Zebrafish ISG15 exerts a strong antiviral activity against RNA and DNA viruses and regulates the interferon response.

ISG15, a 15-kDa interferon-induced protein that participates in antiviral defenses of mammals, is highly conserved among vertebrates. In fish, as in mammals, viral infection and interferon treatment induce isg15 expression. The two ubiquitin-like domains of ISG15 and the presence of a consensus LRLRGG sequence in the C-terminal region, which is required for the covalent conjugation to a substrate protein, are also conserved in fish. Our data demonstrate that overexpression of zebrafish ISG15 (zf-ISG15) in EPC cells is sufficient to inhibit viral infection by RNA viruses belonging to the genera Novirhabdovirus and Birnavirus and by DNA viruses of the genus Iridovirus. In coexpression experiments with IHNV proteins, we demonstrate specific ISGylation of phosphoprotein and nonvirion protein. Mutation of the glycine residues in the consensus LRLRGG motif abolishes zf-ISG15 conjugation to these proteins and the cellular protection against viral infection, thus connecting ISGylation and ISG15-dependent viral restriction. Additionally, zf-ISG15 overexpression triggers induction of the rig-I and viperin genes as well as, to a lesser extent, the IFN gene. Overall, our data demonstrate the antiviral effect of a fish ISG15 protein, revealing the conservation among vertebrates of an ISGylation mechanism likely directed against viruses. Furthermore, our findings indicate that zf-ISG15 affects the IFN system at several levels, and its study shall shed further light on the evolution of the complex regulation of the innate antiviral response in vertebrate cells.

Restricted growth of U-type infectious haematopoietic necrosis virus (IHNV) in rainbow trout cells may be linked to casein kinase II activity.

Previously, we demonstrated that a representative M genogroup type strain of infectious haematopoietic necrosis virus (IHNV) from rainbow trout grows well in rainbow trout-derived RTG-2 cells, but a U genogroup type strain from sockeye salmon has restricted growth, associated with reduced genome replication and mRNA transcription. Here, we analysed further the mechanisms for this growth restriction of U-type IHNV in RTG-2 cells, using strategies that assessed differences in viral genes, host immune regulation and phosphorylation. To determine whether the viral glycoprotein (G) or non-virion (NV) protein was responsible for the growth restriction, four recombinant IHNV viruses were generated in which the G gene of an infectious IHNV clone was replaced by the G gene of U- or M-type IHNV and the NV gene was replaced by NV of U- or M-type IHNV. There was no significant difference in the growth of these recombinants in RTG-2 cells, indicating that G and NV proteins are not major factors responsible for the differential growth of the U- and M-type strains. Poly I:C pretreatment of RTG-2 cells suppressed the growth of both U- and M-type IHNV, although the M virus continued to replicate at a reduced level. Both viruses induced type 1 interferon (IFN1) and the IFN1 stimulated gene Mx1, but the expression levels in M-infected cells were significantly higher than in U-infected cells and an inhibitor of the IFN1-inducible protein kinase PKR, 2-aminopurine (2-AP), did not affect the growth of U- or M-type IHNV in RTG-2 cells. These data did not indicate a role for the IFN1 system in the restricted growth of U-type IHNV in RTG-2 cells. Prediction of kinase-specific phosphorylation sites in the viral phosphoprotein (P) using the NetPhosK program revealed differences between U- and M-type P genes at five phosphorylation sites. Pretreatment of RTG-2 cells with a PKC inhibitor or a p38MAPK inhibitor did not affect the growth of the U- and M-type viruses. However, 100 µm of the casein kinase II (CKII) inhibitor, 5,6-dichloro-1-ß-d-ribofuranosylbenzimidazole (DRB), reduced the titre of the U type 8.3-fold at 24 h post-infection. In contrast, 100 µm of the CKII inhibitor reduced the titre of the M type only 1.3-fold at 48 h post-infection. Our data suggest that the different growth of U- and M-type IHNV in RTG-2 cells may be linked to a differential requirement for cellular protein kinases such as CKII for their growth.

Priming with tat-deleted caprine arthritis encephalitis virus (CAEV) proviral DNA or live virus protects goats from challenge with pathogenic CAEV.

We previously reported that infection of goats with caprine arthritis encephalitis virus (CAEV) tat- proviral DNA or virus results in persistent infection, since the animals seroconverted and direct virus isolation from cultures of blood-derived macrophages was positive. In this study we wanted to determine whether goats injected with CAEV tat- proviral DNA or virus were protected against challenge with the pathogenic homologous virus and to investigate whether CAEV tat- was still pathogenic. All animals injected with CAEV tat- became infected as indicated by seroconversion and virus isolation. Challenge at 8 or 9 months postinfection demonstrated protection in four of four animals injected with CAEV tat- but did not in three of three mock-inoculated challenged goats. Challenge virus was undetectable in the blood macrophages of protected animals during a period of 6 or 10 months postchallenge. In two of four protected animals, however, we were able to detect the challenge wild-type virus by reverse transcriptase PCR on RNA directly extracted from synovial membrane cells surrounding the inoculation site. This result suggests that protection was achieved without complete sterilizing immunity. Animals injected with CAEV tat- and mock challenged developed inflammatory lesions in the joints, although these lesions were not as severe as those in CAEV wild-type-injected goats. These results confirm the dispensable role of Tat in CAEV replication in vivo for the establishment of infection and pathogenesis and demonstrate in another lentivirus infection model the efficacy of live attenuated viruses to induce resistance to superinfection.

Genetic evaluation of suspected cases of transient HIV-1 infection of infants.

