Analysis of the Viral Elements Required in the Nuclear Import of HIV-1 DNA

HIV-1 possesses an exquisite ability to infect cells independently from their cycling status by undergoing an active phase of nuclear import through the nuclear pore. This property has been ascribed to the presence of karyophilic elements present in viral nucleoprotein complexes, such as the matrix protein (MA); Vpr; the integrase (IN); and a cis-acting structure present in the newly synthesized DNA, the DNA flap. However, their role in nuclear import remains controversial at best. In the present study, we carried out a comprehensive analysis of the role of these elements in nuclear import in a comparison between several primary cell types, including stimulated lymphocytes, macrophages, and dendritic cells. We show that despite the fact that none of these elements is absolutely required for nuclear import, disruption of the central polypurine tract-central termination sequence (cPPT-CTS) clearly affects the kinetics of viral DNA entry into the nucleus. This effect is independent of the cell cycle status of the target cells and is observed in cycling as well as in nondividing primary cells, suggesting that nuclear import of viral DNA may occur similarly under both conditions. Nonetheless, this study indicates that other components are utilized along with the cPPT-CTS for an efficient entry of viral DNA into the nucleus.Lentiviruses display an exquisite ability to infect dividing and nondividing cells alike that is unequalled among Retroviridae. This property is thought to be due to the particular behavior or composition of the viral nucleoprotein complexes (NPCs) that are liberated into the cytoplasm of target cells upon virus-to-cell membrane fusion and that allow lentiviruses to traverse an intact nuclear membrane (17, 28, 29, 39, 52, 55, 67, 79). In the case of the human immunodeficiency type I virus (HIV-1), several studies over the years identified viral components of such structures with intrinsic karyophilic properties and thus perfect candidates for mediation of the passage of viral DNA (vDNA) through the nuclear pore: the matrix protein (MA); Vpr; the integrase (IN); and a three-stranded DNA flap, a structure present in neo-synthesized viral DNA, specified by the central polypurine tract-central termination sequence (cPPT-CTS). It is clear that these elements may mediate nuclear import directly or via the recruitment of the host''s proteins, and indeed, several cellular proteins have been found to influence HIV-1 infection during nuclear import, like the karyopherin α2 Rch1 (38); importin 7 (3, 30, 93); the transportin SR-2 (13, 20); or the nucleoporins Nup98 (27), Nup358/RANBP2, and Nup153 (13, 56).More recently, the capsid protein (CA), the main structural component of viral nucleoprotein complexes at least upon their cytoplasmic entry, has also been suggested to be involved in nuclear import or in postnuclear entry steps (14, 25, 74, 90, 92). Whether this is due to a role for CA in the shaping of viral nucleoprotein complexes or to a direct interaction between CA and proteins involved in nuclear import remains at present unknown.Despite a large number of reports, no single viral or cellular element has been described as absolutely necessary or sufficient to mediate lentiviral nuclear import, and important controversies as to the experimental evidences linking these elements to this step exist. For example, MA was among the first viral protein of HIV-1 described to be involved in nuclear import, and 2 transferable nuclear localization signals (NLSs) have been described to occur at its N and C termini (40). However, despite the fact that early studies indicated that the mutation of these NLSs perturbed HIV-1 nuclear import and infection specifically in nondividing cells, such as macrophages (86), these findings failed to be confirmed in more-recent studies (23, 33, 34, 57, 65, 75).Similarly, Vpr has been implicated by several studies of the nuclear import of HIV-1 DNA (1, 10, 21, 43, 45, 47, 64, 69, 72, 73, 85). Vpr does not possess classical NLSs, yet it displays a transferable nucleophilic activity when fused to heterologous proteins (49-51, 53, 77, 81) and has been shown to line onto the nuclear envelope (32, 36, 47, 51, 58), where it can truly facilitate the passage of the viral genome into the nucleus. However, the role of Vpr in this step remains controversial, as in some instances Vpr is not even required for viral replication in nondividing cells (1, 59).Conflicting results concerning the role of IN during HIV-1 nuclear import also exist. Indeed, several transferable NLSs have been described to occur in the catalytic core and the C-terminal DNA binding domains of IN, but for some of these, initial reports of nuclear entry defects (2, 9, 22, 46, 71) were later shown to result from defects at steps other than nuclear import (60, 62, 70, 83). These reports do not exclude a role for the remaining NLSs in IN during nuclear import, and they do not exclude the possibility that IN may mediate this step by associating with components of the cellular nuclear import machinery, such as importin alpha and beta (41), importin 7 (3, 30, 93, 98), and, more recently, transportin-SR2 (20).The central DNA flap, a structure present in lentiviruses and in at least 1 yeast retroelement (44), but not in other orthoretroviruses, has also been involved in the nuclear import of viral DNA (4, 6, 7, 31, 78, 84, 95, 96), and more recently, it has been proposed to provide a signal for viral nucleoprotein complexes uncoating in the proximity of the nuclear pore, with the consequence of providing a signal for import (8). However, various studies showed an absence or weakness of nuclear entry defects in viruses devoid of the DNA flap (24, 26, 44, 61).Overall, the importance of viral factors in HIV-1 nuclear import is still unclear. The discrepancies concerning the role of MA, IN, Vpr, and cPPT-CTS in HIV-1 nuclear import could in part be explained by their possible redundancy. To date, only one comprehensive study analyzed the role of these four viral potentially karyophilic elements together (91). This study showed that an HIV-1 chimera where these elements were either deleted or replaced by their murine leukemia virus (MLV) counterparts was, in spite of an important infectivity defect, still able to infect cycling and cell cycle-arrested cell lines to similar efficiencies. If this result indicated that the examined viral elements of HIV-1 were dispensable for the cell cycle independence of HIV, as infections proceeded equally in cycling and arrested cells, they did not prove that they were not required in nuclear import, because chimeras displayed a severe infectivity defect that precluded their comparison with the wild type (WT).Nuclear import and cell cycle independence may not be as simply linked as previously thought. On the one hand, there has been no formal demonstration that the passage through the nuclear pore, and thus nuclear import, is restricted to nondividing cells, and for what we know, this passage may be an obligatory step in HIV infection in all cells, irrespective of their cycling status. In support of this possibility, certain mutations in viral elements of HIV affect nuclear import in dividing as well as in nondividing cells (4, 6, 7, 31, 84, 95). On the other hand, cell cycle-independent infection may be a complex phenomenon that is made possible not only by the ability of viral DNA to traverse the nuclear membrane but also by its ability to cope with pre- and postnuclear entry events, as suggested by the phenotypes of certain CA mutants (74, 92).Given that the cellular environment plays an important role during the early steps of viral infection, we chose to analyze the role of the four karyophilic viral elements of HIV-1 during infection either alone or combined in a wide comparison between cells highly susceptible to infection and more-restrictive primary cell targets of HIV-1 in vivo, such as primary blood lymphocytes (PBLs), monocyte-derived macrophages (MDM), and dendritic cells (DCs).In this study, we show that an HIV-1-derived virus in which the 2 NLSs of MA are mutated and the IN, Vpr, and cPPT-CTS elements are removed displays no detectable nuclear import defect in HeLa cells independently of their cycling status. However, this mutant virus is partially impaired for nuclear entry in primary cells and more specifically in DCs and PBLs. We found that this partial defect is specified by the cPPT-CTS, while the 3 remaining elements seem to play no role in nuclear import. Thus, our study indicates that the central DNA flap specifies the most important role among the viral elements involved thus far in nuclear import. However, it also clearly indicates that the role played by the central DNA flap is not absolute and that its importance varies depending on the cell type, independently from the dividing status of the cell.     

