- Volume 75, Issue 6, 1994

A quantal assay, based on syncytium formation in the human T cell leukaemia-derived C8166 cell line, was used to determine the kinetics of human immunodeficiency virus type 1 (HIV-1) strain IIIB neutralization. Three rat monoclonal antibodies (MAbs) were used, under physiological conditions of temperature and antibody concentration. MAb ICR39·3b (IgG2b) neutralized virus with no lag period while the other two MAbs, ICR39·13g (IgG2b) and ICR41· 1i (IgG2a), neutralized with lag periods of 5 min and 15 min respectively. It was calculated that the latter two MAbs mediated neutralization by about two and three molecules of IgG per virion respectively. The highest neutralization rate constant (for MAb ICR 41· li) was over 300-fold less than that of MAbs specific for the haemagglutinin of the enveloped influenza virus type A and for poliovirus type 1.

We have analysed the capacity of the trophoblast-derived malignant cell lines BeWo, JAR and JEG-3, and primary cultures of highly purified trophoblast cells to support the basal and Tat-mediated trans-activation-enhanced transcriptional activity of two distinct human immunodeficiency virus type 1 (HIV-1) isolates. Kinetic studies based on expression of long terminal repeat (LTR)-chloramphenicol acetyltransferase (CAT) constructs revealed that LTRs of both the prototype strain 3B and the highly cytopathic Zairean variant NDK were activated significantly in all target cells. Overall, the strongest activation was observed in primary trophoblasts. A novel modification of quantitative PCR was used to normalize LTR expression for transfection efficiency, enabling the calculation of specific expression rates in terms of µU CAT enzyme per fmol of transfected DNA. Using the latter criterion we determined that LTRs of both viruses were activated in decreasing order from trophoblasts to JAR, JEG-3 and BeWo cells; furthermore, the expression of HIV-1 3B LTR always significantly surpassed that of HIV-1 NDK. The effects of trans-activation on either of the LTRs, when assayed in cotransfection assays with various amounts of HIV-1 NDK-Tat expression vector, increased in a dose-dependent fashion and were comparable in a particular neoplastic cell line. Furthermore, the cell-specific LTR activity patterns did not correspond to the abundance of transcription factors binding specifically to the viral NFκB and SP1 motifs. Unlike SP1-binding proteins which were relatively abundant, substantially smaller amounts of proteins with NFκB specificity were found in all cells. Despite this apparent deficit in NFκB activity, trophoblasts supported a high basal activity of both LTRs. These data indicate that an insufficiency of basal or Tat-trans-activated LTR activity cannot account for the low level of HIV-1 replication in this important cell type.

Sequential overlapping Gag protein-derived oligopeptides of human immunodeficiency virus type 1 (HIV-1) 22 to 24 amino acids long, were synthesized and tested in vitro for antiviral activity. Two synthetic peptides, one derived from the matrix protein p17 (NPGLLETSEGCRQ, amino acids 47 to 59) and one located in the capsid protein p24 (PAATLEEMMTA, amino acids 339 to 349) inhibited the production of infectious virus when added to HIV-l-infected cultures when used in the range of 20 to 200 μg/ml. As shown by thin section electron microscopy, peptide treatment resulted in the release of immature, deformed virus particles suggesting that the two peptides interfered with assembly and maturation. Other Gag protein-derived oligopeptides had little or no influence on virus production. To characterize further the functionally active regions we synthesized peptide derivatives with three consecutive amino acids substituted by alanine; they did not cause inhibition. Therefore the regions responsible for inhibition were located between amino acids 50 to 61 in pl7, and 342 to 350 in p24. These observations might lead to the development of a new antiviral strategy affecting the late stage of virus replication.

Erythroid progenitor cells are the main target for B19 parvovirus infection. The UT7 cell line demonstrates a marked erythroid differentiation on induction by erythropoietin (EPO) (UT7/EPO cells) and therefore appears to be a potential target for B19 parvovirus. We aimed to evaluate the presence and localization of B19 nucleic acids in UT7/EPO cells by in situ hybridization.

Three digoxigenin-labelled probes were used: two recognized specifically the non-structural region of the B19 genome and one probe was structural region- specific. In our experiment UT7/EPO cells were not permissive to B19 infection. Transcription led to nonstructural and structural gene transcripts without DNA replication or capsid protein synthesis.

