Efficient induction of focus formation in a subclone of NIH3T3 cells by c-myc and its inhibition by serum and by growth factors.

In both experimental and spontaneous tumors, c-myc expression is often enhanced following its amplification or its rearrangement adjacent to a strong promotor/enhancer. However, c-myc by itself does not induce foci efficiently in fibroblast cultures. The effect of high levels of c-myc expression from a retroviral construct was investigated in several rodent fibroblast cell lines grown in medium containing 10% fetal calf serum or in serum-free PC-1 medium. c-myc-infected NIH3T3 clone 7 cells exhibited efficient quantitative focus formation when grown in PC-1 medium, whereas foci were not detected when grown in serum-supplemented medium. NIH3T3 clone 7 was the only cell line found to be sensitive to c-myc; other clones of NIH3T3 or other rodent fibroblast cell lines proved to be resistant to c-myc focus formation. At least two major types of morphologically distinct c-myc-induced foci were observed; the first was similar to ras-transformed foci induced in NIH3T3 and other fibroblast cell lines, and the second type was composed of adipocyte-like cells similar to NIH3T3 L1 cells. The c-myc infected cells cloned from these two types of foci expressed high levels of retrovirus-derived c-myc RNA and exhibited elevated levels of immunoreactive myc protein, as detected by immunofluorescent staining with an anti-myc polyclonal antibody. c-myc-transformed clones displayed only a limited ability to grow in soft-agar in the presence of serum and were not tumorigenic in nude mice. Focus formation by c-myc was quantitatively inhibited by the addition of interferon alpha + beta (INF alpha, beta), tumor necrosis factor alpha (TNF alpha) or transforming growth factor beta 1 (TGF beta 1) to the serum-free PC-1 medium, and, in correlation, NIH3T3 clone 7 cells produced the lowest level of endogenous TGF beta of the various cell lines tested.

Nuclear localization of poliovirus capsid polypeptide VP1 expressed as a fusion protein with SV40-VP1.

The poliovirus cDNA fragment coding for capsid polypeptide VP1 was inserted between the EcoRI and BamHI sites of SV40 DNA, generating a chimaeric gene in which the sequence of the 302 amino acids (aa) of poliovirus capsid polypeptide VP1 was placed downstream from that of the 94 N-terminal aa of SV40 capsid polypeptide VP1. The resulting defective, hybrid virus, SV40-delta 1 polio, was propagated in CV1 cells using an early SV40 mutant, am404, as a helper. Cells doubly infected by SV40-delta 1 polio and am404 expressed a 50-kDal fusion protein which was specifically immunoprecipitated by polyclonal and/or monoclonal antibodies raised against poliovirus capsids or against poliovirus polypeptide VP1. Examination of the infected cells by immunofluorescence after staining with anti-poliovirus VP1 immune sera revealed that the fusion protein was mostly located in the intra- and perinuclear space of the cells, in contrast to the exclusively intracytoplasmic location of genuine poliovirus VP1 polypeptide that was observed in poliovirus-infected cells. This suggests that the N-terminal part of the SV40-VP1 polypeptide could contain an important sequence element acting as a migration signal for the transport of proteins from the cytoplasm to the nucleus.

The S segment of the Germiston virus RNA genome can code for three proteins.

The complete sequence of the S segment of Germiston bunyavirus has been determined from plasmids containing S cDNA inserts. The S segment is 980 nucleotides long with the first 15 bases at the 3' end complementary to the first 15 bases at the 5' end. Three overlapping open reading frames (ORF) were identified in the viral complementary RNA strand. The first ORF codes for a polypeptide of 233 amino acids (Mr 26,600) which is the nucleoprotein N. The second ORF codes for a polypeptide of 109 amino acids (Mr 11,800) which corresponds to the NSS protein, also called p12. Following this ORF, in the same frame, a third ORF which could encode a polypeptide of 75 amino acids was identified. Such a polypeptide has not yet been detected in infected cells. The N and NSS proteins of Germiston virus were compared with the corresponding proteins of La Crosse, snowshoe hare, and Aino viruses, and show a high extent of homology.

