An intron endonuclease facilitates interference competition between coinfecting viruses.

Introns containing homing endonucleases are widespread in nature and have long been assumed to be selfish elements that provide no benefit to the host organism. These genetic elements are common in viruses, but whether they confer a selective advantage is unclear. In this work, we studied intron-encoded homing endonuclease gp210 in bacteriophage ΦPA3 and found that it contributes to viral competition by interfering with the replication of a coinfecting phage, ΦKZ. We show that gp210 targets a specific sequence in ΦKZ, which prevents the assembly of progeny viruses. This work demonstrates how a homing endonuclease can be deployed in interference competition among viruses and provide a relative fitness advantage. Given the ubiquity of homing endonucleases, this selective advantage likely has widespread evolutionary implications in diverse plasmid and viral competition as well as virus-host interactions.

Targeting Conserved Sequences Circumvents the Evolution of Resistance in a Viral Gene Drive against Human Cytomegalovirus.

Gene drives are genetic systems designed to efficiently spread a modification through a population. They have been designed almost exclusively in eukaryotic species, especially in insects. We recently developed a CRISPR-based gene drive system in herpesviruses that relies on similar mechanisms and could efficiently spread into a population of wild-type viruses. A common consequence of gene drives in insects is the appearance and selection of drive-resistant sequences that are no longer recognized by CRISPR-Cas9. In this study, we analyzed in cell culture experiments the evolution of resistance in a viral gene drive against human cytomegalovirus. We report that after an initial invasion of the wild-type population, a drive-resistant population is positively selected over time and outcompetes gene drive viruses. However, we show that targeting evolutionarily conserved sequences ensures that drive-resistant viruses acquire long-lasting mutations and are durably attenuated. As a consequence, and even though engineered viruses do not stably persist in the viral population, remaining viruses have a replication defect, leading to a long-term reduction of viral levels. This marks an important step toward developing effective gene drives in herpesviruses, especially for therapeutic applications. IMPORTANCE The use of defective viruses that interfere with the replication of their infectious parent after coinfecting the same cells-a therapeutic strategy known as viral interference-has recently generated a lot of interest. The CRISPR-based system that we recently reported for herpesviruses represents a novel interfering strategy that causes the conversion of wild-type viruses into new recombinant viruses and drives the native viral population to extinction. In this study, we analyzed how targeted viruses evolved resistance against the technology. Through numerical simulations and cell culture experiments with human cytomegalovirus, we showed that after the initial propagation, a resistant viral population is positively selected and outcompetes engineered viruses over time. We show, however, that targeting evolutionarily conserved sequences ensures that resistant viruses are mutated and attenuated, which leads to a long-term reduction of viral levels. This marks an important step toward the development of novel therapeutic strategies against herpesviruses.

Herpes Simplex Virus 1 Coinfection Modifies Adeno-associated Virus Genome End Recombination.

Wild-type adeno-associated virus (AAV) can only replicate in the presence of helper factors, which can be provided by coinfecting helper viruses such as adenoviruses and herpesviruses. The AAV genome consists of a linear, single-stranded DNA (ssDNA), which is converted into different molecular structures within the host cell. Using high-throughput sequencing, we found that herpes simplex virus 1 (HSV-1) coinfection leads to a shift in the type of AAV genome end recombination. In particular, open-end inverted terminal repeat (ITR) recombination was enhanced, whereas open-closed ITR recombination was reduced in the presence of HSV-1. We demonstrate that the HSV-1 protein ICP8 plays an essential role in HSV-1-mediated interference with AAV genome end recombination, indicating that the previously described ICP8-driven mechanism of HSV-1 genome recombination may be underlying the observed changes. We also provide evidence that additional factors, such as products of true late genes, are involved. Although HSV-1 coinfection significantly changed the type of AAV genome end recombination, no significant change in the amount of circular AAV genomes was identified. IMPORTANCE Adeno-associated virus (AAV)-mediated gene therapy represents one of the most promising approaches for the treatment of genetic diseases. Currently, various GMP-compatible production methods can be applied to manufacture clinical-grade vector, including methods that employ helper factors derived from herpes simplex virus 1 (HSV-1). Yet, to date, we do not fully understand how HSV-1 interacts with AAV. We observed that HSV-1 modulates AAV genome ends similarly to the genome recombination events observed during HSV-1 replication and postulate that further improvements of the HSV-1 production platform may enhance packaging of the recombinant AAV particles.