Monotypic Human Immunodeficiency Virus Type 1 Genotypes across the Uterine Cervix and in Blood Suggest Proliferation of Cells with Provirus

Understanding the dynamics and spread of human immunodeficiency virus type 1 (HIV-1) within the body, including within the female genital tract with its central role in heterosexual and peripartum transmission, has important implications for treatment and vaccine development. To study HIV-1 populations within tissues, we compared viruses from across the cervix to those in peripheral blood mononuclear cells (PBMC) during effective and failing antiretroviral therapy (ART) and in patients not receiving ART. Single-genome sequences of the C2-V5 region of HIV-1 env were derived from PBMC and three cervical biopsies per subject. Maximum-likelihood phylogenies were evaluated for differences in genetic diversity and compartmentalization within and between cervical biopsies and PBMC. All subjects had one or more clades with genetically identical HIV-1 env sequences derived from single-genome sequencing. These sequences were from noncontiguous cervical biopsies or from the cervix and circulating PBMC in seven of eight subjects. Compartmentalization of virus between genital tract and blood was observed by statistical methods and tree topologies in six of eight subjects, and potential genital lineages were observed in two of eight subjects. The detection of monotypic sequences across the cervix and blood, especially during effective ART, suggests that cells with provirus undergo clonal expansion. Compartmentalization of viruses within the cervix appears in part due to viruses homing to and/or expanding within the cervix and is rarely due to unique viruses evolving within the genital tract. Further studies are warranted to investigate mechanisms producing monotypic viruses across tissues and, importantly, to determine whether the proliferation of cells with provirus sustain HIV-1 persistence in spite of effective ART.Immune and drug pressures modify the human immunodeficiency virus type 1 (HIV-1) population within an individual and, along with physical barriers in the body, may result in uneven selective pressures between tissues, allowing the evolution of tissue-specific viral variants. Whether unique viruses evolve within the genital tract, the tissue involved in most cases of HIV-1 transmission, is relevant to developing vaccines and other interventions to reduce HIV-1 transmission. Compartmentalization is the term applied to genetically distinct HIV-1 populations in different tissues. Compartmentalization of genital tract compared to blood HIV-1 has been observed in 50 to 75% of men and women (6, 9, 12, 13, 19, 21, 22, 29, 31, 33, 34, 41, 42, 48).In a study of compartmentalization in the female genital tract, we noted that HIV-1 DNA sequences from the female genital tract clustered in phylograms, suggestive of a burst of replication with spread to adjacent cells in the cervix (M. E. Bull, G. H. Learn, I. Genowati, J. L. McKernan, J. Hitti, D. Lockhart, S. Holte, J. Dragavon, R. W. Coombs, J. I. Mullins, and L. M. Frenkel, submitted for publication). We suspected that the low viral diversity was due to the small size of the biopsies, taken from a localized area of the cervix that sampled only small foci of the virus population, as observed in a macaque model (25) and in splenic white pulps of humans (5, 8, 17). We hypothesized that sampling multiple sites in the uterine cervix would reveal greater viral diversity than a single biopsy or the blood. We anticipated that if the viral “swarm” transmitted during acute infection was diverse (33, 37), multiple cervical biopsies would reveal more diverse viral variants archived in the genital DNA than in peripheral blood mononuclear cells (PBMC). Since there are few physical barriers, we also hypothesized that genital tract viruses would not appear compartmentalized from viruses in the blood. To evaluate these hypotheses, three cervical biopsies and a peripheral blood sample were obtained at a single study visit. Multiple single-genome sequences (SGS) derived from each specimen were used to evaluate the phylogenetic relationships, including diversity, and compartmentalization of the HIV-1 virus populations within and across specimens from each subject.     

African Swine Fever Virus Protein p17 Is Essential for the Progression of Viral Membrane Precursors toward Icosahedral Intermediates

