Both VP2 and VP3 are synthesized from each of the alternative spliced late 19S RNA species of simian virus 40.

The late 19S RNAs of simian virus 40 consist of a family of alternatively spliced RNAs, each of which contains open reading frames corresponding to all three of the virion proteins. Two approaches were used to test the hypothesis that each alternatively spliced 19S RNA species is translated to synthesize preferentially only one of the virion proteins. First, we analyzed the synthesis of virion proteins in simian virus 40 mutant-infected monkey cells that accumulate predominantly either only one spliced 19S RNA species or only the 19S RNAs. Second, we determined the virion proteins synthesized in a rabbit reticulocyte lysate programmed with specific, in vitro-transcribed 19S RNA species. These results indicated that VP2 and VP3, but not VP1, are synthesized from all 19S RNA species. Quantitative analysis of these data indicated that individual 19S RNA species containing a translation initiation signal upstream of the VP2 AUG codon were translated in a cell extract three- to fivefold less efficiently than were 19S RNA species lacking this signal and that the precise rate of synthesis of VP2 relative to VP3 varied somewhat with the sequence of the leader region. These data are consistent with the synthesis of VP2 and VP3 occurring by a leaky scanning mechanism in which initiation of translation at a specific AUG codon is affected by both (i) the intrinsic efficiency of ribosomes recognizing the sequences surrounding the AUG codon as an initiation signal and (ii) partial interference from 5'-proximal initiation signals and their corresponding open reading frames.     

Leader-encoded open reading frames modulate both the absolute and relative rates of synthesis of the virion proteins of simian virus 40.

Numerous viral and cellular RNAs are polycistronic, including several of the late mRNA species encoded by simian virus 40 (SV40). The functionally bicistronic major late 16S and functionally tricistronic major late 19S mRNA species of SV40 contain the leader-encoded open reading frames (ORFs) LP1, located upstream of the sequence encoding the virion protein VP1, and LP1*, located upstream of the sequence encoding the virion proteins VP2 and VP3. To determine how these leader ORFs affect synthesis of the virion proteins, monkey cells were transfected with viral mutants in which either the leader-encoded translation initiation signal was mutated or the length and overlap of the leader ORF relative to the ORFs encoding the virion proteins were altered. The levels of initiation at and leaky scanning past each initiation signal were determined directly by quantitative analysis of the viral proteins synthesized in cells transfected with these mutants. Novel findings from these experiments included the following. (i) At least one-third of ribosomes bypass the leader-encoded translation initiation signal, GCCAUGG, on the SV40 major late 16S mRNA. (ii) At least 20% of ribosomes bypass even the consensus translation initiation signal, ACCAUGG, when it is situated 10 bases from the 5' end on the major late 16S mRNA. (iii)O The presence of the leader ORF on the bicistronic 16S mRNA species reduces VP1 synthesis threefold relative to synthesis from a similar RNA that lacks it. (iv) At least half and possibly all VP1 synthesized from the bicistronic 16S mRNA species is made by a leaky scanning mechanism. (v) LP1 and VP1 are synthesized from the bicistronic 16S mRNA species at approximately equal molar ratios. (vi) Approximately half of the VP1 synthesized in SV40-infected cells is synthesized from the minor, monocistronic 16S mRNA even though it accounts for only 20% of the 16S mRNA present. (vii) The presence and site of termination of translation of the leader ORF on the late 19S mRNAs affect the relative as well as absolute rates of synthesis of VP2 and VP3.     

Mechanisms of synthesis of virion proteins from the functionally bigenic late mRNAs of simian virus 40.

The late 19S RNAs of simian virus 40 (SV40) are functionally polycistronic, i.e., all encode both VP2 and VP3. The VP3-coding sequences are situated in the same reading frame as the VP2-coding sequences, within the carboxy-terminal two-thirds of the VP2-coding sequences. To test whether VP3 is produced by proteolytic processing of VP2, we introduced a variety of deletion and insertion mutations within the amino-terminal end of the VP2-coding sequences. Genetic and biochemical analysis of the proteins synthesized in cells transfected with these mutants indicated that VP2 and VP3 were synthesized independently of each other. A leaky scanning model for the synthesis of VP3 was tested by the insertion of a strong initiation signal (CCAACATGG) upstream of the VP3-coding sequences. When the signal was placed in the same reading frame as VP3, synthesis of VP3 was reduced by a factor of 10 to 20, whereas synthesis of the expected VP3-related fusion protein occurred at a rate similar to that observed for VP3 in cells transfected with wild-type SV40 DNA. Insertion of this strong initiation signal at the same site, but in a different reading frame, resulted in the synthesis of VP3 at one-third of the wild-type rate. Mutation of the VP2 initiator AUG resulted in a small but reproducible (1.6-fold) increase in VP3 accumulation. From these experiments we conclude that (i) VP3 is synthesized predominantly by independent initiation of translation via a leaky scanning mechanism, rather than by proteolytic processing of VP2 or direct internal initiation of translation; (ii) a strong initiation signal 5' of the VP3-coding sequences can significantly inhibit synthesis of VP3, but does not act as an absolute barrier to scanning ribosomes; (iii) approximately 70% of scanning ribosomes bypass the VP2 initiator AUG, which is present in a weak context (GGUCCAUGG), and initiate at the VP3 initiation signal located downstream; and (iv) reinitiation of translation appears to occur on the SV40 late 19S mRNAs at an efficiency of 25 to 50%.     