Defective Interfering Particles of Negative-Strand RNA Viruses.

Viral defective interfering particles (DIPs) were intensely studied several decades ago but research waned leaving open many critical questions. New technologies and other advances led to a resurgence in DIP studies for negative-strand RNA viruses. While DIPs have long been recognized, their exact contribution to the outcome of acute or persistent viral infections has remained elusive. Recent studies have identified defective viral genomes (DVGs) in human infections, including respiratory syncytial virus and influenza, and growing evidence indicates that DVGs influence disease severity and may contribute to viral persistence. Further, several studies have advanced our understanding of key viral and host factors that regulate DIP formation and activity. Here we review these discoveries and highlight key questions moving forward.

Kinetics of the establishment of HIV-1 viral interference and comprehensive analysis of the contribution of viral genes.

Viral interference defines the reduced susceptibility of an infected cell to reinfection. For HIV-1, both receptor-dependent and independent pathways were described. The relative importance of different receptor-independent pathways has not been addressed. We have used reporter viruses to quantify the percentage of single- and double-infected cells, as a function of the delay between the two infections. For co-infection experiments, the frequency of double infected cells was higher than expected for independent events. By delaying the second infection, this frequency progressively diminished, resulting in significant interference after 18h. Interference measured here was largely receptor-independent. By individually deleting viral genes or expressing them in isolation, we demonstrate that the viral protein Rev plays a dominant role, while other viral proteins contributes to optimal interference. Our study defines the kinetics of early HIV-1 interference, describing the transition from higher susceptibility to double-infection to viral interference, and identifies Rev as its dominant effector.

Multiple virus infections occur in individual polygyne and monogyne Solenopsis invicta ants.

Concurrent infections of Solenopsis invicta colonies with S. invicta virus 1 (SINV-1), SINV-2, and SINV-3 has been reported. However, whether individual ants were capable of supporting multiple virus infections simultaneously was not known, nor whether the social form of the colony (polygyne or monogyne) had an influence on the occurrence of multiple infection rates in individual ants. S. invicta field populations were sampled sequentially to establish whether multiple virus infections co-occurred in individual worker ants. In addition, the intra-colony virus infection rates were compared in monogyne and polygyne field colonies to determine whether social form played a role in the viral infection prevalence. All combinations of virus infection (SINV-1, SINV-2, or SINV-3 alone, SINV-1 & SINV-2, SINV-1 & SINV-3, SINV-2 & SINV-3, and SINV-1, SINV-2 & SINV-3) were detected in individual worker ants as well as queens in the field. Thus, individual S. invicta ants can be infected simultaneously with all combinations of the S. invicta viruses. Colony social form did have an influence on the intra-colony prevalence of multiple S. invicta virus infections. Polygyne colonies exhibited significantly greater intra- and inter-colony single and multiple virus infections compared with monogyne colonies.

Genetic variation and virulence of nucleopolyhedroviruses isolated worldwide from the heliothine pests Helicoverpa armigera, Helicoverpa zea, and Heliothis virescens.

To assess the diversity and relationships of baculoviruses found in insects of the heliothine pest complex, a PCR-based method was used to classify 90 samples of nucleopolyhedrovirus (NPV; Baculoviridae: Alphabaculovirus) obtained worldwide from larvae of Heliothis virescens, Helicoverpa zea, and Helicoverpa armigera. Partial nucleotide sequencing and phylogenetic analysis of three highly conserved genes (lef-8, lef-9, and polh) indicated that 67 of these samples contained isolates of the H. zea-H. armigera single nucleopolyhedrovirus (Hz/HaSNPV) species group. Eighteen of the samples contained isolates of a multiple NPV from H. armigera, HearMNPV, and five of the samples contained isolates of Autographa californica MNPV (AcMNPV). Sequencing and analysis of an additional seven loci (orf5/orf5b, hr3-orf62, orf26, orf79, orf124/orf117a, orf42, and a part of the region between hr2 and hr3) in the Hz/HearSNPV isolates further classified these viruses into two groups of HearSNPV variants mostly from India and China and a third group of HzSNPV variants. Some of the samples contained isolates of more than one virus. In bioassays of a selection of isolates against H. zea, the commercially available Gemstar® isolate of HzSNPV killed larvae faster than most other Hz/HaSNPV and HearMNPV isolates. Gemstar® and two HearMNPV isolates exhibited significantly higher LC(50)s than the Hz/HearSNPV isolates tested. This study expands significantly on what we know about the variation of heliothine NPV populations, provides novel information on the distinct groups in which these NPVs occur, and contributes to the knowledge required for improvement of heliothine baculoviruses as biological control agents.