The 15 amino acid residues preceding the amino terminus of the envelope protein in the yellow fever virus polyprotein precursor act as a signal peptide.

The 15 amino acids which precede the sequence of the envelope (E) protein in the yellow fever virus (YFV) polyprotein precursor have been proposed to function as a signal peptide for the E protein (P. Desprès A. Cahour, C. Wychowski, M. Girard and M. Bouloy; Ann. Inst. Pasteur/Virol., 139, 59-67, 1988). To confirm this hypothesis, recombinant SV40 genomes were constructed in which the sequence of the E protein, or that of the poliovirus VP0 capsid polypeptide were placed immediately downstream of and in frame with the sequence of the putative signal peptide, under the control of the late SV40 promoter. The E protein expressed by the hybrid virus SV-E was recognized by two neutralizing monoclonal antibodies directed against the YFV envelope protein. In this construct, the E protein was deleted of its C-terminal transmembrane zone. Therefore, as expected, the protein appeared to be efficiently transported along the exocytic pathway and excreted into the cell culture medium. In addition, when the putative signal peptide was fused in frame with poliovirus polypeptide VP0, the expressed chimeric polypeptide was targeted to the endoplasmic reticulum where it underwent glycosylation.

Hepatitis C Virus p7 membrane protein quasispecies variability in chronically infected patients treated with interferon and ribavirin, with or without amantadine.

A clinical study was carried out to compare the response rate of two groups of non-responder (NR) hepatitis C virus (HCV) genotype 1 chronically infected patients treated with interferon and ribavirin, with or without amantadine. The viral load decreased more markedly in the group treated by tritherapy including amantadine, but the response rate at the end of treatment was not significantly different between bitherapy and tritherapy. As amantadine could have an antiviral effect on the ion channel activity of the p7 HCV protein, the p7 quasispecies was characterized by cloning and sequencing. Sequence data were analyzed to determine the pattern and significance of p7 genetic heterogeneity and a possible relationship with therapy. Subtype differences were confirmed between p7 HCV genotypes 1a and 1b, and quasispecies analysis showed a reduction of genetic diversity in subtype 1a, but not 1b, during tritherapy. However, the absence of changes at numerous positions, as well as the conservative changes at other positions, indicated the high conservation of the p7 structure. Residue His-17, proposed to interact with amantadine, was fully conserved in both subtypes 1a and 1b, independently of amantadine administration. In conclusion, although the analysis of the p7 sequences revealed a selective pressure during therapy, no specific residues appeared to be linked to the effect of amantadine on viral decline. These results suggest that the potential antiviral effect of amantadine might be non-specific and related to a reduction in endosomal acidification and therefore reduced viral entry of HCV via its pH-dependent pathway.

A domain of SV40 capsid polypeptide VP1 that specifies migration into the cell nucleus.

In order to identify the determinants responsible for the nuclear migration of simian virus 40 (SV40) polypeptide VP1, the 5'-terminal portion of the SV40 VP1 gene was fused with the complete cDNA sequence of poliovirus capsid polypeptide VP1 and the hybrid gene was inserted into an SV40 vector in place of the normal SV40 VP1 gene. Deletions of various length were generated in the SV40 VP1 portion of the hybrid gene, resulting in a set of truncated genes encoding 2-40 NH2-terminal amino acids from SV40 VP1, followed by poliovirus VP1. Monkey kidney cells were infected by the deleted hybrid viruses in the presence of an early SV40 amber mutant as helper, and the subcellular localization of the fusion proteins was determined by indirect immunofluorescence using an anti-poliovirus VP1 immune serum. The presence of the first 11 NH2-terminal amino acids from SV40 VP1 was found to be sufficient to target the fusion protein to the cell nucleus. Deletions extending from the NH2- towards the COOH-terminal end of the protein were next generated. Transport of the SV40 VP1-poliovirus VP1 fusion polypeptide to the nucleus was abolished when the first eight amino acids from SV40 VP1 were deleted. Thus the sequence of the first eight NH2-terminal amino acids of SV40 VP1 appears to contain a nuclear migration signal which is sufficient to target the protein to the cell nucleus.