The first morphological evidence of African swine fever virus (ASFV) assembly is the appearance of precursor viral membranes, thought to derive from the endoplasmic reticulum, within the assembly sites. We have shown previously that protein p54, a viral structural integral membrane protein, is essential for the generation of the viral precursor membranes. In this report, we study the role of protein p17, an abundant transmembrane protein localized at the viral internal envelope, in these processes. Using an inducible virus for this protein, we show that p17 is essential for virus viability and that its repression blocks the proteolytic processing of polyproteins pp220 and pp62. Electron microscopy analyses demonstrate that when the infection occurs under restrictive conditions, viral morphogenesis is blocked at an early stage, immediately posterior to the formation of the viral precursor membranes, indicating that protein p17 is required to allow their progression toward icosahedral particles. Thus, the absence of this protein leads to an accumulation of these precursors and to the delocalization of the major components of the capsid and core shell domains. The study of ultrathin serial sections from cells infected with BA71V or the inducible virus under permissive conditions revealed the presence of large helicoidal structures from which immature particles are produced, suggesting that these helicoidal structures represent a previously undetected viral intermediate.African swine fever virus (ASFV) (61, 72) is the only known DNA-containing arbovirus and the sole member of the Asfarviridae family (24). Infection by this virus of its natural hosts, the wild swine warthogs and bushpigs and the argasid ticks of the genus Ornithodoros, results in a mild disease, often asymptomatic, with low viremia titers, that in many cases develops into a persistent infection (3, 43, 71). In contrast, infection of domestic pigs leads to a lethal hemorrhagic fever for which the only available methods of disease control are the quarantine of the affected area and the elimination of the infected animals (51).The ASFV genome is a lineal molecule of double-stranded DNA of 170 to 190 kbp in length with convalently closed ends and terminal inverted repeats. The genome encodes more than 150 open reading frames, half of which lack any known or predictable function (16, 75).The virus particle, with an overall icosahedral shape and an average diameter of 200 nm (11), is organized in several concentric layers (6, 11, 15) containing more than 50 structural proteins (29). Intracellular particles are formed by an inner viral core, which contains the central nucleoid surrounded by a thick protein coat, referred to as core shell. This core is enwrapped by an inner lipid envelope (7, 34) on top of which the icosahedral capsid is assembled (26, 27, 31). Extracellular virions possess an additional membrane acquired during the budding from the plasma membrane (11). Both forms of the virus, intracellular and extracellular, are infective (8).The assembly of ASFV particles occurs in the cytoplasm of the infected cell, in viral factories located close to the cell nucleus (6, 13, 49). ASFV factories possess several characteristics similar to those of the cellular aggresomes (35), which are accumulations of aggregates of cellular proteins that form perinuclear inclusions (44).Current models propose that ASFV assembly begins with the modification of endoplasmic reticulum (ER) membranes, which are subsequently recruited to the viral factories and transformed into viral precursor membranes. These ER-derived viral membranes represent the precursors of the inner viral envelope and are the first morphological evidence of viral assembly (7, 60). ASFV viral membrane precursors evolve into icosahedral intermediates and icosahedral particles by the progressive assembly of the outer capsid layer at the convex face of the precursor membranes (5, 26, 27, 31) through an ATP- and calcium-dependent process (19). At the same time, the core shell is formed underneath the concave face of the viral envelope, and the viral DNA and nucleoproteins are packaged and condensed to form the innermost electron-dense nucleoid (6, 9, 12, 69). However, the assembly of the capsid and the internal envelope appears to be largely independent of the components of the core of the particle, since the absence of the viral polyprotein pp220 during assembly produces empty virus-like particles that do not contain the core (9).Comparative genome analysis suggests that ASFV shares a common origin with the members of the proposed nucleocytoplasmic large DNA viruses (NCLDVs) (40, 41). The reconstructed phylogeny of NCLDVs as well as the similitude in the structures and organizations of the genomes indicates that ASFV is more closely related to poxviruses than to other members of the NCLDVs. A consensus about the origin and nature of the envelope of the immature form of vaccinia virus (VV), the prototypical poxvirus, seems to be emerging (10, 17, 20, 54). VV assembly starts with the appearance of crescent-shaped structures within specialized regions of the cytoplasm also known as viral factories (21, 23). The crescent membranes originate from preexisting membranes derived from some specialized compartment of the ER (32, 37, 52, 53, 67), and an operative pathway from the ER to the crescent membrane has recently been described (38, 39). VV crescents apparently grow in length while maintaining the same curvature until they become closed circles, spheres in three dimensions, called immature virions (IV) (22). The uniform curvature is produced by a honeycomb lattice of protein D13L (36, 70), which attaches rapidly to the membranes so that nascent viral membranes always appear to be coated over their entirety. The D13L protein is evolutionarily related to the capsid proteins of the other members of the NCLDV group, including ASFV, but lacks the C-terminal jelly roll motif (40). This structural difference is probably related to the fact that poxviruses are the only member of this group without an icosahedral capsid; instead, the spherical D13L coat acts as a scaffold during the IV stage but is discarded in subsequent steps of morphogenesis (10, 28, 46, 66). Thus, although crescents in VV and precursors of the inner envelope in ASFV are the first morphogenetic stages discernible in the viral factories of these viruses, they seem to be different in nature. Crescents are covered by the D13L protein and are more akin to the icosahedral intermediates of ASFV assembly, whereas ASFV viral membrane precursors are more similar to the naked membranes seen when VV morphogenesis is arrested by rifampin treatment (33, 47, 48, 50) or when the expression of the D13L and A17L proteins are repressed during infection with lethal conditional VV viruses (45, 55, 56, 68, 74, 76).Although available evidence strongly supports the reticular origin of the ASFV inner envelope (7, 60), the mechanism of acquisition remains unknown, and the number of membranes present in the inner envelope is controversial. The traditional view of the inner envelope as formed by two tightly opposed membranes derived from ER collapsed cisternae (7, 59, 60) has recently been challenged by the careful examination of the width of the internal membrane of viral particles and the single outer mitochondrial membrane, carried out using chemical fixation, cryosectioning, and high-pressure freezing (34). The results suggest that the inner envelope of ASFV is a single lipid bilayer, which raises the question of how such a structure can be generated and stabilized in the precursors of the ASFV internal envelope. In the case of VV, the coat of the D13L protein has been suggested to play a key role in the stabilization of the single membrane structure of the crescent (10, 17, 36), but the ASFV capsid protein p72 is not a component of the viral membrane precursors. The identification and functional characterization of the proteins involved in the generation of these structures are essential for the understanding of the mechanisms involved in these early stages of viral assembly. For this reason, we are focusing our interest on the study of abundant structural membrane proteins that reside at the inner envelope of the viral particle. We have shown previously that one of these proteins, p54, is essential for the recruitment of ER membranes to the viral factory (59). Repression of protein p54 expression has a profound impact on virus production and leads to an early arrest in virion morphogenesis, resulting in the virtual absence of membranes in the viral factory.Protein p17, encoded by the late gene D117L in the BA71V strain, is an abundant structural protein (60, 65). Its sequence, which is highly conserved among ASFV isolates (16), does not show any significant similarity with the sequences present in the databases. Protein p17 is an integral membrane protein (18) that is predicted to insert in membranes with a Singer type I topology and has been localized in the envelope precursors as well as in both intracellular and extracellular mature particles (60), suggesting that it resides at the internal envelope, the only membranous structure of the intracellular particles.In this work, we analyze the role of protein p17 in viral assembly by means of an IPTG (isopropyl-β-d-thiogalactopyranoside)-dependent lethal conditional virus. The data presented indicate that protein p17 is essential for viral morphogenesis. The repression of this protein appears to block assembly at the level of viral precursor membranes, resulting in their accumulation at the viral factory.From the electron microscopy analysis of serial sections of viral factories at very early times during morphogenesis, we present experimental evidence that suggests that, during assembly, viral precursor membranes and core material organize into large helicoidal intermediates from which icosahedral particles emerge. The possible role of these structures during ASFV morphogenesis is discussed.     