Limited interference at the early stage of infection between two recombinant novirhabdoviruses: viral hemorrhagic septicemia virus and infectious hematopoietic necrosis virus.

The genome sequence of a hypervirulent novirhabdovirus, viral hemorrhagic septicemia virus (VHSV) French strain 23-75, was determined. Compared to the genome of the prototype Fil3 strain, a number of substitutions, deletions, and insertions were observed. Following the establishment of a plasmid-based minigenome replication assay, recombinant VHSV (rVHSV) was successfully recovered. rVHSV exhibits wild-type-like growth properties in vitro as well as in vivo in rainbow trout. The dispensable role of NV for the novirhabdovirus replication was confirmed by generating rVHSV-DeltaNV, in which the NV gene was deleted. This deletion mutant was shown to be as debilitated as that previously described for infectious hematopoietic necrosis virus (IHNV), a distantly related novirhabdovirus (S. Biacchesi, M. I. Thoulouze, M. Bearzotti, Y. X. Yu, and M. Bremont, J. Virol. 74:11247-11253, 2000). Recombinant VHSV and IHNV expressing tdTomato and GFP(max) reporter genes, respectively, were generated, demonstrating the potential of these rhabdoviruses to serve as viral vectors. Interestingly, rIHNV-GFP(max) could be recovered using the replicative complex proteins of either virus, whereas rVHSV-Tomato could be recovered only by using its own replicative complex, reflecting that the genome signal sequences of VHSV are relatively distant from those of IHNV and do not allow their cross-recognition. Moreover, the use of heterologous protein combinations underlined the importance of strong protein-protein interactions for the formation of a functional ribonucleoprotein complex. The rIHNV-GFP(max) and rVHSV-Tomato viruses were used to simultaneously coinfect cell monolayers. It was observed that up to 74% of the cell monolayer was coinfected by both viruses, demonstrating that a limited interference phenomenon exists during the early stage of primary infection, and it was not mediated by a cellular antiviral protein or by some of the viral proteins.

Expression of Nef downregulates CXCR4, the major coreceptor of human immunodeficiency virus, from the surfaces of target cells and thereby enhances resistance to superinfection.

Lentiviral Nef proteins are key factors for pathogenesis and are known to downregulate functionally important molecules, including CD4 and major histocompatibility complex class I (MHC-I), from the surfaces of infected cells. Recently, we demonstrated that Nef reduces cell surface levels of the human immunodeficiency virus type 1 (HIV-1) entry coreceptor CCR5 (N. Michel, I. Allespach, S. Venzke, O. T. Fackler, and O. T. Keppler, Curr. Biol. 15:714-723, 2005). Here, we report that Nef downregulates the second major HIV-1 coreceptor, CXCR4, from the surfaces of HIV-infected primary CD4 T lymphocytes with efficiencies comparable to those of the natural CXCR4 ligand, stromal cell-derived factor-1 alpha. Analysis of a panel of mutants of HIV-1(SF2) Nef revealed that the viral protein utilized the same signature motifs for downmodulation of CXCR4 and MHC-I, including the proline-rich motif P(73)P(76)P(79)P(82) and the acidic cluster motif E(66)E(67)E(68)E(69.) Expression of wild-type Nef, but not of specific Nef mutants, resulted in a perinuclear accumulation of the coreceptor. Remarkably, the carboxy terminus of CXCR4, which harbors the classical motifs critical for basal and ligand-induced receptor endocytosis, was dispensable for the Nef-mediated reduction of surface exposure. Functionally, the ability of Nef to simultaneously downmodulate CXCR4 and CD4 correlated with maximum-level protection of Nef-expressing target cells from fusion with cells exposing X4 HIV-1 envelopes. Furthermore, the Nef-mediated downregulation of CXCR4 alone on target T lymphocytes was sufficient to diminish cells' susceptibility to X4 HIV-1 virions at the entry step. The downregulation of chemokine coreceptors is a conserved activity of Nef to modulate infected cells, an important functional consequence of which is an enhanced resistance to HIV superinfection.

