Mutations in the nonstructural region 5B of hepatitis C virus genotype 1b: their relation to viral load, response to interferon, and the nonstructural region 5A

The nonstructural 5B (NS5B) protein of hepatitis C virus possesses RNA-dependent RNA polymerase activity and plays an essential role in viral replication. The mutations in NS5B were determined and the correlation with viral load and response to interferon (IFN) were assessed. The entire NS5B region in 33 patients and its thumb domain in 62 patients was sequenced. The number of amino acid substitutions in the NS5B protein, that in thumb domain and the substitution at aa 389 was correlated with viral load and the response to IFN. Multivariate analysis selected only mutation in IFN sensitivity determining region (ISDR) as a factor associated with the viral load and response to IFN. The number of substitutions in the thumb domain and the substitution at aa 389 correlated with the number of substitutions in the ISDR. These results suggest that mutations in NS5B, especially in the thumb domain and at aa 389, have an important effect on viral load and the response to IFN, although they were dependent on mutations in ISDR. Further studies on the relationship between NS5B and NS5A (ISDR) are necessary to elucidate the mechanism of the correlation with viral load and the response to IFN.     

The influenza C virus NS gene: evidence for a spliced mRNA and a second NS gene product (NS2 protein)

It has previously been shown that the shortest RNA (RNA 7) of influenza C viruses codes for a nonstructural (NSI) protein (Nakada et al. (1985) J. Virol. 56, 221-226). Experiments reported here indicate that RNA 7 also directs the synthesis of a second nonstructural protein via a spliced mRNA. The amino terminal 62 codons of this NS2 protein appear to be shared with the NS1 protein and the carboxyl terminal 59 amino acids are unique (derived from a +1 open reading frame in the mRNA). Although the size of the C virus NS2 protein is comparable to that of the A and B virus NS2 proteins, the overall arrangement of the C virus NS gene is quite different from that of the A and B virus NS genes. The second (+1) open reading frame of the C virus NS gene is completely overlapped by that of the NS1 protein, whereas the second (+1) open reading frame of the A and B virus NS genes extends to the 3' end of the RNA and only partially (or in some strains not at all) overlaps the NS1 open reading frame.     

A novel luciferase and GFP dual reporter virus for rapid and convenient evaluation of hepatitis C virus replication

Herein, we describe the development of a monocistronic dual reporter virus for monitoring hepatitis C virus (HCV) replication. The recombinant construct encodes for the humanized Renilla luciferase (hRLuc) reporter gene inserted upstream of the viral open reading frame and a green fluorescent protein (GFP) gene inserted into the C-terminus of non-structural protein 5A (NS5A) of the JFH1 viral genome. The viral RNA replicated efficiently in transfected cells and infectious virions could be produced without obvious attenuation of viral replication. The viral titer of the dual reporter virus was comparable to that of single reporter viruses. The expression levels of these two reporter genes correlated well with HCV replication in the presence or absence of antiviral agents. Moreover, because of the direct visibility of GFP fluorescence and the correlation between GFP positive cell numbers and hRLuc activity, the optimal time for measuring hRLuc activity was determined. This novel infectious system is a time saving and cost effective method for studying the interaction between viruses and host cells as well as for screening anti-HCV drugs.     

Nonstructural protein 5A does not contribute to the resistance of hepatitis C virus replication to interferon alpha in cell culture

The hepatitis C virus (HCV) subgenomic replicon system was used to study a possible involvement of nonstructural protein 5A (NS5A) in the mechanisms of HCV resistance to interferon alpha (IFN-alpha). A series of chimeric HCV replicons was constructed. In these replicons, the NS5A gene in the backbone of the Con1 replicon was swapped by corresponding fragments obtained from four IFN-alpha responder and four IFN-alpha nonresponder patients that had been infected with the same HCV AD78 strain. Experiments with transfected Huh7 cells did not reveal significant differences in sensitivity of HCV RNA replication to IFN-alpha in cell clones, bearing chimeric Con1/AD78 replicons with NS5A sequences from IFN responders and nonresponders. Thus, these data provide no evidence that the NS5A protein contributes to the resistance of HCV replication to IFN-alpha.     