In many cases, virus-encoded acylproteins appear to localize to specific cellular and viral membranes and to be directly involved with the processes of virus morphogenesis and/or egress from the infected cell. It was therefore of interest to determine whether the major vaccinia virus (W) myristylprotein, L1R, is specifically associated with one or more of the membranes enveloping various infectious forms of W virions. To this end, single-membraned intracellular virions (INV) and extracellular enveloped virions (EEV), which are surrounded by at least two distinct membranes, were purified from W-infected cell lysates. The location of the VV L1R protein was determined by using a monospecific anti-L1R serum to detect the L1R protein by immunoblot in INV- and EEV-containing fractions, by examining the proteinase K sensitivity of the L1R protein in intact INV and EEV particles, and by immunoelectron microscopy. The data obtained clearly indicate that although the L1R protein is a constituent of both the INV and EEV particles, it is exclusively found in the inner INV-specific membrane. These results are discussed with regard to the potential role of the W LIR protein in the primary intracellular envelopment of infectious W particles.

The antigenic determinants for the main hepatitis B vims (HBV) subtypes adw, adr, ayw and ayr lie in the S (surface) polypeptide. Two amino acid residues in particular, encoded by the S gene at codon positions 122 and 160, have been postulated to determine the different antigenic subtypes. In contrast, the 165 nucleotide pre- S2 gene encodes an immunodominant region common to all subtypes that can give rise to neutralizing antibodies. We have characterized the pre-S2 gene sequences of 29 HBV strains of the three main subtypes, adw, ayw and adr. Seven base positions showed variation that was entirely subtype-specific, with six of these variations leading to subtype-specific amino acid differences. This finding affords the possibility of using pre-S2 sequences for genetic subtyping. Two ayw strains from unrelated patients infected in the Middle East had identical pre-S2 sequences with a block of 12 nucleotides deleted. A geographical correlation with subtype observed from serological results was also apparent from phylogenetic analysis of DNA identities within the pre- S2 region. The results support the concept that the main HBV subtypes truly represent families of phylogen- etically different strains.

cDNA copies of the M and N genes of equine arteritis virus (EAV) isolates were synthesized by reverse transcription followed by polymerase chain reaction amplification. The cDNA was subjected to a cycle sequencing strategy using Taq polymerase, and the nucleotide and derived amino acid sequences of 10 virus isolates were compared. The M and N genes of all isolates had the same initiation and termination sites as the prototype Bucyrus strain and the encoded proteins were conserved between viruses. Comparison of nucleotide sequence homologies and phylogenetic tree analysis implied the existence of three EAV variants originating from the U.S.A. (Bucyrus), Austria (Vienna) and Switzerland (Bibuna), and suggested that RNA recombination between EAV isolates may have occurred.

We have analysed the cDNA coding for the envelope glycoprotein (El) gene and the terminal non-translated regions (NTRs) of the avirulent Semliki Forest virus (SFV) A774 (A7) variant. The El gene exhibited 98·5 % identity to the SFV prototype strain L10 (WT) sequence at the nucleotide level. Of the 34 single base substitutions, six led to a change in the deduced amino acid sequence. The 3′ NTR of A7 consisted of a 101 nucleotide sequence, not found in WT, followed by five tandemly arranged sequence motifs, two of which were truncated forms of the others. One full-length and one truncated repeat are found at the 3′ NTR of WT. The repeats of A7 were followed by a non-repeating sequence, very similar to the equivalent region in WT. Owing to the unique sequence motif and the tandem repeats, the 3′ NTR of A7 is 334 nucleotides longer than that of WT. Each of the repeats had an internal 12 nucleotide motif complementary to a conserved sequence in the 5′ -terminal non-structural protein 1- encoding region, thought to be important in alphavirus RNA replication. In the 5′ NTR, three point mutations were found. The conserved sequence binding to the repeated 3 ′ motifs was identical in A7 and WT.

Nucleotide sequences of the 5′ non-coding region and the structural protein genes of the live, attenuated Japanese encephalitis vaccine virus strains SA14-2-8 and SA14-5-3 and the wild-type parental strain SA14/ USA were determined. SA14-2-8 differed from SA14/ USA by 13 nucleotides and eight amino acids whereas SA14-5-3 differed from SA14/USA by 15 nucleotides and eight amino acids. A comparison of the 5′ noncoding region and amino acid sequences of the structural proteins of these two attenuated vaccine strains and of vaccine strains SA14-14-2/PHK and SA14-14-2/PDK with three sequences of their wild-type parent SA14 virus was performed. This revealed only two common amino acid substitutions at positions 138 and 176 in the envelope (E) protein. The substitution at E138 was predicted to cause a change in the secondary structure of the E protein. These two amino acid substitutions in the E protein may contribute to attenuation of the Japanese encephalitis vaccine viruses.