The intranuclear location of simian virus 40 polypeptides VP2 and VP3 depends on a specific amino acid sequence.

A cDNA fragment coding for poliovirus capsid polypeptide VP1 was inserted into a simian virus 40 (SV40) genome in the place of the SV40 VP1 gene and fused in phase to the 3' end of the VP2-VP3 genes. Simian cells were infected with the resulting hybrid virus in the presence of an early SV40 mutant used as a helper. Indirect immunofluorescence analysis of the infected cells using anti-poliovirus VP1 immune serum revealed that the SV40/poliovirus fusion protein was located inside the cell nucleus. Deletions of various lengths were generated in the SV40 VP2-VP3 portion of the hybrid gene using BAL31 nuclease. The resulting virus genomes expressed spliced fusion proteins whose intracellular location was either intranuclear or intracytoplasmic, depending on the presence or absence of VP2 amino acid residues 317 to 323 (Pro-Asn-Lys-Lys-Lys-Arg-Lys). This was confirmed by site-directed mutagenesis of the Lys residue at position 320. Modification of Lys-320 into either Thr or Asn abolished the nuclear accumulation of the fusion protein. It is concluded that at least part of the sequence of VP2 amino acids 317 to 323 allows VP2 and VP3 to remain stably located inside the cell nucleus. The proteins are most probably transported from the cell cytoplasm to the cell nucleus by interaction, with VP1 acting as a carrier.

Highly infectious plasmids carrying poliovirus cDNA are capable of replication in transfected simian cells.

We examined events leading to production of infectious poliovirus upon transfection of simian cells with plasmids carrying poliovirus cDNA and simian virus 40 transcription and replication signals. The nature of the simian virus 40 promoter upstream from the poliovirus cDNA had no influence on its infectivity. A high specific infectivity was correlated with plasmid replication, dependent on expression of T antigen either encoded by the plasmid or present in host cells (COS-1).

Involvement of endoplasmic reticulum chaperones in the folding of hepatitis C virus glycoproteins.

The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2) which interact noncovalently to form a heterodimer (E1-E2). During the folding and assembly of HCV glycoproteins, a large portion of these proteins are trapped in aggregates, reducing the efficiency of native E1-E2 complex assembly. To better understand this phenomenon and to try to increase the efficiency of HCV glycoprotein folding, endoplasmic reticulum chaperones potentially interacting with these proteins were studied. Calnexin, calreticulin, and BiP were shown to interact with E1 and E2, whereas no interaction was detected between GRP94 and HCV glycoproteins. The association of HCV glycoproteins with calnexin and calreticulin was faster than with BiP, and the kinetics of interaction with calnexin and calreticulin were very similar. However, calreticulin and BiP interacted preferentially with aggregates whereas calnexin preferentially associated with monomeric forms of HCV glycoproteins or noncovalent complexes. Tunicamycin treatment inhibited the binding of HCV glycoproteins to calnexin and calreticulin, indicating the importance of N-linked oligosaccharides for these interactions. The effect of the co-overexpression of each chaperone on the folding of HCV glycoproteins was also analyzed. However, the levels of native E1-E2 complexes were not increased. Together, our data suggest that calnexin plays a role in the productive folding of HCV glycoproteins whereas calreticulin and BiP are probably involved in a nonproductive pathway of folding.

Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites.

