in the E2 71-to-77 region, including mutations in residues critical for HS binding by the WT virus. The natural variant viruses, which possessed either a mutation from lysine to glutamine at E2 71, a mutation from lysine to threonine at E2 71, or a mutation from threonine to lysine at E2 72, exhibited altered interactions with heparan sulfate and cell surfaces and altered virulence in a mouse model of EEEV disease. An electrostatic map of the EEEV E1/E2 heterotrimer based upon the recent Chikungunya virus crystal structure (J. E. Voss, M. C. Vaney, S. Duquerroy, C. Vonrhein, C. Girard-Blanc, E. Crublet, A. Thompson, G. Bricogne, and F. A. Rey, Nature, 468:709-712, 2010) showed the HS binding site to be at the apical surface of E2, with variants affecting the electrochemical nature of the binding site. Together, these results suggest that natural variation in the EEEV HS binding domain may arise during EEEV sylvatic cycles and that this variation may influence receptor interaction and the severity of EEEV disease.

A single polymorphic residue in humans underlies species-specific restriction of HSV-1 by the antiviral protein MxB.

Myxovirus resistance proteins (MxA and MxB) are interferon-induced proteins that exert antiviral activity against a diverse range of RNA and DNA viruses. In primates, MxA has been shown to inhibit myxoviruses, bunyaviruses, and hepatitis B virus, whereas MxB restricts retroviruses and herpesviruses. As a result of their conflicts with viruses, both genes have been undergoing diversifying selection during primate evolution. Here, we investigate how MxB evolution in primates has affected its restriction of herpesviruses. In contrast to human MxB, we find that most primate orthologs, including the closely related chimpanzee MxB, do not inhibit HSV-1 replication. However, all primate MxB orthologs tested restrict human cytomegalovirus. Through the generation of human and chimpanzee MxB chimeras we show that a single residue, M83, is the key determinant of restriction of HSV-1 replication. Humans are the only primate species known to encode a methionine at this position, whereas most other primate species encode a lysine. Residue 83 is also the most polymorphic residue in MxB in human populations, with M83 being the most common variant. However, ∼2.5% of human MxB alleles encode a threonine at this position, which does not restrict HSV-1. Thus, a single amino acid variant in MxB, which has recently risen to high frequency in humans, has endowed humans with HSV-1 antiviral activity. Importance: Herpesviruses present a major global disease burden. Understanding the host cell mechanisms that block viral infections as well as how viruses can evolve to counteract these host defenses is critically important for understanding viral disease pathogenesis, and for developing therapeutic tools aimed at treating or preventing viral infections. Additionally, understanding how these host and viral mechanisms adapt to counter one another can aid in identifying the risks of, and barriers to, cross-species transmission events. As highlighted by the recent SARS-CoV-2 pandemic, episodic transmission events can have severe consequences for human health. This study reveals that the major human variant of the antiviral protein MxB inhibits the human pathogen HSV-1, whereas human minor variants and orthologous MxB genes from even closely related primates do not. Thus, in contrast to the many antagonistic virus-host interactions in which the virus is successful in thwarting the defense systems of their native hosts, in this case the human gene appears to be at least temporarily winning at this interface of the primate-herpesviral evolutionary arms race. Our findings further show that a polymorphism at amino acid 83 in a small fraction of the human population is sufficient to abrogate MxB's ability to inhibit HSV-1, which could have important implications for human susceptibility to HSV-1 pathogenesis.

Exposing the invader.

A restriction factor induced by interferons blocks the replication of herpesviruses by disassembling the capsid proteins surrounding their genome.

Adaptation by copy number variation in monopartite viruses.

Viruses evolve rapidly in response to host defenses and to exploit new niches. Gene amplification, a common adaptive mechanism in prokaryotes, archaea, and eukaryotes, has also contributed to viral evolution, especially of large DNA viruses. In experimental systems, gene amplification is one mechanism for rapidly overcoming selective pressures. Because the amplification generally incurs a fitness cost, emergence of adaptive point mutations within the amplified locus or elsewhere in the genome can enable collapse of the locus back to a single copy. Evidence of gene amplification followed by subfunctionalization or neofunctionalization of the copies is apparent by the presence of families of paralogous genes in many DNA viruses. These observations suggest that copy number variation has contributed broadly to virus evolution.