Effects of Route of Inoculation and Viral Genetic Variation on Antibody Responses to Polyomavirus SV40 in Syrian Golden Hamsters

Genetic variants of polyomavirus SV40 are powerful agents with which to define viral effects on cells and carcinogenesis pathways. We hypothesized that differences in biologic variation among viral strains affect the process of viral infection and are reflected in antibody responses to the viral nonstructural large T-antigen (TAg) protein but not in neutralizing antibody responses against the inoculated viral particles. We analyzed the production of TAg antibody and neutralizing antibody in Syrian golden hamsters that were inoculated with SV40 viral strains by intracardiac, intravenous, or intraperitoneal routes and remained tumor free. Compared with the intraperitoneal route, intravascular (that is, intravenous, intracardiac) inoculation resulted in increased frequency of responsiveness to TAg but not in higher TAg antibody titers. The intravascular route was superior both for eliciting neutralizing antibody responses and for higher titers of those responses. Viruses with complex regulatory regions induced TAg antibody more often than did viruses with simple regulatory regions after intraperitoneal but not intravascular injections, with no differences in antibody titers. This viral genetic variation had no effect on neutralizing antibody production after intraperitoneal or intravascular inoculations or on neutralizing antibody titers achieved. These findings confirm that SV40 variants differ in their biologic properties. Route of inoculation combined with viral genetic variation significantly influence the development of serum antibodies to SV40 TAg in tumor-free hamsters. Route of inoculation—but not viral genetic variation—is an important factor in production of neutralizing antibody to SV40.Abbreviations: TAg, large T antigenPolyomavirus SV40 was discovered as an unrecognized contaminant of early poliovaccines³⁸ and was shown promptly to be an oncogenic virus.^9,15,17,18 Syrian golden hamsters are the small animal model that is susceptible to SV40 tumorigenicity.^{7,9-13,18,27,35} Since its discovery, SV40 has proven to be an outstanding tool for the discovery of mechanisms underlying carcinogenesis and for viral influences on cellular processes.^{1,3,5,19,23,30}Genetic variants of SV40 exist.^{16,20,26,28,33,34,36,37,41} This variation typically occurs in the viral regulatory region, in which some strains have duplications or rearrangements (or both) in the enhancer region, and in the C-terminal region of the large T-antigen (TAg) gene, in which nucleic acid variations may result in amino acid changes in the protein. TAg is an essential viral replication protein and the major viral oncoprotein. An important question is whether SV40 viral variants differ in their biologic properties, including in host responses to infections, as this could affect the spectrum of viral disease pathogenesis. We previously have shown that the viral regulatory region influences SV40 tumor induction in hamsters^35,40 and vertical transmission of the virus in hamsters²⁹ and that the route of inoculation influences SV40-mediated carcinogenesis.²⁷ Because TAg is not a component of the virus particle but instead is synthesized in virus-infected cells, we hypothesized that differences in antibody responses to TAg reflect biologic variation among viral strains with respect to the process of viral infection. In contrast, we expected that neutralizing antibody responses arise primarily against the injected viral particles, which represent a single serotype, and therefore are less informative about viral variation. We report here that an analysis revealed that the route of inoculation—in combination with viral genetic variation—significantly influences the development of serum antibodies to SV40 large TAg but not the titer of those antibodies in virus-exposed, tumor-free hamsters. The route of inoculation—but not viral genetic variation—influenced both the frequency of development and the titers of virus-neutralizing antibodies in the same animals.     

Acquisition of Complement Resistance through Incorporation of CD55/Decay-Accelerating Factor into Viral Particles Bearing Baculovirus GP64

A major obstacle to gene transduction by viral vectors is inactivation by human complement in vivo. One way to overcome this is to incorporate complement regulatory proteins, such as CD55/decay accelerating factor (DAF), into viral particles. Lentivirus vectors pseudotyped with the baculovirus envelope protein GP64 have been shown to acquire more potent resistance to serum inactivation and longer transgene expression than those pseudotyped with the vesicular stomatitis virus (VSV) envelope protein G. However, the molecular mechanisms underlying resistance to serum inactivation in pseudotype particles bearing the GP64 have not been precisely elucidated. In this study, we generated pseudotype and recombinant VSVs bearing the GP64. Recombinant VSVs generated in human cell lines exhibited the incorporation of human DAF in viral particles and were resistant to serum inactivation, whereas those generated in insect cells exhibited no incorporation of human DAF and were sensitive to complement inactivation. The GP64 and human DAF were detected on the detergent-resistant membrane and were coprecipitated by immunoprecipitation analysis. A pseudotype VSV bearing GP64 produced in human DAF knockdown cells reduced resistance to serum inactivation. In contrast, recombinant baculoviruses generated in insect cells expressing human DAF or carrying the human DAF gene exhibited resistance to complement inactivation. These results suggest that the incorporation of human DAF into viral particles by interacting with baculovirus GP64 is involved in the acquisition of resistance to serum inactivation.Gene therapy is a potential treatment option for genetic diseases, malignant diseases, and other acquired diseases. To this end, safe and efficient gene transfer into specific target cells is a central requirement, and a variety of nonviral and viral vector systems have been developed (6, 44). Recombinant viruses can be used for efficient gene transfer. Retroviruses, adeno-associated viruses, and lentiviruses are able to integrate foreign genes into host genomes and are suitable for gene therapeutics by virtue of their permanent expression of the therapeutic genes, whereas adenoviruses, herpesviruses, and baculoviruses can transiently express foreign genes (6, 12, 44). Pseudotype particles bearing other viral envelope proteins have been developed to improve transduction efficiency and the safety of viral vectors, including retrovirus (4, 7), lentivirus (25), vesicular stomatitis virus (VSV) (29), and baculovirus (17, 42). Pseudotype retroviruses and lentiviruses bearing the baculovirus envelope protein GP64 of Autographa californica nucleopolyhedrosis virus (AcNPV) have been shown to exhibit efficient gene transduction into a wide variety of cells with a lower cytotoxicity compared to those bearing the VSV envelope protein G (VSVG), which is commonly used for pseudotyping (18, 32, 35, 36).However, a drawback of gene transduction by viral vectors is that human sera inactivate the vectors (11, 40). Complement is a major element of the innate immune response and serves to link innate and adaptive immunity (8). Complement activation can occur via classical, lectin, and alternative pathways (2, 8). All pathways invoke several responses, such as virus opsonization, virolysis, anaphylatoxin, and chemotaxin production, as well as others (2, 8). VSV and baculovirus are inactivated by human sera via the classical pathway (1, 11). Because complement activation also induces potential damage to host cells, the complement system is tightly regulated by the complement regulatory proteins (CRPs), including CD55/decay-accelerating factor (DAF), CD46/membrane cofactor protein (MCP), and CD59 (2, 8, 15). DAF and CD46 inhibit activation of C3/C5-converting enzymes, which regulate the activation of classical and alternative pathways, whereas CD59 regulates the assembly of the membrane attack complex (2, 8, 15).Viral vectors can be manipulated to confer resistance to the complement inactivation. Human immunodeficiency virus (HIV) is known to develop resistance to human complement through the incorporation of DAF, CD46, and CD59 to the viral particles (22, 30, 31, 38). Moloney murine leukemia virus vectors produced in HT1080 cells are resistant to complement inactivation (5). Baculovirus and lentivirus vectors bearing DAF or the fusion protein between the functional domains of human DAF and the GP64 were resistant to complement inactivation (9, 13). It has been shown that lentivirus vectors pseudotyped with the GP64 are more resistant to inactivation in the sera of mice and rats (14, 32) and are capable of executing longer expression of the transgenes in nasal epithelia compared to those pseudotyped with the VSVG (35, 36). However, the precise mechanisms underlying the resistance to complement inactivation by pseudotyping of the GP64 is not known.To clarify the molecular mechanisms underlying the resistance of the viral vectors pseudotyped with the GP64 to the complement inactivation, we produced pseudotype and recombinant VSVs bearing the GP64. The recombinant VSVs carrying the gp64 gene generated in human cells but not in insect cells exhibited incorporation of human DAF on the viral particles and were resistant to the complement inactivation. Furthermore, production of the gp64 pseudotype VSV in the DAF knockdown human cells impaired serum resistance, whereas production of the gp64 recombinant VSV in the CHO cell lines stably expressing human DAF and the recombinant baculoviruses in the insect cells stably expressing human DAF or encoding the DAF gene in the genome conferred resistance to the complement inactivation. These results suggest that DAF incorporation into viral particles bearing baculovirus GP64 confers resistance to serum inactivation.     