Characterization of homologous defective interfering RNA during persistent infection of Vero cells with Japanese encephalitis virus.

It has been suggested that defective interfering (DI) RNA contributes to the persistence of Japanese en-cephalitis virus (JEV). In this study, we characterized molecular and biological aspects of the DI RNA and its relation to viral persistence. We identified a homolo-gous DI virus intimately associated with JEV persis-tence in Vero cells. The production of DI RNA during undiluted serial passages of JEV coincided with the appearance of cells refractory to acute infection with JEV. We also established a Vero cell clone with a per-sistent JEV infection in which the DI RNA co-replicated efficiently at the expense of helper virus. The infectious virus yield of the clone fluctuated dur-ing its growth depending upon the amount of DI RNA accumulated in the previous replication cycle. Identifi-cation of the corresponding negative-sense RNA of the DI RNA indicated that the DI RNA functioned as a replication unit. Most of the DI RNA molecules re-tained their open reading frames despite a large dele-tion, encompassing most of the prM, the entire E, and the 5' half of the NS1 gene. Taken together, these ob-servations suggest that the generation of homologous DI RNA during successive JEV acute infections in Vero cells probably participates actively in persistent JEV infection.

Characterization of human immunodeficiency virus (HIV)-2 vector mobilization by HIV-1.

Conditionally replicating human immunodeficiency virus type 2 (crHIV-2) vectors can compete with HIV-1 for packaging in HIV-1-infected cells, indicating that the mobilization of vectors could selectively target as well as protect reservoirs susceptible to HIV-1 infection. The incorporation of HIV-1-specific antiviral transgenes in crHIV-2 vectors, although increasing the direct antiviral effect, may decrease mobilization and transmission to surrounding cells. To investigate how HIV-1-specific catalytic RNA cassettes (ribozymes) affect this balance between antiviral activity and mobilization, crHIV-2 vectors shown to display anti-HIV-1 activity were packaged by HIV-2 and used to transduce cells previously infected with HIV-1 or to transduce uninfected cells that were subsequently challenged with HIV-1. Vector mobilization was greater when HIV-1-infected cells were transduced with vector than when transduced cells were infected with HIV-1, and approximately 3-fold lower vector production was observed in cultures transduced with vectors expressing anti-HIV-1 ribozymes. Vector and antiviral effects could be transferred to new cultures by passaging supernatants to fresh cultures. No evidence of recombination with HIV-1 was observed. Vector mobilization and protection from HIV-1 infection were also demonstrated in human peripheral blood mononuclear cells. These data suggest that strategies employing vector mobilization for HIV-1 gene therapy should use vectors with maximal antiviral potency, despite resulting reductions in mobilization of the vector.

The vir gene of bacteriophage MAV1 confers resistance to phage infection on Mycoplasma arthritidis.

Lysogenization of Mycoplasma arthritidis with the MAV1 bacteriophage increases the virulence of the mycoplasma in rats. The MAV1 vir gene is one of only two constitutively transcribed phage genes in the lysogen. We show here that Vir is a lipoprotein and is located on the outer surface of the cell membrane. To investigate whether Vir is a virulence factor, the vir gene was cloned into the transposon vector Tn4001T and inserted in the genome of the nonlysogen strain 158. The virulence of the resulting transformants was no different from that of the parent strain. Interestingly, all vir-containing transformants were resistant to infection by MAV1. Vir had no effect on MAV1 adsorption. We conclude that Vir is not a virulence factor but functions to exclude superinfecting phage, possibly by blocking the injection of phage DNA into the bacterial cytoplasm.

Interference between avian endogenous ev/J 4.1 and exogenous ALV-J retroviral envelopes.