Processing of the hepatitis C virus (HCV) H strain polyprotein yields at least nine distinct cleavage products: NH2-C-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B-CO OH. As described in this report, site-directed mutagenesis and transient expression analyses were used to study the role of a putative serine proteinase domain, located in the N-terminal one-third of the NS3 protein, in proteolytic processing of HCV polyproteins. All four cleavages which occur C terminal to the proteinase domain (3/4A, 4A/4B, 4B/5A, and 5A/5B) were abolished by substitution of alanine for either of two predicted residues (His-1083 and Ser-1165) in the proteinase catalytic triad. However, such substitutions have no observable effect on cleavages in the structural region or at the 2/3 site. Deletion analyses suggest that the structural and NS2 regions of the polyprotein are not required for the HCV NS3 proteinase activity. NS3 proteinase-dependent cleavage sites were localized by N-terminal sequence analysis of NS4A, NS4B, NS5A, and NS5B. Sequence comparison of the residues flanking these cleavage sites for all sequenced HCV strains reveals conserved residues which may play a role in determining HCV NS3 proteinase substrate specificity. These features include an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position.

Analysis of neutralization-escape mutants selected from a mouse virulent type 1/type 2 chimeric poliovirus: identification of a type 1 poliovirus with antigenic site 1 deleted.

A chimeric type 1/type 2 poliovirus (v510), in which the antigenic site 1 (Ag1) of poliovirus type 1 (PV-1) Mahoney was replaced by the corresponding site of poliovirus type 2 (PV-2) Lansing, is known to be neurovirulent for mice and neutralized by both type 1 and type 2 monoclonal antibodies. Neutralization-escape mutants to monoclonal antibodies specifically recognizing the PV-2 sequence were obtained from v510. The nucleotide sequence and the mouse neurovirulence of mutants were determined. Amino acid substitutions obtained inside the replaced sequence, at positions 95 and 99, and outside this site, at positions 93 or 104, rendered the virus attenuated for mice. One of the escape mutants harboured a deletion of the entire substituted nonapeptide sequence in v510. This particular virus, which is a PV-1 Mahoney lacking the natural Ag1 loop, does not react with PV-2-specific monoclonal antibodies, has a ts phenotype, is heat-labile and is devoid of neurovirulence for mice.

Charged residues in the transmembrane domains of hepatitis C virus glycoproteins play a major role in the processing, subcellular localization, and assembly of these envelope proteins.

For most membrane proteins, the transmembrane domain (TMD) is more than just an anchor to the membrane. The TMDs of hepatitis C virus (HCV) envelope proteins E1 and E2 are extreme examples of the multifunctionality of such membrane-spanning sequences. Indeed, they possess a signal sequence function in their C-terminal half, play a major role in endoplasmic reticulum localization of E1 and E2, and are potentially involved in the assembly of these envelope proteins. These multiple functions are supposed to be essential for the formation of the viral envelope. As for the other viruses of the family Flaviviridae, these anchor domains are composed of two stretches of hydrophobic residues separated by a short segment containing at least one fully conserved charged residue. Replacement of these charged residues by an alanine in HCV envelope proteins led to an alteration of all of the functions performed by their TMDs, indicating that these functions are tightly linked together. These data suggest that the charged residues of the TMDs of HCV glycoproteins play a key role in the formation of the viral envelope.

Identification and site-directed mutagenesis of the primary (2A/2B) cleavage site of the hepatitis A virus polyprotein: functional impact on the infectivity of HAV RNA transcripts.

The junction between 2A and 2B proteins of the hepatitis A virus (HAV) polyprotein is processed by the virus-encoded 3C protease to liberate the precursor for capsid proteins, but details of this cleavage remain poorly defined. We identified the location of this primary cleavage by a novel approach involving expression of HAV polypeptides in eukaryotic cells via recombinant vaccinia viruses. A substrate polyprotein spanning the putative HAV 2A/2B site was fused at its C-terminus to a poliovirus VP1 reporter sequence. This substrate was cleaved efficiently in trans by protease 3C derived from another recombinant vaccinia virus expressing a 3C precursor protein. N-terminal sequencing of the 2B-poliovirus VP1 fusion product identified the site of cleavage as the Gln836/Ala837 dipeptide, 144 residues upstream of the originally predicted site. Two mutations were introduced at the P1 position of the 2A/2B site. Gln836-->Asn, and Gln836-->Arg. Asn substitution at the P1 residue reduced the efficiency of cleavage in the vaccinia expression system and resulted in a small replication focus phenotype of virus rescued from infectious HAV RNA transcripts. Arg substitution abolished cleavage and was lethal to HAV replication. In addition to identifying the site of the primary HAV polyprotein cleavage, these results shed light on the in vivo specificities of the HAV 3C protease.