N-terminal region of the PB1-F2 protein is responsible for increased expression of influenza A viral protein PB1

Influenza A virus (IAV) PB1-F2 protein is encoded by an alternative reading frame (+1) within the PB1 gene. PB1-F2 has been shown to contribute to the pathogenesis of influenza virus infection as well as to the secondary bacterial infection. More recently has been shown that PB1-F2 protein may regulate a viral RNA (vRNA) polymerase activity by the interaction with PB1 protein. We proved that PB1-F2 protein increased the level of expression of PB1 protein and vRNA in the infected cells. Moreover, we demonstrated that a higher level of vRNA expression resulted in the increase of expression of multiple viral proteins, including NP, M1, and NS1. Finally, we used plasmids expressing N-terminal (1-50 aa) or C-terminal (51-87 aa) region of the PB1-F2 molecule for transfection of MDCK cells co-infected with influenza A/Puerto Rico/8/34 (H1N1) virus deficient in the PB1-F2 protein expression (PR8ΔPB1-F2). These experiments clearly showed that N-terminal region of PB1-F2 protein was responsible for the increase in PB1 protein expression. C-terminal region of PB1-F2 protein had no effect. Thus, we have identified the important function for N-terminal region of PB1-F2 protein.     

Transient resistance of influenza virus to interferon action attributed to random multiple packaging and activity of NS genes.

Interferon (IFN) action survival curves for an avian influenza virus (AIV) in chicken or quail cells showed that 40-60% of the virions in a stock of virus were highly sensitive to the inhibitory effects of chicken IFN-alpha (ChIFN-alpha), whereas the rest were up to 100 times less sensitive. This greater resistance to IFN was transient, that is, was not a stable characteristic, in that virus stocks grown from plaques that formed in the presence of 50-800 U/ml IFN gave rise to virus populations that contained both sensitive and resistant virions. If AIV was serially passaged several times in the presence of IFN, the proportion of transiently IFN-resistant virus was greater. We propose a model to account for this transient resistance of AIV to IFN action based on the reported inactivation of the dsRNA-dependent protein kinase (PKR) and its activator dsRNA by the NS1 protein of influenza virus and also on the increase in the survival of AIV in IFN-treated cells exposed to 2-aminopurine, a known inhibitor of PKR. We suggest that IFN-resistant AIV is generated from a random packaging event that results in virions that contain two or more copies of RNA segment 8, the gene segment that encodes the NS1 protein of AIV, and that these virions will produce correspondingly elevated levels of NS1. The experimental data fit well to theoretical curves based on this model and constructed from the fraction of virus in the population expected by chance to contain one, two, or three copies of the NS gene when packaging an average of 12 influenza gene segments that include the 8 segments essential for infectivity.     

The hepatitis C virus NS5A protein and response to interferon α: mutational analyses in patients with chronic HCV genotype 3a infection from India

The hepatitis C virus (HCV) non-structural (NS)5A protein is linked to interferon α resistance in vitro and higher numbers of NS5A amino acid (aa) variations in HCV 1a/b isolates are associated with virologic response to interferon α-based therapy in vivo. Here, we aimed to study NS5A aa variations in Indian patients undergoing interferon α/ribavirin treatment infected with HCV 3a. The NS5A region [aa 2194–2401, comprising interferon sensitivity determining region, protein kinase resource (PKR) binding domain, V3 region] was sequenced from pre-treatment sera of 24 patients with HCV 3a infection. Mean number and physicochemical properties of aa variations (conserved vs. non-conserved) were assessed. Additionally, published NS5A sequences [NS5A region (n = 61), PKR binding domain (n = 111)] of characterized HCV 3a isolates were analyzed. The mean number of NS5A aa variations was not correlated with treatment response in our cohort. When all available NS5A sequences were included, a higher number of non-conserved aa variations within PKR binding domain and an extended V3 region of NS5A was associated with virologic response (P = 0.004 and 0.05, respectively). Mutational analyses of a large number of NS5A sequences suggest, that a higher number of non-conserved aa variations within the PKR binding domain and the extended V3 region is correlated with virologic response in HCV 3a infected patients. Ankur Goyal and Wolf P. Hofmann contributed equally to this work.     

In vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication

Molecular clones of infectious bronchitis virus (IBV), derived from the Vero cell adapted Beaudette strain, were constructed, using an in vitro assembly method. In vitro transcribed RNA from a cDNA template that had been constructed from seven cDNA fragments, encompassing the entire genome of IBV, was electroporated into BHK-21 cells. The cells were overlaid onto the susceptible Vero cells and viable virus was recovered from the molecular clone. The molecularly cloned IBV (MIBV) demonstrated growth kinetics, and plaque size and morphology that resembled the parental Beaudette strain IBV. The recombinant virus was further manipulated to express enhanced green fluorescent protein (EGFP) by replacing an open reading frame (ORF) of the group-specific gene, ORF 5a, with the EGFP ORF. The rescued recombinant virus, expressing EGFP (GIBV), replicated to lower viral titers and formed smaller plaques compared to the parental virus and the MIBV. After six passages of GIBV, a minority of plaques were observed that had reverted to the larger plaque size and virus from these plaques no longer expressed EGFP. Direct sequencing of RT-PCR products derived from cells infected with the plaque-purified virus, which had lost expression of EGFP, confirmed loss of the EGFP ORF. The loss of EGFP expression (Delta5a IBV) was also accompanied by reversion to growth kinetics resembling the standard virus and intact recombinant virus. This study demonstrates that the 5a ORF is not essential for viral multiplication in Vero cells.     

