- Volume 76, Issue 2, 1995

Here we examine by polymerase chain reaction amplification followed by cloning and sequence analyses selected regions of the human herpesvirus 6 (HHV-6) genome which contain human telomeric repeats (TTAGGG). We determine the relative number, arrangement and orientation of the repeats in the unit length genome, in concatemeric replicative intermediates and in heterogeneous (het) regions. We also examine distribution of the repeats in the entire genome (159 kb) and their orientation relative to DNA packaging motifs and the origin of lytic replication. In the prototype orientation the HHV-6 repeat is the related complement, TAACCC. We show that tandem array sof this repeat are present in the right and left long direct repeats (DRL and DRR, 8 kb each) which bound the long unique sequence (UL, 143 kb). Within each DR there is a left terminal imperfect tandem array and a right terminal perfect tandem array (58 copies). In DR they are each adjacent to DNA packaging motifs, pac1 and pac2, described for herpes simplex virus and human cytomegalovirus, in the arrangement pac1-imperfect repeat-7.2 kb-perfect repeat-pac2. Five independent clones were isolated and sequence determined from junctions of concatemeric replicative intermediates which showed adjacent pac2 and pac1 motifs surrounded by telomeric repeats. Favoured cleavage sites for unit length genomes were indicated which avoided cleavage within the repeats. Analyses of the complete genome showed no tandem repeats within UL but did show a polar distribution of monomeric copies and related sequences around the origin of replication, with an effect on the overall base composition. The implications for virus replication are discussed.

The nature of the amino acids in the N-terminal ‘DAGX’ motif of the coat protein of tobacco vein mottling virus (TVMV) that have a direct effect on aphid transmissibility of the virion were further defined by sitedirected mutagenesis. In the first position of the DAGX motif, Asp or Asn are required for aphid transmissibility. In the second position, the nonpolar residue Ala, but not the nonpolar Gly or Val or the polar Thr and Ser, is compatible with transmissibility. In the third position, the small, neutral, nonpolar Gly appears to be critical; even substitution of Ala, with a minimal side-chain, drastically reduces transmissibility. Although the amino acid following the DAG sequence is not highly conserved among potyviruses, the presence of an acidic Glu or Asp residue at this position in the TVMV coat protein drastically reduces or abolishes aphid transmissibility. An attempt was made to test the hypothesis that trypsin cleavage of the N terminus is involved in the aphid inoculation process by destroying a trypsin cleavage site downstream from the DAGX motif. While the predicted decrease in transmission occurred from infected plants, there was no effect on the transmission of purified virus. Of the 23 mutations in the DAGX region of TVMV reported here and previously, only two, substitutions of Lys and Arg for Asp, had a detectable adverse effect other than on aphid transmissibility. These, and perhaps other, residues near the N terminus function in some phase of the TVMV life cycle, in addition to aphid transmission.

In vitro RNA transcripts of both (+) and (-) polarities were obtained from a circularly permuted dimer clone of the small satellite of arabis mosaic virus (sArMV). The transcripts show efficient self-cleavage at the two specific sites in the sequence where the monomers were joined. Autolytic processing of the full-length transcript in both orientations releases promoter-proximate fragments (+) or (-) P, promoter-distal fragments (+) or (-) D, and the monomer fragments (+) or (-) M. The presence of an OH group at their 5′ ends and a 2′,3′ cyclophosphate at their 3′ ends suggests that (+) and (-) M originated via two self-cleavage reactions within the full-length transcript of corresponding polarity. Infectivity assays showed that the (+) M fragment but not the (-) M fragment initiates replication as efficiently as the natural linear sArMV in Chenopodium quinoa. Two (-) fragments were identified which are the result of religation activity: a P-D fragment formed by religation of P and D, and c-M, which is the result of efficient self-ligation of (-) M. In contrast, linear (+) M self-ligates in vitro to a very limited extent but could be circularized enzymically in a wheat germ extract.

Full-length cucumber mosaic cucumovirus (CMV) cDNAs were cloned into a new plasmid vector containing a modified plant virus promoter designed to transcribe the inserted sequence from its first nucleotide. cDNA copies of CMV strain Q (Q-CMV) genomic RNAs 1, 2 and 3 cloned into this vector were infectious when inoculated together, producing symptoms indistinguishable from those caused by wildtype Q-CMV infection. The infectivity of the clones could be substantially increased by excision of the viral insert together with the transcriptional promoter and terminator prior to inoculation. A diagnostic but silent mutation was introduced into the RNA 2 cDNA and found to be stably maintained in viral infection, allowing distinction of the recombinant virus from native contaminants. The infectious cDNA clones supported the replication of CMV satellite RNA when co-inoculated with biologically active Q-CMV satellite RNA transcripts. Using the infectious cDNAs described, it was found that a newly-identified overlapping gene (2b) encoded by Q-CMV RNA 2 was not essential for either systemic viral infection of Nicotiana glutinosa or replication of the satellite RNAs.

The nucleotide sequence of tomato ringspot nepovirus (TomRSV) RNA1 has been determined. TomRSV RNA1 is 8214 nucleotides in length, excluding the 3′ poly(A) tail, and contains a single long open reading frame (ORF) of 6591 nucleotides beginning at the first AUG codon at nucleotide position 78. This ORF accounts for 80% of the RNA1 sequence and would give rise to a polyprotein with a predicted molecular mass of 244 kDa. Amino acid sequence comparisons between portions of the TomRSV RNA1-encoded polyprotein and proteins encoded by several members of the picornavirus superfamily have provided information concerning the genomic organization and putative functions of TomRSV-encoded proteins. The putative TomRSV protease retains a conserved histidine residue present in the proteases encoded by members of the como-, poty- and poliovirus groups which is thought to be involved in dipeptide cleavage site recognition. Interestingly, this histidine residue is replaced by a leucine in the proteases of other sequenced nepoviruses. This suggests that the TomRSV protease shares dipeptide cleavage site specificity with that of como-, poty- and picornaviruses rather than the other nepoviruses.

Faba bean necrotic yellows virus (FBNYV) has a circular ssDNA genome possibly consisting of several components of about 1 kb each. The complete nucleotide sequence of one component of FBNYV (FBNYV DNA 1) containing a putative replicase gene is presented. This component consists of 1002 nucleotides and, in the virion orientation, contains one large open reading frame (ORF1) potentially encoding a 32.3 kDa replicase with the NTP-binding motif GGEGKS. No obvious functions could be assigned to two smaller ORFs (7.4 and 9.3 kDa) occurring in the complementary orientation. Amino acid sequence comparisons of the putative replicase of FBNYV with that of other similar ssDNA viruses yielded higher homologies to subterranean clover stunt virus than to banana bunchy top and coconut foliar decay viruses. A potential stem-loop structure and a TATA box were identified within the noncoding region. Two oligonucleotides derived from FBNYV DNA 1 were used for direct sequencing of the virion ssDNA to determine its virion polarity and for amplifying part of this component by immunocapture PCR in extracts from FBNYV-infected plants.