BALB/e and severe combined immunodeficiency (SCID) mice were inoculated intracerebrally or intraperitoneally with scrapie agent strain ME7 to examine the role of functional lymphocytes and follicular dendritic cells in splenic infectivity and PrPSe accumulation. Intracerebrally inoculated BALB/c and SCID mice developed the clinical signs and microscopic lesions characteristic of scrapie. Spleens from terminally affected BALB/c mice contained PrPSc which was detectable by immuno- blot analysis; SCID mouse spleens did not contain detectable PrPSc. SCID mouse spleens collected during the first 90 days after intraperitoneal infection contained neither infectivity nor PrPSc.

The nucleotide sequence of the helper component protease (HC-Pro) genes of three zucchini yellow mosaic virus (ZYMV) strains has been compared with that of a helper-deficient strain of ZYMV-HC. The comparisons revealed three unique deduced amino acid differences. Two of these mutations were located in regions which are conserved in other potyviruses. The role of these mutations in aphid transmissibility was examined by exchanging DNA fragments of part of the deficient HC- Pro gene with the respective section within the gene of the infectious full-length clone of the aphid-transmissible ZYMV. The first exchange included two of the three mutations, the first coding for a change from Asp to Gly (in a non-eonserved region) and the second coding for a change from Arg to lie [within the Phe-Arg-Asp-Lys (FRNK) conserved box]. This exchange resulted in a reduced transmission (20·6% for the mutated virus compared with 57·4% in the normal ZYMV when acquired from plants and 37·2 % compared with 831 %, respectively, when acquired from membranes). The second exchange incorporated a single mutation [conferring a change from Thr to Ala within the Pro-Thr-Lys (PTK) conserved box]. This single mutation resulted in almost total loss of HC activity in aphid transmission both from plants and from membranes. The Lys residue in the conserved Lys-Ile-Thr-Cys (KITC) box, which is related to loss of HC activity in potato virus Y, tobacco vein mottling virus and in the Michigan strain of ZYMV, is unchanged in the helper-deficient ZYMV. It is therefore proposed that more than one site in HC-Pro may be functionally related to aphid transmissibility. The possible reasons for the role of these mutations in helper activity in aphid transmission of ZYMV are discussed.

The genome of beet yellows virus (BYV), the type representative of the closterovirus group, encodes a homologue of the cellular heat-shock protein (HSP) 70 family. A pair of degenerate primers targeted to motifs A and E, which are highly conserved in HSP70s, was synthesized. Genomes of several definite and possible members of the closterovirus group were screened for the presence of the HSP70 gene with PCR using these degenerate primers. BYV, citrus tristeza virus (CTV), beet yellow stunt virus (BYSV) and carnation necrotic fleck virus templates produced 1 kb amplification products, which were shown by sequencing to represent fragments of the respective HSP70 genes. Further screening was performed with an additional degenerate primer targeted to the motif IV of the putative viral polymerase. This degenerate primer and specific primers complementary to the 5′ region of the HSP70 genes of the respective viruses were used to estimate the distance between polymerase motif IV and the start point of the HSP70 gene for BYV (approximately 1·1 kb), CTV and BYSV (around 2·0 kb) by PCR. The amplified genome regions of CTV (3026 nucleotides) and BYSV (2837 nucleotides) were cloned and sequenced. CTV and BYSV were found to encode the gene for an additional 3OK (BYSV) or 33K (CTV) protein between the polymerase and the small hydrophobic protein genes, which was absent in BYV. These two 30K proteins displayed very weak similarity to each other, unlike the highly conserved polymerases, hydrophobic proteins and HSP70s of BYV, CTV and BYSV. Degenerate primer- mediated PCR proved to be an efficient tool for rapid screening and subsequent cloning of the viral genomes.

The coat protein gene of pea early browning virus (PEBV) was replaced with that of another tobravirus, tobacco rattle virus (TRV strain PPK20). The recombinant virus multiplied efficiently in the systemic host Nicotiana benthamiana and, on the local lesion host Phaseolus vulgaris, produced symptoms typical of PEBV rather than TRV showing that viral coat protein is not a determinant for lesion morphology. Both viral RNAs were encapsidated by TRV coat protein although the shorter particles (encapsidated RNA-2) did not form a discrete population. Evidence is presented to suggest involvement of nucleotide sequences upstream of the coat protein gene in virus particle assembly.