Artesunate induces necrotic cell death in schwannoma cells

Established as a potent anti-malaria medicine, artemisinin-based drugs have been suggested to have anti-tumour activity in some cancers. Although the mechanism is poorly understood, it has been suggested that artemisinin induces apoptotic cell death. Here, we show that the artemisinin analogue artesunate (ART) effectively induces cell death in RT4 schwannoma cells and human primary schwannoma cells. Interestingly, our data indicate for first time that the cell death induced by ART is largely dependent on necroptosis. ART appears to inhibit autophagy, which may also contribute to the cell death. Our data in human schwannoma cells show that ART can be combined with the autophagy inhibitor chloroquine (CQ) to potentiate the cell death. Thus, this study suggests that artemisinin-based drugs may be used in certain tumours where cells are necroptosis competent, and the drugs may act in synergy with apoptosis inducers or autophagy inhibitors to enhance their anti-tumour activity.Artemisinin, a sesquiterpene lactone isolated from the Chinese herb Artemisia annua L., has profound activity against malaria.¹ Artemisinin contains an endoperoxide moiety that reacts with iron to produce toxic reactive oxygen species (ROS). When malaria parasite (Plasmodia) consumes iron-rich haemoglobin within its acidic food vacuole in erythrocytes, the exposure of artemisinin to haem-derived iron results in lethal ROS production that exerts fatal toxicity to the parasite.² Therefore, artemisinin, its water-soluble derivative artesunate (ART) and other analogues are potent in killing malarial parasites.^1,3Cancer cells contain substantial free iron, resulting from their higher-rate iron uptake via transferrin receptors compared with normal cells. Therefore, artemisinin-based drugs such as ART possess selective toxicity to cancer cells.^{4, 5, 6} Importantly, the pharmacokinetics and tolerance of ART as an anti-malarial drug have been well documented, with clinical studies showing excellent safety. Collectively, these properties make artemisinin-based compounds attractive drug candidates for cancer chemotherapy. Artemisinin and ART have been shown to induce cell death in multiple cancer cells, including colon, breast, ovarian, prostate,⁷ pancreatic⁸ and leukaemia⁹ cancer cells. Preliminary in vivo experiments also indicate the therapeutic potential for these drugs as anti-cancer treatments. In animal models, artemisinin or ART has shown promising results in Kaposi Sarcoma,¹⁰ pancreatic cancer¹¹ and hepatoma,¹² while compassionate use of ART in uveal melanoma patients fortifies standard chemotherapy potential for the patients.¹³ Currently, ART is on clinical trial for breast cancer treatment (ClinicalTrials.gov ID: {"type":"clinical-trial","attrs":{"text":"NCT00764036","term_id":"NCT00764036"}}NCT00764036).Programmed cell death (PCD) is one of the critical terminal paths for the cells of metazoans. Among PCD, apoptosis has been well studied and it is known that caspase activation is essential in this process.¹⁴ In addition to apoptosis, necroptosis is another form of PCD. The RIP1-RIP3 complex highlights the signals that regulate necroptosis.^{15, 16, 17} Artemisinin derivatives, mostly ART, have been suggested to lead to apoptosis via ROS production in cancer cells. Efforts have been focused on ROS-mediated mitochondrial apoptosis,^9,18,19 and DNA damage²⁰ in cancer cells. Recent data suggest that artemisinin and its derivatives may induce cell death or inhibit proliferation through diverse mechanisms in different cell types. Artemisinin or its analogues were shown to inhibit cell proliferation in multiple cancer cells by regulating cell-cycle arrest^{21, 22, 23} or inducing apoptosis.^24,25 Nevertheless, the detailed molecular mechanisms underlying artemisinin or ART-induced cell death are poorly understood, thus need to be further addressed.Neurofibromatosis 2 (NF2) is caused by the loss of NF2 gene encoding Merlin protein. NF2 gene mutations cause the low grade tumour syndrome, composed of schwannomas, meningiomas and ependymomas.²⁶ All spontaneous schwannomas, the majority of meningiomas and a third of ependymomas are caused by NF2 gene mutations. Notably, approximately 10% of intracranial tumours are schwannomas.²⁷ Interestingly, NF2 gene mutations are also found in a variety of cancers, including breast cancer and mesothelioma.^{28, 29, 30} The low grade tumours caused by NF2 gene mutations do not respond well to current cancer drugs and therapy is restricted to surgery and radiosurgery.²⁶ Therefore, there is a need for drug treatment of the diseases. Here, we show that ART sufficiently induced schwannoma cell death in both RT4 cell line and human primary cells. Importantly, we show, for the first time, that ART-induced cell death is largely dependent on necroptosis. Our data suggest that ART has great potential in schwannoma chemotherapy, especially when used in synergy with an apoptosis-inducing drug and/or an autophagy-inhibitory drug.     