A new family of avian retroviral endogenous sequences designated ev/J or EAV-HP has been identified recently. Here an additional avian ev/J 4.1 endogenous sequence, ev/J 4.1 Rb, is reported. ev/J 4.1 Rb has the most extensive amino acid identity ever described for an endogenous envelope protein with the ALV-J avian leukosis virus. Here, we also demonstrate that ev/J 4.1 Rb functionally pseudotypes murine leukaemia virions and leads to a complete reciprocal interference with ALV-J envelopes. This is the first demonstration of such a high level of envelope interference between endogenous and exogenous avian retroviruses. Our results provide additional clues on the co-evolution of retroviral sequences among vertebrates.

Selective virus resistance conferred by expression of Borna disease virus nucleocapsid components.

Persistent viral infections can render host cells resistant to superinfection with closely related viruses by largely uncharacterized mechanisms. We present evidence for superinfection exclusion in brains of Borna disease virus (BDV)-infected rats and in persistently infected Vero cells, and we suggest that acquired resistance to BDV is due to unbalanced intracellular levels of viral nucleocapsid components. We observed that expression of BDV protein P, N, or X rendered human cells resistant to subsequent challenge with BDV but not with other RNA viruses, indicating that incorrect stoichiometry of nucleocapsid components selectively blocked the polymerase activity of incoming viruses. Vero cells containing high levels of an untranslatable BDV-N transcript remained virus susceptible, demonstrating that viral protein rather than RNA mediated resistance. Transient overexpression of BDV-P in persistently infected Vero cells was also remarkably effective against BDV, indicating that the intracellular balance of viral nucleocapsid components could serve as a target for future therapeutic antiviral strategies.

Single nucleotide insertion in the 5'-untranslated region of hepatitis C virus with clearance of the viral RNA in a liver transplant recipient during acute hepatitis B virus superinfection.

Hepatitis C virus (HCV) infection is an important etiology in patients undergoing orthotopic liver transplantation (OLT) world-wide. Antiviral therapy-related clearance of HCV RNA may occur both in patients with chronic HCV infection and in transplanted patients for HCV-related liver cirrhosis, but the role of the 5'-untranslated region (UTR) of HCV containing the internal ribosome entry site (IRES), which directs the translation of the viral open reading frame has not hitherto been evaluated. We studied the 5'-UTR in an HCV-infected recipient of a liver graft that showed spontaneous clearance of HCV RNA during an acute hepatitis B virus (HBV) superinfection. Sequencing of the 5'-UTR of HCV showed a nucleotide A insertion at position 193 of the IRES.

Characterization of the major control region of Vibrio cholerae bacteriophage K139: immunity, exclusion, and integration.

The temperate bacteriophage K139 is highly associated with pathogenic O1 Vibrio cholerae strains. The nucleotide sequence of the major control region of K139 was determined. The sequences of four (cox, cII, cI, and int) of the six deduced open reading frames and their gene order indicated that K139 is related to the P2 bacteriophage family. Two genes of the lysogenic transcript from the mapped promoter PL encode homologs to the proteins CI and Int, with deduced functions in prophage formation and maintenance. Between the cI and int genes, two additional genes were identified: orf2, which has no significant similarity to any other gene, and the formerly characterized gene glo. Further analysis revealed that Orf2 is involved in preventing superinfection. In a previous report, we described that mutations in glo cause an attenuation effect in the cholera mouse model (J. Reidl and J. J. Mekalanos, Mol. Microbiol. 18:685-701, 1995). In this report, we present strong evidence that Glo participates in phage exclusion. Glo was characterized to encode a 13.6-kDa periplasmic protein which inhibits phage infection at an early step, hence preventing reinfection of vibriophage K139 into K139 lysogenic cells. Immediately downstream of gene int, the attP site was identified. Upon analysis of the corresponding attB site within the V. cholerae chromosome, it became evident that phage K139 is integrated between the flagellin genes flaA and flaC of O1 El Tor and O139 V. cholerae lysogenic strains.

Virus promoters determine interference by defective RNAs: selective amplification of mini-RNA vectors and rescue from cDNA by a 3' copy-back ambisense rabies virus.

