- Volume 78, Issue 3, 1997

Two abundant subgenomic RNAs, designated RNA 3B and RNA 5, were found to be associated with the V strain of tomato aspermy cucumovirus (V-TAV). Sequence determination showed that the 3'-terminal 323 nucleotides (nt) of RNA 3B was identical to RNA 5, whereas its 5'-terminal 163 nt was a direct repeat (one nt difference) of the 5'-half of RNA 5, and that both RNAs are completely homologous to the 3'-terminal untranslated region of TAV RNA 3. TAV RNAs 3B and 5 were also detected in the infection of a pseudorecombinant virus consisting of TAV RNA 3 and RNAs 1 and 2 from cucumber mosaic virus. Furthermore, only RNA 5, not RNA 3B, was detected in a TAV mutant in which one of the repeats was deleted from RNA 3. These genetic studies clearly show that both RNA species are derived from TAV RNA 3. However, in contrast to TAV RNAs 4 and 4A, which encode coat protein and 2b protein, respectively, RNAs 3B and 5 represent a novel class of subgenomic RNAs from TAV that do not function as mRNAs. Possible functional roles for such a class of viral subgenomic RNAs are discussed.

Pea enation mosaic virus (PEMV) is composed of two autonomously replicating virus RNAs related to the genomic RNAs of viruses in the genera Luteovirus and Umbravirus. The transmission of PEMV resembles that of its luteovirus relatives in utilizing circulative aphid transmission. However, unlike its luteovirus counterparts, PEMV can also be mechanically transmitted. Prolonged mechanical passage of PEMV can lead to the loss of aphid transmissibility, a trait that is mirrored by specific changes in the PEMV virion composition. These changes were used to examine the virus contribution to vector transmission and the mechanisms by which it is regulated. Using a local lesion isolation technique, one aphid transmissible and two aphid non-transmissible isolates of PEMV were compared. Structural analysis of a 54 kDa minor structural subunit unique to the aphid transmissible isolate demonstrated that it was a fusion of the 21 kDa virus coat protein and a 33 kDa protein encoded immediately downstream of the 21 kDa ORF, consistent with the formation of the 54 kDa subunit by translational readthrough. Genetic analyses utilizing exchanges between infectious in vitro transcripts of each isolate demonstrated that although the 33 kDa protein was non-essential for infection, its presence was mandatory for aphid transmission, and that specific changes within the 33 kDa ORF were sufficient to confer or abolish aphid transmission. This study also demonstrates that isolates of PEMV exist as mixtures of aphid transmissible and non-transmissible genotypes, and provides insight into the mechanisms used by this virus to down-regulate aphid transmission in response to a specific selection pressure.

The complete sequence of the single-stranded, (+)-sense RNA genome of saguaro cactus carmovirus (SCV) has been determined. The 3879 nucleotide genome contains five open reading frames (ORFs). The 5'-proximal ORF encodes a 26 kDa protein (p26) and terminates with an amber codon which is readthrough into an in-frame p57 ORF to generate an 86 kDa fusion protein (p86). Two small, centrally located ORFs encode a 6 kDa protein (p6) and a 9 kDa protein (p9), respectively. The 3'-proximal ORF encodes a 37 kDa (p37) capsid protein (CP). Analysis of the nucleotide and predicted amino acid sequences supports the classification of SCV in the genus Carmovirus in the family Tombusviridae. All predicted SCV proteins are expressed in an in vitro translation system. SCV p26 and the readthrough fusion protein p86 are synthesized from the genomic RNA while p6, p9 and p37 CP ORFs at the 3' half of the genome are expressed from two subgenomic (sg) RNAs. The 5' termini of both sg RNAs have been mapped. The large 1614 nucleotide sg RNA contains the p6 and p9 ORFs as the first and the second ORFs respectively from its 5' end. It directs the synthesis of abundant p6 but a small amount of p9. While a synthetic transcript with the p9 ORF at the 5' end is a more efficient messenger for p9, no corresponding sg RNA has been identified in vivo. The smaller 1396 nucleotide sg RNA contains only the p37 ORF and directs the synthesis of SCV CP.