Borna Disease Virus Requires Cholesterol in both Cellular Membrane and Viral Envelope for Efficient Cell Entry

Borna disease virus (BDV), the prototypic member of the family Bornaviridae within the order Mononegavirales, provides an important model for the investigation of viral persistence within the central nervous system (CNS) and of associated brain disorders. BDV is highly neurotropic and enters its target cell via receptor-mediated endocytosis, a process mediated by the virus surface glycoprotein (G), but the cellular factors and pathways determining BDV cell tropism within the CNS remain mostly unknown. Cholesterol has been shown to influence viral infections via its effects on different viral processes, including replication, budding, and cell entry. In this work, we show that cell entry, but not replication and gene expression, of BDV was drastically inhibited by depletion of cellular cholesterol levels. BDV G-mediated attachment to BDV-susceptible cells was cholesterol independent, but G localized to lipid rafts (LR) at the plasma membrane. LR structure and function critically depend on cholesterol, and hence, compromised structural integrity and function of LR caused by cholesterol depletion likely inhibited the initial stages of BDV cell internalization. Furthermore, we also show that viral-envelope cholesterol is required for BDV infectivity.Borna disease virus (BDV) is an enveloped virus with a nonsegmented negative-strand RNA genome whose organization (3′-N-p10/P-M-G-L-5′) is characteristic of mononegaviruses (6, 28, 46, 48). However, based on its unique genetics and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales (8, 28, 46, 49).BDV can infect a variety of cell types in cell culture but in vivo exhibits exquisite neurotropism and causes central nervous system (CNS) disease in different vertebrate species, which is frequently manifested in behavioral abnormalities (19, 33, 44, 53). Both host and viral factors contribute to a variable period of incubation and heterogeneity in the symptoms and pathology associated with BDV infection (14, 16, 29, 42, 44). BDV provides an important model for the investigation of both immune-mediated pathological events associated with virus-induced neurological disease and mechanisms whereby noncytolytic viruses induce neurodevelopmental and behavioral disturbances in the absence of inflammation (15, 18, 41). Moreover, serological data and molecular epidemiological studies suggest that BDV, or a BDV-like virus, can infect humans and that it might be associated with certain neuropsychiatric disorders (17, 24), which further underscores the interest in understanding the mechanisms underlying BDV persistence in the CNS and its effect on brain cell functions. The achievement of these goals will require the elucidation of the determinants of BDV cell tropism within the CNS.BDV enters its target cell via receptor-mediated endocytosis, a process in which the BDV G protein plays a central role (1, 5, 13, 14, 39). Cleavage of BDV G by the cellular protease furin generates two functional subunits: GP1 (GP_N), involved in virus interaction with a yet-unidentified cell surface receptor (1, 39), and GP2 (GP_C), which mediates a pH-dependent fusion event between viral and cellular membranes (13). However, a detailed characterization of cellular factors and pathways involved in BDV cell entry remains to be done.Besides cell surface molecules that serve as viral receptors, many other cell factors, including nonproteinaceous molecules, can influence cell entry by virus (52). In this regard, cholesterol, which plays a critical role in cellular homeostasis (55), has also been identified as a key factor required for productive infection by different viruses. Accordingly, cholesterol participates in a variety of processes in virus-infected cells, including fusion events between viral and cellular membranes (3), viral replication (23), and budding (35, 37), as well as maintenance of lipid rafts (LR) (12) as scaffold structures where the viral receptor and coreceptor associate (11, 26, 32, 36). LR are specialized microdomains within cellular membranes constituted principally of proteins, sphingolipids, and cholesterol. LR facilitate the close proximity and interaction of specific sets of proteins and contribute to different processes associated with virus multiplication (38). Cholesterol can also influence virus infection by contributing to the maintenance of the properties of the viral envelope required for virus particle infectivity (21, 54). Here, we show for the first time that cholesterol plays a critical role in BDV infection. Depletion of cellular cholesterol prior to, but not after, BDV cell entry prevented productive BDV infection, likely due to disruption of plasma membrane LR that appear to be the cell entry point for BDV. In addition, we document that cholesterol also plays an essential role in the properties of the BDV envelope required for virus particle infectivity.     

Reduction of Immune Activation with Chloroquine Therapy during Chronic HIV Infection

Increased levels of activated T cells are a hallmark of the chronic stage of human immunodeficiency virus (HIV) infection and are highly correlated with HIV disease progression. We evaluated chloroquine (CQ) as a potential therapy to reduce immune activation during HIV infection. We found that the frequency of CD38⁺ HLA-DR⁺ CD8 T cells, as well as Ki-67 expression in CD8 and CD4 T cells, was significantly reduced during CQ treatment. Our data indicate that treatment with CQ reduces systemic T-cell immune activation and, thus, that its use may be beneficial for certain groups of HIV-infected individuals.Chronic HIV infection is characterized by multifaceted systemic immune activation, including increased frequencies of activated T cells (9, 17) and increased turnover of T cells (5, 12, 18) that correlate directly with disease progression (8, 9). T-cell immune activation is also associated with lower gains in CD4 T-cell count in HIV-infected individuals even while they are on antiretroviral therapy (ART) that appears to suppress viral replication (10). Thus, therapies that reduce immune activation may be of benefit, particularly for such individuals. Three clinical studies have been conducted using hydroxychloroquine monotherapy for patients with HIV infection (6, 21, 22), and the studies showed that hydroxychloroquine-treated patients had decreased viral loads as well as decreased serum interleukin-6 (IL-6) levels, heightened levels of which are correlated with disease progression (13). However, these studies did not examine other parameters of immune activation. Chloroquine (CQ) is known to suppress immune activation by a number of mechanisms, including inhibition of intracellular toll-like receptor (TLR) signaling and inflammatory cytokine secretion (11, 19). In vitro, CQ has been shown to reduce HIV infection-induced T-cell immune activation (14). Here, we report results using samples from a clinical study of HIV-infected individuals treated with CQ monotherapy, where we examine multiple parameters of immune activation during the course of CQ treatment.     