Structural basis for ubiquitin-like ISG 15 protein binding to the NS1 protein of influenza B virus: a protein-protein interaction function that is not shared by the corresponding N-terminal domain of the NS1 protein of influenza A virus

The N-terminal domains of the NS1 protein of influenza B virus (NS1B protein) and the NS1 protein of influenza A virus (NS1A protein) share one function: binding double-stranded RNA (dsRNA). Here we show that the N-terminal domain of the NS1B protein possesses an additional function that is not shared by its NS1A counterpart: binding the ubiquitin-like ISG15 protein that is induced by influenza B virus infection. Homology modeling predicts that the dimeric six-helical N-terminal domain of the NS1B protein differs from its NS1A protein counterpart in containing large loops between helices 1 and 2 (loops 1 and 1') and between helices 2 and 3 (loops 2 and 2'). Mutagenesis establishes that residues located in loop 1/1' together with residues located in polypeptide segment 94-103 form the ISG15 protein-binding site of NS1B protein. Loop 1/1' is not required for dsRNA binding, which instead requires arginine residues R50, R53, R50', and R53' located in antiparallel helices 1 and 1'. Further, we demonstrate that the binding sites for RNA and protein are independent of each other. In particular, ISG15 and dsRNA can bind simultaneously; the binding of the ISG15 protein does not have a detectable effect on the binding of dsRNA, and vice versa.     

Interferon pretreatment enhances the sensitivity of Vero cells to Clostridium perfringens type A enterotoxin

Treatment of Vero (African green monkey kidney) cells with interferon (IFN) before the addition of Clostridium perfringens type A enterotoxin (CPE) significantly increased the sensitivity of these cells to CPE. IFN pretreatment caused a subsequent two- to four-fold increase in CPE-induced membrane permeability alterations and also decreased the time of CPE treatment required before the onset of permeability alterations and morphologic damage. Enhancement of CPE activity was dependent on the amount of IFN added during pretreatment and on the duration of IFN pretreatment incubations. Potentiation of CPE activity was observed following pretreatment of Vero cells with natural human IFN-alpha or IFN-gamma or Roferon recombinant human IFN-alpha. However, pretreatment with mouse IFN did not affect CPE activity. IFN pretreatment did not grossly enlarge the size of the functional hole produced in plasma membranes by CPE. IFN pretreatment of Vero cells slightly increased CPE specific binding, but this effect occurred kinetically after the enhancement of CPE toxicity. These results suggest that IFN pretreatment enhances the action of CPE on IFN pretreated Vero cells by increasing the sensitivity of these cells to the action of CPE rather than by increasing CPE specific binding or by directly activating the CPE molecule. Additional studies are required to further clarify the mechanism by which IFN sensitized Vero cells to CPE.     

Therapy-induced antibodies to interferon-alpha 2a recognise its receptor-binding site.

Fifty-eight patients with chronic hepatitis B (HB) or C (HC) were treated with recombinant human interferon (rIFN)-alpha 2 and their sera were assayed for antibodies to rIFN-alpha 2c. Twelve of these patients produced low titres and two high titres of the antibodies. We localized the region which was recognised by the high-titre therapy-induced antibodies on the IFN molecule by testing the antibodies with a set of murine monoclonal antibodies (MoAbs) to IFN-alpha 2 in a competitive radioimmune assay (RIA). Only MoAbs with epitopes located in the amino-terminal portion of IFN-alpha 2 could inhibit the binding of radiolabelled IFN-alpha 2 by patients' sera. Our data indicate that the therapy-induced antibodies were directed to the receptor-binding domain of IFN-alpha 2 formed by amino acids (aa) 30-53. In accordance with this observation, human anti-IFN sera inhibited the binding of rIFN-alpha 2 to human cells.     