Typical defective interfering (DI) RNAs are more successful in the competition for viral polymerase than the parental (helper) virus, which is mostly due to an altered DI promoter composition. Rabies virus (RV) internal deletion RNAs which possess the authentic RV terminal promoters, and which therefore are transcriptionally active and can be used as vectors for foreign gene expression, are poorly propagated in RV-infected cells and do not interfere with RV replication. To allow DI-like amplification and high-level gene expression from such mini-RNA vectors, we have used an engineered 3' copy-back (ambisense) helper RV in which the strong replication promoter of the antigenome was replaced with the 50-fold-weaker genome promoter. In cells coinfected with ambisense helper virus and mini-RNAs encoding chloramphenicol acetyltransferase (CAT) and luciferase, mini-RNAs were amplified to high levels. This was correlated with interference with helper virus replication, finally resulting in a clear predominance of mini-RNAs over helper virus. However, efficient successive passaging of mini-RNAs and high-level reporter gene activity could be achieved without adding exogenous helper virus, revealing a rather moderate degree of interference not precluding substantial HV propagation. Compared to infections with recombinant RV vectors expressing CAT, the availability of abundant mini-RNA templates led to increased levels of CAT mRNA such that CAT activities were augmented up to 250-fold, while virus gene transcription was kept to a minimum. We have also exploited the finding that internal deletion model RNAs behave like DI RNAs and are selectively amplified in the presence of ambisense helper virus to demonstrate for the first time RV-supported rescue of cDNA after transfection of mini-RNA cDNAs in ambisense RV-infected cells expressing T7 RNA polymerase.

[Virus interaction in HBV and HCV super infection].

OBJECTIVE: To investigate virus interaction in HBV and HCV superinfection. METHODS: The changes of serum markers of 30 patients with HBV and HCV superinfetion were observed. Meanwhile, mutation of HBV precore region at nt 1,896 and the amount of TNF alpha and IL-6 in sera were also investigated. RESULTS: Compared with HBV or HCV single infection, positive rates of HBeAg, HBV DNA and HCV RNA in superinfectim were lower, positive rates of anti-HBe was higher, the average titers of HBsAg, anti-HBcIgG and anti-HCV were significantly lower, too. In some patients, HBsAg were seronegatively convested. Occurrence rate of HBV precore region mutation at nt1896 and levels of TNF alpha and IL-6 in sera were higher in super infection than those in single infection. CONCLUSION: Interference existed between HBV and HCV superinfection, conversion from HBeAg to anti-HBe was not only due to the suppression by HCV but also to the mutation of HBV precore region, and HCV may be a reason causing HBV precore region mutation through increasing the pressure of the body's immunosystem.

The potential of various HIV-1 mutants to inhibit the replication of wild-type virus.

We have previously demonstrated that many of gag mutants of human immunodeficiency virus type 1 (HIV-1) inhibited the replication of wild-type (wt) HIV-1. In this study, various HIV-1 mutants were systematically analyzed with respect to their ability to suppress the replication of wt HIV-1. Sixteen mutants of all eight HIV-1 genes other than gag were evaluated for their inhibitory effects. Only an env mutant designated NL-Hi efficiently interfered with the replication of wt HIV-1 in a single round of infection. The NL-Hi did not affect the late replication processes of wt virus, including transcription, translation, and assembly/release. Virions produced in the presence of the mutant Env were defective for the viral entry process in the early phase of HIV-1 replication cycle.

Coinfection by hepatitis B virus and hepatitis C virus.

Coinfection by hepatotropic viruses can occur due to the fact that hepatitis B virus (HBV) and hepatitis C virus (HCV) share similar routes of transmission. Different clinical features of liver disease can be observed in infected patients, ranging from fulminant, acute and chronic hepatitis to hepatocellular carcinoma (HCC). The relative role of the infecting viruses in determining the final clinical picture is not yet well defined. Several reports indicate that clinical and pathological severity of liver disease among coinfected patients is increased and in patients with HCC, co-occurrence of both viruses is a common event. The potential mechanism of tumour development still remains speculative, although direct and indirect roles for both HBV and HCV have been proposed. At the molecular level, reciprocal interference of virus replication has been repeatedly described and the extent of interference is influenced by the infecting HCV genotype, genotype 1 of HCV having more efficient inhibitory activity on HBV than genotype 2. Sequence similarities between an arginine-rich nucleocapsid motif of both viruses could support these clinical observations. Concerning response rates to interferon therapy, no satisfactory results have been achieved to date, although identification of effective therapeutic schemes, based on virological status of both viruses are warranted.