Translation of equine infectious anemia virus bicistronic tat-rev mRNA requires leaky ribosome scanning of the tat CTG initiation codon.

We have examined the translational regulation of the equine infectious anemia virus (EIAV) bicistronic tat-rev mRNA. Site-directed mutagenesis of the tat leader region followed by expression of the tat-rev cDNA both in vitro and in transiently transfected cells established that tat translation is initiated exclusively at a CTG codon. Increasing the efficiency of tat translation by altering the CTG initiator to ATG resulted in a dramatic decrease in translation of the downstream (rev) cistron, indicating that leaky scanning of the tat CTG initiation codon permitted translation of the downstream rev cistron. Since the tat leader sequences precede the major EIAV splice donor and are therefore present at the 5' termini of both spliced and unspliced viral mRNAs, the expression of all EIAV structural and regulatory proteins is dependent on leaky scanning of the tat initiator.     

貂肠炎病毒的核苷酸序列和基因组结构

本试验测定了已克隆的貂肠炎病毒(MEV)复制型(RF)DNA的核苷酸序列,确定MEV基因组全长约为5064个核苷酸(nucleotides,nt),推测了3'端和5'端结构,在5'端非编码区有3个51 nt的重复。MEV基因组序列与犬细小病毒(CPV)、猫细小病毒(FPV)有很高的同源性,结构基因区的同源性分别达99.1%和99.9%,但在5'端非编码区有较大差异。MEV基因组结构与CPV和FPV基本一致,有两个大的开放阅读框架,分别编码688和722个氨基酸。在map unit(m.u.)3.7和m.u.39处有两个启动子,在m.u.97处有poly A位点。NS2、VP1和VP2的mRNA都发生剪接。     

家蚕浓核病毒 Bm DNV-3(中国株)VD1基因组结构与转录分析

为了进一步认识家蚕浓核病毒BmDNV-3(中国株)VD1基因组的结构和功能,VD1被分离、纯化、克隆到pUC119载体上,完成了基因组全序列的测定。序列分析显示VD1基因组全长为6543个核苷酸,末端拥有224个核苷酸反向重复序列(ITRs)。VD1基因组正链含有3个大的开放阅读框(ORF1-3),负链含有1个大的开放阅读框(ORF4)。比较BmDNV-3的VD1和BmDNV-2(Yamanashiisolate)的VD1基因组全序列,两者同源性为98.4%,并且有107个碱基的替代和1个碱基插入,氨基酸突变集中在VD1ORF3和VD1ORF4。Northern杂交结果显示VD1的左边正链上有1.1kb和1.5kb两个转录本,右边的负链上有一个3.3kb转录本。3′和5′-RACE结果显示1.1kb转录本开始于nt290,结束于nt1437;1.5kb转录本开始于nt1423,结束于nt2931;3.3kb转录本开始于nt6287,结束于nt2922。正链上1.5kb转录本和负链上3.3kb转录本拥有10个核苷酸的3′端的共同序列。研究结果显示该病毒基因转录与已报道的其它浓核病毒存在较大的差异性。     

A premature termination codon in either exon of minute virus of mice P4 promoter-generated pre-mRNA can inhibit nuclear splicing of the intervening intron in an open reading frame-dependent manner.

How premature translation termination codons (PTCs) mediate effects on nuclear RNA processing is unclear. Here we show that a PTC at nucleotide (nt) 385 in the NS1/2 shared exon of P4-generated pre-mRNAs of the autonomous parvovirus minute virus of mice caused a decrease in the accumulated levels of doubly spliced R2 relative to singly spliced R1, although the total accumulated levels of R1 plus R2 remained the same. The effect of this PTC was evident within nuclear RNA, was mediated by a PTC and not a missense transversion mutation at this position, and could be suppressed by improvement of the large intron splice sites and by mutation of the AUG that initiated translation of R1 and R2. In contrast to the PTC at nt 385, the reading frame-dependent effect of the PTC at nt 2018 depended neither on the initiating AUG nor the normal termination codon for NS2; however, it could be suppressed by a single nucleotide deletion mutation in the upstream NS1/2 common exon that shifted the 2018 PTC out of the NS2 open reading frame. This suggested that there was recognition and communication of reading frame between exons on a pre-mRNA in the nucleus prior to or concomitant with splicing.