Infection with “Escaped” Virus Variants Impairs Control of Simian Immunodeficiency Virus SIVmac239 Replication in Mamu-B*08-Positive Macaques

An understanding of the mechanism(s) by which some individuals spontaneously control human immunodeficiency virus (HIV)/simian immunodeficiency virus replication may aid vaccine design. Approximately 50% of Indian rhesus macaques that express the major histocompatibility complex (MHC) class I allele Mamu-B*08 become elite controllers after infection with simian immunodeficiency virus SIVmac239. Mamu-B*08 has a binding motif that is very similar to that of HLA-B27, a human MHC class I allele associated with the elite control of HIV, suggesting that SIVmac239-infected Mamu-B*08-positive (Mamu-B*08⁺) animals may be a good model for the elite control of HIV. The association with MHC class I alleles implicates CD8⁺ T cells and/or natural killer cells in the control of viral replication. We therefore introduced point mutations into eight Mamu-B*08-restricted CD8⁺ T-cell epitopes to investigate the contribution of epitope-specific CD8⁺ T-cell responses to the development of the control of viral replication. Ten Mamu-B*08⁺ macaques were infected with this mutant virus, 8X-SIVmac239. We compared immune responses and viral loads of these animals to those of wild-type SIVmac239-infected Mamu-B*08⁺ macaques. The five most immunodominant Mamu-B*08-restricted CD8⁺ T-cell responses were barely detectable in 8X-SIVmac239-infected animals. By 48 weeks postinfection, 2 of 10 8X-SIVmac239-infected Mamu-B*08⁺ animals controlled viral replication to <20,000 viral RNA (vRNA) copy equivalents (eq)/ml plasma, while 10 of 15 wild-type-infected Mamu-B*08⁺ animals had viral loads of <20,000 vRNA copy eq/ml (P = 0.04). Our results suggest that these epitope-specific CD8⁺ T-cell responses may play a role in establishing the control of viral replication in Mamu-B*08⁺ macaques.A few individuals spontaneously control the replication of human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) to very low levels. The precise mechanisms underlying this control are of great interest, as a clear understanding of what constitutes a successful immune response may aid in developing an AIDS vaccine. Particularly pressing questions for vaccine design include which proteins to use as immunogens, the extent to which increasing the breadth and magnitude of responses is advantageous, how immunodomination affects T-cell responses, and if biasing the immune response toward particular effector profiles is beneficial. Characterization of immune responses made by elite controllers (ECs) may reveal patterns that can then be applied to vaccine formulation and evaluation.HIV ECs are generally not infected with grossly unfit viruses (6, 42). Instead, elite control of immunodeficiency virus replication is correlated with the presence of particular major histocompatibility complex class I (MHC-I) alleles (11, 12, 18, 32, 41, 55). The association of MHC-I alleles with the control of viremia implicates CD8⁺ T cells as being mediators of this immune containment. Several lines of evidence support this hypothesis. These lines of evidence include the correlation between the appearance of CD8⁺ T-cell responses and the resolution of peak viremia during acute infection (7, 29), the finding that alleles associated with viral control restrict dominant acute-phase CD8⁺ T-cell responses (3), and the finding that responses directed against epitopes restricted by these alleles frequently select for viral escape variants (4, 27, 38). Perhaps most compelling is the observation that for a few HIV-infected individuals, the selection of escape variants by an immunodominant HLA-B27-restricted T-cell response temporally preceded substantial increases in viremia (17, 21, 53). While viruses exhibiting escape variants in epitopes restricted by protective alleles are often detectably less fit in vitro (10, 38, 43, 51), recent data have found normal, high levels of replication in vivo upon the transmission of some of these variants (15).The association of control with MHC-I alleles does not, of course, implicate solely CD8⁺ T cells. MHC-I molecules are also ligands for killer immunoglobulin receptors (KIRs), which are predominantly expressed on natural killer (NK) cells. Genetic studies of HIV-infected humans suggest a model in which individuals with particular KIR/HLA combinations are predisposed to control HIV replication more readily than those with other KIR/HLA combinations (36, 37). These data were supported by functional studies of this KIR/HLA pairing in vitro, which demonstrated an inhibition of HIV replication by such NK cells (2). The relative contributions of NK and CD8⁺ T-cell responses to control have yet to be elucidated and may be closely intertwined.Previously, the experimental depletion of circulating CD8⁺ cells from SIVmac239-infected ECs resulted in a sharp spike in viremia, which resolved as CD8⁺ cells repopulated the periphery (19). During the reestablishment of control of SIV replication, CD8⁺ T cells targeting multiple epitopes restricted by alleles associated with elite control expanded in frequency, providing strong circumstantial evidence for their role in maintaining elite control (19, 31). However, CD8 depletion antibodies used in macaques also remove NK cells, which, at least in vitro, also inhibit SIV replication (19). It was therefore difficult to make definitive conclusions regarding the separate contributions of these subsets to maintaining the control of SIV replication in vivo.Here we investigate elite control in the rhesus macaque model for AIDS. We focused on the macaque MHC-I allele most tightly associated with the control of SIVmac239, Mamu-B*08. Approximately 50% of Mamu-B*08-positive (Mamu-B*08⁺) animals infected with SIVmac239 become ECs (32). Peptides presented by Mamu-B*08 share a binding motif with peptides presented by HLA-B27. Although these two MHC-I genes are dissimilar in domains that are important for peptide binding, each molecule can bind peptides that are presented by the other molecule (33). This striking similarity suggests that the elite control of SIVmac239 in Mamu-B*08⁺ animals is a good model for the elite control of HIV.Seven SIVmac239 epitopes restricted by Mamu-B*08 accrue variation in Mamu-B*08⁺ rhesus macaques (30, 31). For an eighth Mamu-B*08-restricted epitope, which is also restricted by Mamu-B*03 (Mamu-B*03 differs from Mamu-B*08 by 2 amino acids in the α1 and α2 domains [9, 32]), escape has been documented only for SIV-infected Mamu-B*03⁺ macaques (16). Variation in these CD8⁺ T-cell epitopes accumulates with different kinetics, starting during acute infection for those targeted by high-magnitude responses.In this study, we addressed the question of whether the elite control of SIVmac239 in Mamu-B*08⁺ animals is mediated by the known high-frequency CD8⁺ T-cell responses targeting Mamu-B*08-restricted epitopes. To this end, we introduced point mutations into eight epitopes, with the goal of reducing or abrogating immune responses directed against these epitopes during acute infection. We hypothesized that Mamu-B*08⁺ macaques would be unable to control SIV replication without these Mamu-B*08-restricted T-cell responses.     