Chromosome 19 microRNAs exert antiviral activity independent from type III interferon signaling.

INTRODUCTION: Cultured primary human trophoblasts (PHT), derived from term placentas, are relatively resistant to infection by diverse viruses. The resistance can be conferred to non-trophoblastic cells by pre-exposing them to medium that was conditioned by PHT cells. This antiviral effect is mediated, at least in part, by microRNAs (miRNA) expressed from the chromosome 19 microRNA cluster (C19MC). Recently we showed that PHT cells and cells pre-exposed to PHT medium are also resistant to infection by Zika virus (ZIKV), an effect mediated by the constitutive release of the type III interferons (IFN) IFN lambda-1 and IFN lambda-2 in trophoblastic medium. We hypothesized that trophoblastic C19MC miRNA are active against ZIKV, and assessed the interaction of this pathway with IFN lambda-1 - mediated resistance. METHODS: Term PHT cells were cultured using standard techniques. An osteosarcoma cell line (U2OS) was used as non-trophoblastic cells, which were infected with either ZIKV or vesicular stomatitis virus (VSV). Trophoblastic extracellular vesicles (EVs) were produced by gradient ultracentrifugation. RT-qPCR was used to determine viral infection, cellular or medium miRNA levels and the expression of interferon-stimulated genes. RESULTS: We showed that C19MC miRNA attenuate infection of U2OS cells by ZIKV, and that C19MC miRNA or exosomes that contain C19MC miRNA did not influence the type III IFN pathway. Similarly, cell exposure to recombinant IFN lambda-1 had no effect on miRNA expression, and these pathways did not exhibit synergistic interaction. DISCUSSION: PHT cells exert antiviral activity by at least two independent mechanisms, mediated by C19MC miRNA and by type III IFNs.

Type III Interferons Produced by Human Placental Trophoblasts Confer Protection against Zika Virus Infection.

During mammalian pregnancy, the placenta acts as a barrier between the maternal and fetal compartments. The recently observed association between Zika virus (ZIKV) infection during human pregnancy and fetal microcephaly and other anomalies suggests that ZIKV may bypass the placenta to reach the fetus. This led us to investigate ZIKV infection of primary human trophoblasts (PHTs), which are the barrier cells of the placenta. We discovered that PHT cells from full-term placentas are refractory to ZIKV infection. In addition, medium from uninfected PHT cells protects non-placental cells from ZIKV infection. PHT cells constitutively release the type III interferon (IFN) IFNλ1, which functions in both a paracrine and autocrine manner to protect trophoblast and non-trophoblast cells from ZIKV infection. Our data suggest that for ZIKV to access the fetal compartment, it must evade restriction by trophoblast-derived IFNλ1 and other trophoblast-specific antiviral factors and/or use alternative strategies to cross the placental barrier.

Isolation of human trophoblastic extracellular vesicles and characterization of their cargo and antiviral activity.

INTRODUCTION: Primary human trophoblasts release a repertoire of extracellular vesicles (EVs). Among them are nano-sized exosomes, which we found to suppress the replication of a wide range of diverse viruses. These exosomes contain trophoblastic microRNAs (miRNAs) that are expressed from the chromosome 19 miRNA cluster and exhibit antiviral properties. Here, we report our investigation of the cargo of placental EVs, focusing on the composition and the antiviral properties of exosomes, microvesicles, and apoptotic blebs. METHODS: We isolated EVs using ultracentrifugation and defined their purity using immunoblotting, electron microscopy, and nanoparticle tracking. We used liquid chromatography-electrospray ionization-mass spectrometry, protein mass spectrometry, and miRNA TaqMan card PCR to examine the phospholipids, proteins, and miRNA cargo of trophoblastic EVs and an in vitro viral infection assay to assess the antiviral properties of EVs. RESULTS: We found that all three EV types contain a comparable repertoire of miRNA. Interestingly, trophoblastic exosomes harbor a protein and phospholipid profile that is distinct from that of microvesicles or apoptotic blebs. Functionally, trophoblastic exosomes exhibit the highest antiviral activity among the EVs. Consistently, plasma exosomes derived from pregnant women recapitulate the antiviral effect of trophoblastic exosomes derived from in vitro cultures of primary human trophoblasts. DISCUSSION: When compared to other trophoblastic EVs, exosomes exhibit a unique repertoire of proteins and phospholipids, but not miRNAs, and a potent viral activity. Our work suggests that human trophoblastic EVs may play a key role in maternal-placental-fetal communication.