Yellow fever 5' noncoding region as a potential element to improve hepatitis C virus production through modification of translational control.

The lengthy 5' noncoding region (5' NCR) of hepatitis C virus (HCV) RNA forms a highly ordered secondary structure, very conserved among different strains. It includes an internal ribosome entry site (IRES) element, responsible for the cap-independent translation initiation of HCV RNA. Similarly to the IRES of hepatitis A virus (HAV), another human hepatitis virus, HCV IRES, activity in internal initiation of translation is weak. Furthermore, both viruses exhibit a poor growth phenotype that may result at least partially from an inhibitory control of translation. To enhance HCV translation, as a preliminary step in designing constructs for improvement in viral production, we sought to evaluate a chimeric construct containing the yellow fever virus (YFV) 5' NCR fused to the initiation codon of the HCV coding sequence. YF viral RNA, as the majority of eukaryotic messenger RNAs, is translated by a ribosome scanning mechanism in a cap-dependent manner. The efficiency of translation initiation of the parental HCV construct was compared in vitro in rabbit reticulocyte lysates with that of the chimeric construct containing YFV 5' NCR. Surprisingly, the related distanced YFV 5' NCR was fivefold more active than was the wild-type HCV IRES in directing that function. Furthermore, chimeric transcripts were shown to be effective in vivo after transfection of eukaryotic cells. Taken together, these results raise the following question: why has the HCV genus evolved to the acquisition of an IRES element within its 5' NCR among the Flaviviridae family?

A retention signal necessary and sufficient for endoplasmic reticulum localization maps to the transmembrane domain of hepatitis C virus glycoprotein E2.

The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2). These glycoproteins interact to formin a noncovalent heterodimeric complex which is retained in the endoplasmic reticulum (ER). To identify whether E1 and/or E2 contains an ER-targeting signal potentially involved in ER retention of the E1-E2 complex, these proteins were expressed alone and their intracellular localization was studied. Due to misfolding of E1 in the absence of E2, no conclusion on the localization of its native form could be drawn from the expression of E1 alone. E2 expressed in the absence of E1 was shown to be retained in the ER similarly to E1-E2 complex. Chimeric proteins in which E2 domains were exchanged with corresponding domains of a protein normally transported to the plasma membrane (CD4) were constructed to identify the sequence responsible for its ER retention. The transmembrane domain (TMD) of E2 (C-terminal 29 amino acids) was shown to be sufficient for retention of the ectodomain of CD4 in the ER compartment. Replacement of the E2 TMD by the anchor signal of CD4 or a glycosyl phosphatidylinositol (GPI) moiety led to its expression on the cell surface. In addition, replacement of the E2 TMD by the anchor signal of CD4 or a GPI moiety abolished the formation of E1-E2 complexes. Together, these results suggest that, besides having a role as a membrane anchor, the TMD of E2 is involved in both complex formation and intracellular localization.

Processing of the E1 glycoprotein of hepatitis C virus expressed in mammalian cells.

The structural part of the hepatitis C virus (HCV) genome encodes a capsid protein, C and two envelope glycoproteins, E1 and E2, released from the virus polyprotein precursor by signalase(s) cleavage(s). The processing of E1 was investigated by infecting simian cells with recombinant vaccinia viruses expressing parts of the HCV structural proteins. When the predicted E1 sequence was expressed alone (amino acid residues 174-370 of the polyprotein) or with the capsid protein gene (residues 1-370). it showed an apparent molecular mass of 35 kDa as measured by SDS-PAGE analysis. However, when E1 was expressed as part of a truncated C-E1-truncated E2 polypeptide (residues 132-383), the processed E1 product had the expected apparent molecular mass of 31 kDa, suggesting that flanking sequences are necessary for the generation of the mature 31 kDa El form. The N-terminal sequence of the two E1 forms was found to be the same. Analysis of the glycosylation pattern showed that, in both species, only four of the five potential N-linked glycosylation sites were recognized, indicating that glycosylation was not involved in the molecular mass difference. We showed that expression of E1 with or without the hydrophobic stretch of amino acids residues 371-383, defined as the E2 signal sequence, may be responsible for the difference in electrophoretic mobility of the two E1 species. In vitro translation assays and site-directed mutagenesis experiments suggest that this sequence remains part of the 31 kDa E1 mature protein.