Detection of Infectious Adenoviruses in Environmental Waters by Fluorescence-Activated Cell Sorting Assay

Methods for rapid detection and quantification of infectious viruses in the environment are urgently needed for public health protection. A fluorescence-activated cell-sorting (FACS) assay was developed to detect infectious adenoviruses (Ads) based on the expression of viral protein during replication in cells. The assay was first developed using recombinant Ad serotype 5 (rAd5) with the E1A gene replaced by a green fluorescent protein (GFP) gene. Cells infected with rAd5 express GFP, which is captured and quantified by FACS. The results showed that rAd5 can be detected at concentrations of 1 to 10⁴ PFU per assay within 3 days, demonstrating a linear correlation between the viral concentration and the number of GFP-positive cells with an r² value of >0.9. Following the same concept, FACS assays using fluorescently labeled antibodies specific to the E1A and hexon proteins, respectively, were developed. Assays targeting hexon showed greater sensitivity than assays targeting E1A. The results demonstrated that as little as 1 PFU Ads was detected by FACS within 3 days based on hexon protein, with an r² value greater than 0.9 over a 4-log concentration range. Application of this method to environmental samples indicated positive detection of infectious Ads in 50% of primary sewage samples and 33% of secondary treated sewage samples, but none were found in 12 seawater samples. The infectious Ads ranged in quantity between 10 and 165 PFU/100 ml of sewage samples. The results indicate that the FACS assay is a rapid quantification tool for detecting infectious Ads in environmental samples and also represents a considerable advancement for rapid environmental monitoring of infectious viruses.Waterborne viral infection is one of the most important causes of human morbidity in the world. There are hundreds of different types of human viruses present in human sewage, which, if improperly treated, may become the source of contamination in drinking and recreational waters (6, 12, 19). Furthermore, as water scarcity intensifies in the nation, so has consideration of wastewater reuse as a valid and essential alternative for resolving water shortages (31).Currently, routine viral monitoring is not required for drinking or recreational waters, nor is it required for wastewater that is discharged into the environment. This lack of a monitoring effort is due largely to the lack of methods that can rapidly and sensitively detect infectious viruses in environmental samples. In the past 20 years, tremendous progress has been made in detection of viruses in the environment based on molecular technology (32, 33, 35). PCR and quantitative real-time PCR (qPCR) methods have improved both the speed and sensitivity of viral detection compared with detection by the traditional tissue culture method (2, 11, 17, 18). However, they provide little information on viral infectivity, which is crucial for human health risk assessment (22-24, 35). Our previous work using a real-time PCR assay to detect human adenoviruses (Ads) in sewage could not differentiate the infectious viruses in the secondary treated sewage from those killed by chlorination disinfection (15). In this research, we pursued an innovative approach to detecting infectious viruses in water using fluorescence-activated cell sorting (FACS). This method is rapid and sensitive, with an established record in microbiological research (29, 34, 39).FACS is a specialized type of flow cytometry which provides a method for counting and sorting a heterogeneous mixture of biological cells into two or more kinds, one cell at a time, based upon the specific light-scattering and fluorescent characteristics of each cell (4, 25, 34, 38). It is a useful method since it provides fast and quantitative recording of fluorescent signals from individual cells (14, 16, 34, 47). The FACS viral assay is based on the expression of viral protein inside the recipient cell during viral replication (16). Specific antibody labeled with fluorescence is bound to the target viral protein, which results in fluorescence emission from infected cells. Viral particles outside the cell will not be captured, because the size of virus is below the detection limit of flow cytometry. Therefore, detection of cells, which can be captured with fluorescently labeled viral antibody, is a definitive indication of the presence of infectious virus.This research used human Ads as the target for development of the FACS method. The rationale for this choice is as follows. (i) Ads are important human pathogens that may be transmitted by water consumption and water spray (aerosols) (26, 32). The health hazard associated with exposure to Ads has been demonstrated by epidemiological data and clinical research (1, 7, 9, 35, 40, 43). (ii) Ads are among the most prevalent human viruses identified in human sewage and are frequently detected in marine waters and the Great Lakes (17, 32, 33, 35). (iii) Ads are more resistant to UV disinfection than any other bacteria or viruses (3, 5, 10, 24, 41, 42, 44). Thus, they may survive wastewater treatment as increasing numbers of wastewater treatment facilities switch from chlorination to UV to avoid disinfection by-products. (iv) Some serotypes of Ads, including enteric Ad 40 and 41, are fastidious. They are difficult to detect by plaque assay, and a routine assay of infectivity takes 7 to 14 days (8, 20).In this study, recombinant Ad serotype 5 (rAd5) with the E1A gene (the first transcribed gene after infection) replaced by a green fluorescent protein (GFP) gene was first used to test for sensitivity and speed of the assay. Two other viral proteins were then used as targets for development of FACS assays using Ad serotype 2 (Ad2) and Ad41. This study demonstrated the feasibility, sensitivity, and reliability of the assay for detection of infectious Ads in environmental samples.     

New-Onset Diabetes Mellitus After Transplantation in a Cynomolgus Macaque (Macaca fasicularis)

A 5.5-y-old intact male cynomolgus macaque (Macaca fasicularis) presented with inappetence and weight loss 57 d after heterotopic heart and thymus transplantation while receiving an immunosuppressant regimen consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone to prevent graft rejection. A serum chemistry panel, a glycated hemoglobin test, and urinalysis performed at presentation revealed elevated blood glucose and glycated hemoglobin (HbA1c) levels (727 mg/dL and 10.1%, respectively), glucosuria, and ketonuria. Diabetes mellitus was diagnosed, and insulin therapy was initiated immediately. The macaque was weaned off the immunosuppressive therapy as his clinical condition improved and stabilized. Approximately 74 d after discontinuation of the immunosuppressants, the blood glucose normalized, and the insulin therapy was stopped. The animal''s blood glucose and HbA1c values have remained within normal limits since this time. We suspect that our macaque experienced new-onset diabetes mellitus after transplantation, a condition that is commonly observed in human transplant patients but not well described in NHP. To our knowledge, this report represents the first documented case of new-onset diabetes mellitus after transplantation in a cynomolgus macaque.Abbreviations: NODAT, new-onset diabetes mellitus after transplantationNew-onset diabetes mellitus after transplantation (NODAT, formerly known as posttransplantation diabetes mellitus) is an important consequence of solid-organ transplantation in humans.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} A variety of risk factors have been identified including increased age, sex (male prevalence), elevated pretransplant fasting plasma glucose levels, and immunosuppressive therapy.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} The relationship between calcineurin inhibitors, such as tacrolimus and cyclosporin, and the development of NODAT is widely recognized in human medicine.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} Cynomolgus macaques (Macaca fasicularis) are a commonly used NHP model in organ transplantation research. Cases of natural and induced diabetes of cynomolgus monkeys have been described in the literature;^14,43,45 however, NODAT in a macaque model of solid-organ transplantation has not been reported previously to our knowledge.