The positive-sense RNA genome of beet yellows closterovirus (BYY) encompasses open reading frames (ORFs) for the viral capsid protein (CP, ORF 6) and for a CP homologue (p24, ORF 5). The sequences of the ORFs 5 and 6 were inserted into an Escherichia coli expression vector, pQE-9, under the control of the bacteriophage T5 promoter. The proteins were expressed in bacteria, purified, and used for antiserum production in rabbits. The recombinant BYV CP and p24 showed serological cross-reactions when probed with each antiserum on Western blots. The crossreactions of the anti-p24 serum with the CP, and of the anti-CP serum with the p24, were abolished by preadsorption with the heterologous antigens, suggesting that CP and p24 share a common epitope(s) resistant to SDS denaturation. Cross-reactivity of the soluble CP and p24 was also observed in indirect plate-trapped antigen ELISA, whereas virtually none was encountered in double-antibody sandwich ELISA. Using a polyclonal anti-p24 serum preadsorbed with the recombinant CP, the p24 was detected in BYV-infected plants. Analysis of subcellular fractions of BYV-infected Tetragonia expansa indicated that both proteins are predominantly located in the soluble fraction of the host cells. Primer extension analysis of the individual double-stranded forms of the subgenomic RNAs bearing the CP and p24 genes allowed them to be mapped and their 5′ start sites to be located at nucleotide positions 13588 and 12815, respectively, in the complete genome sequence. This corresponds to the 5′ untranslated regions of 52 and 105 nucleotides in the subgenomic RNAs for CP and p24, respectively. The data obtained indicate that the synthesis of both subgenomic RNAs is initiated on a negative RNA strand at an adenosine residue found within the conserved sequence 5′ CCAUUUPyA (shown as positive-sense), which may thus represent a core element of the subgenomic promoter. This conserved sequence also resembles the sequences at the 5′ ends of the CP subgenomic RNAs of tobamoviruses and the Bromoviridae family members, the viruses evolutionarily most closely related to BYV.

The complete nucleotide sequence of apple mosaic ilarvirus RNA 4 was obtained from cloned cDNAs and direct sequencing of the 5′ -terminal RNA region. The sequence is 891 nucleotides long and can encode a protein of 226 amino acids (M r 25171) that, by analogy to alfalfa mosaic virus (A1MV) and tobacco streak virus (TSY), should correspond to the coat protein (CP). Database comparisons showed that no significant similarity to other proteins was apparent. Analysis of the CP sequence revealed a putative ‘zinc finger’ domain and a region rich in basic residues at the amino-terminal portion of the protein, similar to that of TSY. The secondary structure proposed for the 3′-terminal region of RNA 4 shows the presence of three hairpin structures flanked by the tetranucleotide AUGC that are highly similar to those previously described in the RNA 4 species from A1MV and TSV. These results support the idea that both features (metal-binding domain and highly conserved hairpin structures) are characteristics of ilarviruses and are probably involved in the peculiar ‘genome activation’ phenomenon described in these viruses.

The complete nucleotide (nt) sequence of the original coleus yellow viroid (CYYd) from Solenostemon scutellarioides, ‘Golden Bedder’, has been determined. The covalently closed single-stranded CYYd RNA molecule consists of 248 nt residues which assumes a rod-like secondary structure when folded in the model of lowest free energy. The sequence was determined by direct sequencing of RNA and from three overlapping cDNA clones. Comparison of the CYYd sequence with that of Coleus blumei viroid 1 (CbVd 1) from Germany demonstrated that they are closely related. The differences observed in the genome organization of CYVd relative to CbVd 1 were at three sites: position 25 (one U deletion), position 26 (a U was replaced by an A) and position 241 (one A insertion). The first two mutations were detected in one A-rich segment of eight nt (between positions 25 and 34). Northern blot hybridization of partially purified nucleic acids from the leaf tissue of S. scutellarioides ‘Frilled Fantasy ’ inoculated with doublestranded cDNA, demonstrated that this fragment was infectious. These data enable CYYd to be assigned to the viroid class of plant pathogens, based on its biological properties and molecular structure. This work also gives additional support to the present classification system, in which the viroids isolated from S. scutellarioides form a distinct subgroup.