A poliovirus type 1 neutralization epitope is located within amino acid residues 93 to 104 of viral capsid polypeptide VP1.

Using nuclease Bal31, deletions were generated within the poliovirus type 1 cDNA sequences, coding for capsid polypeptide VP1, within plasmid pCW119. The fusion proteins expressed in Escherichia coli by the deleted plasmids reacted with rabbit immune sera directed against poliovirus capsid polypeptide VP1 (alpha VP1 antibodies). They also reacted with a poliovirus type 1 neutralizing monoclonal antibody C3, but reactivity was lost when the deletion extended up to VP1 amino acids 90-104. Computer analysis of the protein revealed a high local density of hydrophilic amino acid residues in the region of VP1 amino acids 93-103. A peptide representing the sequence of this region was chemically synthesized. Once coupled to keyhole limpet hemocyanin, this peptide was specifically immunoprecipitated by C3 antibodies. The peptide also inhibited the neutralization of poliovirus type 1 by C3 antibodies. We thus conclude that the neutralization epitope recognized by C3 is located within the region of amino acids 93-104 of capsid polypeptide VP1.

Engineering a poliovirus type 2 antigenic site on a type 1 capsid results in a chimaeric virus which is neurovirulent for mice.

Poliovirus type 2 (PV-2) Lansing strain produces a fatal paralytic disease in mice after intracerebral injection, whereas poliovirus type 1 (PV-1) Mahoney strain causes disease only in primates. Atomic models derived from the three-dimensional crystal structure of the PV-1 Mahoney strain have been used to locate three antigenic sites on the surface of the virion. We report here the construction of type 1-type 2 chimaeric polioviruses in which antigenic site 1 from the PV-1 Mahoney strain was substituted by that of the PV-2 Lansing strain by nucleotide cassette exchange in a cloned PV-1 cDNA molecule. These chimaeras proved to have mosaic capsids with composite type 1 and type 2 antigenicity, and induced a neutralizing response against both PV-1 and PV-2 when injected into rabbits. Moreover, a six-amino-acid change in PV-1 antigenic site 1 was shown to be responsible for a remarkable host-range mutation in so far as one of the two type 1-type 2 chimaera was highly neurovirulent for mice.

Construction of a chimaeric type 1/type 2 poliovirus by genetic recombination.

A chimaeric poliovirus carrying a type-2-specific neutralization epitope on a type 1 capsid was created by site-directed mutagenesis of the Mahoney strain of poliovirus type 1. An EcoRV and a HindIII restriction sites were first constructed in the cDNA of poliovirus type 1 at nucleotide positions 2756 and 2786, respectively, i.e. on either side of the sequence encoding neutralization epitope C3 (VP1 amino acids 93-103), which is part of neutralization site NImI. The cDNA sequence framed by the two sites was next taken out and replaced by custom-made oligonucleotides encoding the equivalent region of VP1 from the Lansing strain of poliovirus type 2. The DNA from the plasmid carrying such a hybrid construct was transfected onto CV1 cells generating a chimaeric virus, v510. Neutralization of v510 with a panel of monoclonal antibodies showed that v510 has lost the poliovirus type 1 C3 epitope but acquired a new, poliovirus type-2-specific neutralization epitope. Preliminary results indicate that v510 also shows neurovirulence for mice, which is a specific trait of the Lansing strain of poliovirus type 2.