Detection of human immunodeficiency virus-type 1 (HIV-1) on only one or a few occasions in infants born to infected mothers has been interpreted to indicate that infection may be transient rather than persistent. Forty-two cases of suspected transient HIV-1 viremia among 1562 perinatally exposed seroreverting infants and one mother were reanalyzed. HIV-1 env sequences were not found in specimens from 20; in specimens from 6, somatic genetic analysis revealed that specimens were mistakenly attributed to an infant; and in specimens from 17, phylogenetic analysis failed to demonstrate the expected linkage between the infant's and the mother's virus. These findings argue that transient HIV-1 infection, if it exists, will only rarely be satisfactorily documented.

Requirement of caprine arthritis encephalitis virus vif gene for in vivo replication.

Replication of vif-caprine arthritis encephalitis virus (CAEV) is highly attenuated in primary goat synovial membrane cells and blood-derived macrophages compared to the wild-type (wt) virus. We investigated the requirement for CAEV Vif for in vivo replication and pathogenicity in goats by intra-articular injection of either infectious proviral DNA or viral supernatants. Wild-type CAEV DNA or virus inoculation induced persistent infection resulting in severe inflammatory arthritic lesions in the joints. We were unable to detect any sign of virus replication in vif- CAEV DNA inoculated goats, while vif- CAEV virus inoculation resulted in the seroconversion of the goats. However, virus isolation and RT-PCR analyses on blood-derived macrophage cultures remained negative throughout the experiment as well as in joint or lymphoid tissues taken at necropsy. No pathologic lesions could be observed in joint tissue sections examined at necropsy. Goats inoculated with the vif- virus demonstrated no protection against a pathogenic virus challenge. These results demonstrate that CAEV Vif is absolutely required for efficient in vivo virus replication and pathogenicity and provide additional evidence that live attenuated lentiviruses have to establish a persistent infection to induce efficient protective immunity.

The caprine arthritis encephalitis virus tat gene is dispensable for efficient viral replication in vitro and in vivo.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus closely related to visna virus and more distantly to other lentiviruses, such as human immunodeficiency virus. The genomes of visna virus and CAEV contain a tat gene encoding a protein able to weakly transactivate its own long terminal repeat, suggesting that transactivation may be a dispensable function for viral replication. Three different tat gene mutants of an infectious molecular clone of CAEV were used to study their replication after transfection or infection of primary goat synovial membrane cells and of blood-derived mononuclear cells or macrophages. Our results showed no difference between replication of the wild type and either the complete tat deletion mutant or the tat stop point mutant, whereas slower growth kinetics and lower levels of expression of the partial tat deletion mutant that of the wild type were obtained in these cells. Quantitative PCR and reverse transcription-PCR analyses of the different steps of a single replicative cycle revealed an identical pattern of retrotranscription, transcription, and viral production, whereas time course analysis demonstrated that the intracellular level of viral genomic RNA was affected by the partial tat deletion at later time points. We then compared the infectious properties of the wild-type and tat mutant viruses in vivo by direct inoculation of proviral DNAs into the joints of goats. All the animals seroconverted between 27 and 70 days postinoculation. Moreover, we were able to isolate tat mutant CAEV from blood-derived macrophages that was still able to infect synovial membrane cells in vitro. This study clearly demonstrates that the tat gene of CAEV is dispensable for viral replication in vitro and in vivo.

The vif gene is essential for efficient replication of caprine arthritis encephalitis virus in goat synovial membrane cells and affects the late steps of the virus replication cycle.

Complex retrovirus genomes contain a variable number of accessory genes, among which is the vif gene. We investigated in vitro the role of the vif gene of caprine arthritis encephalitis virus (CAEV) by studying the phenotype of five vif mutants after infection of primary goat synovial membrane (GSM) cells and blood-derived monocytes/macrophages. Any deletion introduced into the vif gene resulted in slow and low viral replication and production of virions with an infectious titer lower than that of wild-type viral particles. The wild-type phenotype could be restored by the trans expression of the vif gene in a complementation assay. Quantitative PCR and reverse transcription-PCR analyses were performed in order to determine which stage of the replicative cycle was impaired by the vif deletion. Our results demonstrated that CAEV Vif did not act at the level of reverse transcription or transcription but rather at the late stage of virus formation and/or release, as lower amounts of virus were produced after a single replicative cycle. The vif-deleted CAEV produced after 24 h of infection was still able to infect GSM cells, indicating that the vif gene is not essential for virus infectivity but is required for efficient virus production.

Peripheral blood mononuclear cells produce normal amounts of defective Vif- human immunodeficiency virus type 1 particles which are restricted for the preretrotranscription steps.

Previous studies have demonstrated the absence of viral replication of Vif- mutants in stimulated primary blood mononuclear cells (PBMC). Human immunodeficiency virus type 1 strain NDK Vif- mutants were propagated on the semipermissive CEM cell line, and the viral stock obtained was compared with the wild-type virus during a single cycle in PBMC. The Vif- virus was able to enter PBMC with the same efficiency as the wild type, as demonstrated by quantification of the strong-stop cDNA, and retrotranscription was observed for both viruses within 4 h postinfection. Using a PCR assay with an Alu-long terminal repeat pair of primers, we detected integration for both the wild-type and Vif- viruses. We then used qualitative and quantitative reverse transcription-mediated PCR techniques to study the steady-state level of intracellular and extracellular viral RNAs. All mRNA species were detected in PBMC infected with the wild-type virus or with the Vif- virus 36 h postinfection. Furthermore, quantification of viral RNA released from infected cells demonstrated similar levels of virus produced after a unique cycle of replication. However, the Vif- virus obtained after one replication cycle in PBMC was unable to initiate retrotranscription in permissive target cells. These data strongly suggest that the failure to infect target cells is due to a defect in the formation of the viral particle in PBMC.