The Function of TrophomiRs and Other MicroRNAs in the Human Placenta.

In eutherian organisms, the placenta interfaces the fetal and maternal environments. Located at the placental villous surface, in direct contact with maternal blood, is the trophoblast layer, which mediates the crucial maternal-fetal exchange of gases, nutrients, and waste products, produces hormones that support the pregnancy, and provides immunologic defense. Discovery of microRNAs (miRNAs) and their role in development, differentiation, and homeostatic resilience has increased our understanding of genomic and epigenomic networks that regulate placental function. Moreover, unique miRNA species, which are expressed by human trophoblasts and are termed "trophomiRs," may show specialized functions during normal and pathological pregnancies. Placental miRNAs, packaged within exosomes and other vesicles or bound in protein complexes, are capable of communicating distinctive signals to maternal and/or fetal tissues. Additional research may usher in the use of circulating miRNAs as pregnancy-related disease biomarkers, providing new diagnostic and therapeutic options during pregnancy.

The role of trophoblastic microRNAs in placental viral infection.

During the past decade, various types of small non-coding RNAs were found to be expressed in all kingdoms and phyla of life. Intense research efforts have begun to shed light on their biological functions, although much remains to be determined in order to fully characterize their scope of biological action. Typically, small RNAs provide sequence specificity to a protein complex that is driven to silence a long target RNA. MicroRNAs (miRNAs) are small RNAs that are coded in the genome of most eukaryotes, and contribute to the cellular identity by regulating cell-specific gene networks by translational repression or degradation of mRNA. These effects commonly fine-tune gene expression associated with developmental or environmental cues. Different cell types can be characterized by their distinctive cellular miRNA landscape. The human placenta expresses a unique set of miRNAs, a high proportion of which is derived from a large cluster located on chromosome 19, (termed chromosome 19 miRNA cluster, or C19MC). Interestingly, a fraction of these placenta-enriched miRNAs are released to the extracellular environment through exosomes that were recently found to induce an antiviral immunity. In this review, we explore relevant placental viral infections and discuss the antiviral role of exosome-packaged placental C19MC miRNAs in this context.

Autophagy as a mechanism of antiviral defense at the maternal-fetal interface.

Mechanisms to protect against viral infections are crucial during pregnancy as maternal-fetal transmission can have serious pathological outcomes, including fetal infection and its sequelae, such as growth restriction, birth defects, and/or fetal death. The trophoblast forms the interface between the feto-placental unit and the maternal blood, and is therefore a critical physical and immunological barrier to restrict the spread of pathogens into the fetal microenvironment. Recently, we found that primary human placental trophoblast (PHT) cells are highly resistant to infection by diverse viruses. In this study, we also found that conditioned medium from PHT cell cultures transferred viral resistance to nonplacental recipient cells, suggesting that a component secreted by trophoblasts and present within the conditioned medium is responsible for this antiviral effect. We found that specific miRNAs from a unique primate- and placental-specific locus?the C19MC (chromosome 19 miRNA cluster)?are packaged within exosomes produced by PHT cells and confer viral resistance in nonplacental recipient cells. In addition to conveying viral resistance, we found that PHT-derived exosomes and select miRNA members of the C19MC family strongly induce autophagy, which is involved in recipient cell viral resistance. Our findings establish an exciting and novel mechanism by which placental trophoblasts exploit exosome-dependent transfer of placental-specific miRNAs to influence autophagic induction and antiviral immunity at the maternal?fetal interface.