The positive-strand RNA genome of beet yellows closterovirus (BYV) encodes a 65 kDa protein (p65) related to the HSP70 family of cell chaperones. The full-sized BYV p65, and N- and C-terminal fragments, with (His)6 tails, were overexpressed in bacteria and purified by metal-chelate chromatography. Using a polyclonal antiserum raised against the C-terminal fragment of p65, evidence was obtained for expression of the viral protein in planta. Purified recombinant p65 and its N-terminal 40 kDa fragment exhibited Mg2+-dependent ATPase activity in vitro. However, unlike its cellular HSP70 homologues, p65 was unable to bind to denatured protein and its ATPase activity was not stimulated by synthetic peptides which are known to stimulate HSP70 ATPases. Hence, the BYV p65, although being a chaperone-type ATPase, may have a distinct substrate specificity and function in BYV-infected cells.

The nucleotide sequence of the large (L) genome segment of impatiens necrotic spot virus (INSV) has been determined, and herewith the complete nucleotide sequence of the whole, tripartite genome of this important tospovirus has been elucidated. The L RNA is 8776 nucleotides long and of negative polarity, containing one large ORF on the viral complementary strand. Comparison of the deduced amino acid sequence of the INSV L RNA primary translation product (330.3 kDa) with those of the L RNAs of other members of the family Bunyaviridae reveals that this protein represents the putative viral RNA-dependent RNA polymerase. A cluster dendrogram of the (putative) RNA polymerases indicates that the genera Tospovirus and Tenuivirus, though both encompassing ambisense plant-infecting viruses, have different affinities to the animal-infecting Bunyaviridae, tospoviruses being most closely related to the genus Bunyavirus, and tenuiviruses to the genus Phlebovirus.

The large (L) RNA segment of Lassa fever virus (LAS) encodes a putative RNA-dependent RNA polymerase (RdRp or L protein). Similar to other arenaviruses, the LAS L protein is encoded on the genome-complementary strand and is predicted to be 2218 amino acids in length (253 kDa). It has an unusually large non-coding region adjacent to its translation start site. The LAS L protein contains six motifs of conserved amino acids that have been found among arenavirus L proteins and core RdRp of other segmented negative-stranded (SNS) viruses (Arena-, Bunya- and Orthomyxoviridae). Phylogenetic analyses of the RdRp of 20 SNS viruses reveals that arenavirus L proteins represent a distinct cluster divided into LAS-lymphocytic choriomeningitis and Tacaribe-Pichinde virus lineages. Monospecific serum against a synthetic peptide corresponding to the most conserved central domain precipitates a 250 kDa product from LAS and lymphocytic choriomeningitis virus-infected cells.

H1N1 influenza A viruses isolated from pigs in Europe since 1981 were examined both antigenically and genetically and compared with H1N1 viruses from other sources. H1N1 viruses from pigs and birds could be divided into three groups: avian, classical swine and 'avian-like' swine viruses. Low or no reactivity of 'avian-like' swine viruses in HI tests with monoclonal antibodies raised against classical swine viruses was associated with amino acid substitutions within antigenic sites of the haemagglutinin (HA). Phylogenetic analysis of the HA gene revealed that classical swine viruses from European pigs are most similar to each other and are closely related to North American swine strains, whilst the 'avian-like' swine viruses cluster with avian viruses. 'Avian-like' viruses introduced into pigs in the UK in 1992 apparently originated directly from strains in pigs in continental Europe at that time. The HA genes of the swine viruses examined had undergone limited variation in antigenic sites and also contained fewer potential glycosylation sites compared to human H1N1 viruses. The HA exhibited antigenic drift which was more marked in 'avian-like' swine viruses than in classical swine strains. Genetic analyses of two recent 'avian-like' swine viruses indicated that all the RNA segments are related most closely to those of avian influenza A viruses.

When MDCK cells in a semiconfluent monolayer were infected with 5 p.f.u. per cell of influenza virus A/PR/8/34 (H1N1), a majority of the cells continued to grow stably upon subsequent cultivation with a growth medium containing 50% foetal calf serum. While growing, the cells spontaneously excreted virus, the amount of which declined gradually as the passage number of the cells increased. The extent of virus shedding was significantly increased when the cells were subsequently maintained in a medium containing 0.2% bovine serum albumin. Within the cells, viral messenger RNAs for all eight genes of A/PR/8 were demonstrated by PCR indicating that endogenous viral genes were constitutively transcribed. However, viral proteins as well as viral genes were not demonstrable by radioimmunoprecipitation or ribonuclease protection assays, respectively.

The importance of the receptor-destroying enzyme of influenza C virus for inactivation of inhibitors was analysed. Using three different inhibitors (rat serum, bovine submandibulary mucin and bovine brain gangliosides) inhibition of virus infection was observed only at an inhibitor concentration that was about 100-fold higher than the maximum concentration of inhibitor that could be inactivated by the receptor-destroying enzyme of a given amount of virus. From our data and other observations we conclude that the receptor-destroying enzyme is not required to inactivate inhibitors.