VSV replication in neurons is inhibited by type I IFN at multiple stages of infection

Vesicular stomatitis virus (VSV) is a rhabdovirus which causes acute encephalitis in mice after intranasal infection. Because type I interferon (IFN) has been shown to be a potent inhibitor of VSV, we investigated the role of type I IFN in viral replication in neurons in culture. Pre-treatment of NB41A3 neuroblastoma cells or primary neuron cultures with IFN-beta or IFN-alpha strongly inhibits virus replication, with 1000-fold inhibition of infectious virus release occurring at 7 h post-infection, and maximum inhibition of 14,000-fold occurring at 14 h. Type I IFN inhibited both viral protein and RNA synthesis, but not enough to account for the inhibition of infectious virus yield. The influenza virus protein NS1 binds dsRNA and antagonizes induction of PKR activity, an IFN-inducible antiviral protein which phosphorylates and inactivates the elongation factor eIF-2alpha, resulting in cessation of translation. In NS1-expressing neuroblastoma cells, VSV replication was inhibited by IFN-beta as well as in control NB41A3 cells, and eIF-2alpha phosphorylation was blocked, suggesting that PKR activity was not involved in inhibition of viral protein synthesis. Similarly, inhibition of VSV by IFN-beta was not affected by addition of inhibitors of nitric oxide synthase, indicating that IFN-beta activity is not mediated by nitric oxide or superoxide. This contrasts with the essential role of NOS-1 in inhibition of VSV replication when neurons are treated with IFN-gamma. Analysis of cell culture supernatants revealed suppression of release of VSV particles from both NB41A3 cells and primary neurons treated with IFN. The inhibition of virion release closely matched the overall suppression of infectious VSV particle release, suggesting that type I IFN plays a role in inhibition of VSV assembly.     

Influenza virus NS vectors expressing the mycobacterium tuberculosis ESAT-6 protein induce CD4+ Th1 immune response and protect animals against tuberculosis challenge

Infection with Mycobacterium tuberculosis remains a major cause of morbidity and mortality all over the world. Since the effectiveness of the only available tuberculosis vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), is suboptimal, there is a strong demand to develop new tuberculosis vaccines. As tuberculosis is an airborne disease, the intranasal route of vaccination might be preferable. Live influenza virus vaccines might be considered as potential vectors for mucosal immunization against various viral or bacterial pathogens, including M. tuberculosis. We generated several subtypes of attenuated recombinant influenza A viruses expressing the 6-kDa early secretory antigenic target protein (ESAT-6) of M. tuberculosis from the NS1 reading frame. We were able to demonstrate the potency of influenza virus NS vectors to induce an M. tuberculosis-specific Th1 immune response in mice. Moreover, intranasal immunization of mice and guinea pigs with such vectors induced protection against mycobacterial challenge, similar to that induced by BCG vaccination.     

Direct random insertion of an influenza virus immunologic determinant into the NS1 glycoprotein of a vaccine flavivirus

A live chimeric vaccine virus against Japanese encephalitis (JE), ChimeriVax-JE, was used to define methods for optimal, random insertion of foreign immunologic determinants into flavivirus glycoproteins. The conserved M2e peptide of influenza A virus was randomly inserted into the yellow fever-specific NS1 glycoprotein of ChimeriVax-JE. A technique combining plaque purification with immunostaining yielded a recombinant virus that stably expressed M2e at NS1-236 site. The site was found permissive for other inserts. The insertion inhibited NS1 dimerization in vitro, which had no significant effect on virus replication in vitro and immunogenicity in vivo. Two different NS1-specific monoclonal antibodies and a polyclonal antibody efficiently recognized only the NS1 protein dimer, but not monomer. Adaptation of the virus to Vero cells resulted in two amino acid changes upstream from the insert which restored NS1 dimerization. Immunized mice developed high-titer M2e-specific antibodies predominantly of the IgG2A isotype indicative of a Th1-biased response.     

Interferon antagonist function of Japanese encephalitis virus NS4A and its interaction with DEAD-box RNA helicase DDX42

The interferon (IFN) antagonists of Japanese encephalitis virus (JEV) proteins contribute to the JE pathogenesis. Most flavivirus non-structural (NS) proteins correlate with virus-induced inflammation and immune escape. NS4A proteins of West Nile virus and dengue type 2 virus have been demonstrated to inhibit IFN signaling. In this study, JEV NS4A without the C-terminal 2K domain has been demonstrated to partially block activation of an IFN-stimulated response element (ISRE)-based cis-reporter by IFN-α/β. In addition, JEV NS4A significantly inhibited the phosphorylation levels of STAT1 and STAT2, but not TYK2 in the IFN-treated cells. Moreover, the N-terminus of a RNA helicase DDX42 protein identified using a phage display human brain cDNA library have been demonstrated to specifically bind to JEV NS4A in vitro using a co-immunoprecipitation assay. The interaction between JEV NS4A and RNA helicase DDX42 showed partial co-localization in human medulloblastoma TE-671 cells by confocal microscopy. Importantly, the expression of N-terminal DDX42 is able to overcome JEV-induced antagonism of IFN responses. Therefore, these results show that JEV NS4A without the C-terminal 2K domain is associated with modulation of the IFN response and the interaction of JEV NS4A with RNA helicase DDX42 could be important for JE pathogenesis.