This paper corrects the previously published sequence of the L gene of canine distemper virus (CDV). Errors in the published sequence (M. S. Sidhu et al., 1993, Virology 193, 50-65) led to frame shifts between residues 1021-1032, 1190-1219 and 1645-1650; a deletion of 21 amino acids between residues 1684-1705, and a single residue deletion at residue 1478. Residue 237 is now found to be glycine rather than tryptophan and residue 1626 proline instead of threonine. The sequence of the L gene of phocine distemper virus (PDV) was also determined. Alignment of the morbillivirus L proteins showed that PDV and CDV are more closely related to each other than to rinderpest virus and measles virus. Two regions of low identity are proposed to function as hinge regions between three highly conserved domains (I-III) in the morbillivirus L proteins. New sequence motifs have been identified on the basis of conservation in the morbilliviruses and the Paramyxovirinae.

Comparative sequence analysis of a 354 nt fragment of the NS5 region of hepatitis G virus (HGV) isolates was performed to assess two levels of HGV genomic variability: (1) heterogeneity of HGV within an infected individual, and (2) heterogeneity of different HGV isolates. Comparison of nucleotide sequences of DNA clones from two virus isolates demonstrated that in each infected individual HGV is represented by a population of virions with closely related but heterogeneous genomes (quasi-species). Phylogenetic analysis of nucleotide sequences of 42 isolates collected from 14 countries revealed less significant genome variability of HGV as compared to hepatitis C virus. Sequences of all HGV isolates fell into one group of distribution of evolutionary distances. On a phylogenetic tree all HGV sequences segregated into numerous branches. All sequences of isolates from Africa, South and South-East Asia, however, were clustered together and were separated from those of other isolates collected in Europe, North America and Central Asia.

Haemophilic patients exposed to unsterilized clotting factor concentrates prior to 1985 have become infected with hepatitis C virus (HCV). We have studied the sequence evolution of the 5'UTR and a region of NS4 over 12 years in one human immunodeficiency virus (HIV) positive haemophilic patient and 14 years for one HIV negative haemophilic patient. One sample each year from the date of HCV infection to 1994 was analysed for genotype, virus load and nucleotide sequence of the two genetic loci. Both patients were infected with HCV genotype 1 throughout the study period. The virus load profiles were similar except that the profile for the HIV infected patient was displaced 4 years earlier relative to the other patient. Mean divergence of the quasispecies at both the 5'UTR and NS4 loci was higher in the HIV coinfected patient. Phylogenetic analysis indicated that evolution of the 5'UTR was host independent, whereas the NS4 region containing a CD8 restricted CTL epitope evolved in a host specific fashion.

An increase in the incidence of acute flaccid paralysis cases associated with wild-type 1 poliovirus occurred in children in the city of Bangui, Central African Republic (CAR), in 1993 and 1994. Genetic relationships of 33 isolates were analysed by restriction fragment length polymorphism and by sequencing the VP1/2A junction region (150 nucleotides) of the viral genome. Two distinct genotypes, A and B, were co-circulating in 1993, while in 1994 only a third genotype, C, was observed. Comparison of the sequences found, with those of the sequences from isolates from neighbouring and other endemic countries revealed that genotype A isolates were related to strains from Egypt (90.7% identity), genotype B isolates to strains from Kenya (96.7% identity), Sudan and Egypt, and genotype C isolates to strains from various countries in western and southern Africa (89.0% identity). Genotypic diversity and genetic linkage with strains from neighbouring countries indicate intense poliovirus circulation and transmission that does not respect national borders. Therefore, eradication of poliomyelitis from CAR can only be achieved by a coordinated multinational strategy that stops poliovirus circulation in the whole of Africa.

Clonal populations of foot-and-mouth disease virus have been serially passaged in cell culture to analyse variation in the absence of immune selection at different antigenic sites of the virus. Mutant frequencies at the RNA regions encoding two independent antigenic sites (sites C and D) were more than twentyfold lower than for antigenic site A (the G-H loop of VP1). Correspondingly, fixation of amino acid substitutions was very restricted in sites C and D. In spite of such a restriction, neutralization assays using fractionated anti-virus polyclonal antibodies has provided direct evidence of significant antigenic variation in the absence of immune selection at sites unrelated to site A. It is proposed that the degree of tolerance to acceptance of amino acid replacements may modulate the variation at different antigenic epitopes of the same virus.

A monoclonal antibody which blocks infection with feline immunodeficiency virus (FIV) was found previously to react with the cell surface molecule CD9, implicating CD9 in the process of virus entry. We report here that inhibition by anti-CD9 antibody does not operate at the level of virus entry but at a subsequent stage in the virus life-cycle. Moreover, inhibition of infection is independent of the passage history of the virus or the virus subtype. Inhibition of FIV infection by anti-CD9 antibody does not operate in 3201 cells, which do not express this surface antigen. However, ectopic expression of CD9 on 3201 cells enhances infection with FIV, suggesting that the role of CD9 may be direct rather than via cellular signalling pathways. These results suggest a novel control point in the lentivirus life-cycle which might be susceptible to modulation by natural antagonists.

Human immunodeficiency virus (HIV) type 1 encodes three genes, Vpu, Env and Nef, that decrease cellular CD4. Vpu and Env act cooperatively to accelerate degradation of CD4 in the endoplasmic reticulum. Here we report that Vpu/Env-induced CD4 degradation is inhibited by lactacystin, a specific inhibitor of the proteasome, and by other proteasome inhibitors, but not by non-proteasome protease inhibitors. We also note that Vpu has amino acid sequence homology with a segment of IkappaB known to be involved in proteasome-mediated degradation, suggesting that HIV-1 could have transduced cellular sequences to enhance down-regulation of CD4.

In order to facilitate analyses of the molecular function of the human immunodeficiency virus type 1 (HIV-1) Vif protein, we have developed a cell culture model-system which allows permanent production of genotypically and phenotypically vif-defective HIV-1 virions in 'non-permissive' H9 cells. Using recombinant, replication-competent HIV-1 proviruses coding for a selectable marker gene (gpt) instead of nef, two stably infected H9 subclones named M2 (vif-mutant) and WX (wild-type), respectively, were generated. Virions released from cell line M2--displaying the expected vif-defective phenotype--are produced permanently, and in an at least 50 times higher amount than virus particles from acutely vif-negative HIV-1-infected H9 cells. Analysis of viral protein composition and the electron-microscopic morphology of vif-mutant virions did not reveal any detectable differences in comparison to wild-type virions.

In order to gain insight into the role of the major homology region (MHR) in the infectious potential of bovine leukaemia virus (BLV), mutations were introduced into the capsid gene of an infectious molecular clone. A provirus that was designed to contain only a slightly modified version of the MHR (substitution of phenylalanine 147 with a tyrosine) was still infectious in vivo. Furthermore, the provirus loads were not significantly different from those obtained with a wild-type virus. A second mutant was designed to analyse a mild modification of the MHR at the level of arginine 150. The substitution of this residue with a lysine completely destroyed the infectious potential of the recombinant virus. Finally, a third mutant that was deleted in the MHR region was unable to infect the host. Thus it appears that the integrity of the MHR domain is essential for BLV infectivity in vivo.

Antisense RNA-mediated inhibition of gene expression has the potential for gene therapy of virus infections. We studied the inhibitory effect of antisense RNA directed against the hepatitis B virus (HBV) genome on the expression of the HBV surface antigen (HBsAg). Three prokaryotic antisense RNA expression constructs were produced which expressed antisense RNA complementary to the entire coding region (1.4 kb) and to 1.0 kb and 582 bp of the 5' region of HBsAg mRNA, respectively. In an in vitro translation system, all three antisense RNAs showed concentration-dependent inhibitory effects on translation of HBsAg mRNA. In a coupled in vitro transcription and translation system, concentration-dependent inhibition of HBsAg synthesis was observed for all above mentioned antisense RNAs. Three mammalian antisense RNA expression vectors were then constructed, expressing the same antisense RNAs as used above. Transfection of the vectors into Hep3B cells (an HBsAg secreting cell line) resulted in almost complete blockage of HBsAg production, whereas control vector transfected cells secreted high levels of HBsAg. The inhibitory effect lasted for more than 10 months post-transfection. To examine the possible mechanism of the antisense RNA effect in the cell line, we measured HBV mRNA levels in the transfected cells and found that the mRNA levels in the antisense RNA expressing cells were much lower than those in the control cells. Therefore, in Hep3B cells, the antisense RNAs inhibited HBsAg synthesis, at least partially, through the reduction of HBV mRNA levels.