A novel mechanism for the initiation of Tacaribe arenavirus genome replication.

The ends of arenavirus genome and antigenome RNAs are highly conserved and where determined directly, always contain a 3' G (referred to as position +1). However, primers extended to the 5' ends of Tacaribe virus genomes and antigenomes extend to position -1. When genomes and antigenomes are annealed either inter or intramolecularly and treated with RNase A or T1, there appears to be a single unpaired G at the 5' ends of the hybrids. A single extra G is also found by cloning the 5' ends of S antigenomes, and studies with capping enzyme detect (p)ppG at the 5' ends of genome and antigenome chains. A model is proposed in which genome replication initiates with pppGpC to create the nontemplated extra G. In contrast, the nontemplated bases at the 5' ends of the N mRNAs, which extend to positions -1 to -5, were found to be capped and also heterogeneous in sequence.     

5'-proximal hot spot for an inducible positive-to-negative-strand template switch by coronavirus RNA-dependent RNA polymerase

Coronaviruses have a positive-strand RNA genome and replicate through the use of a 3' nested set of subgenomic mRNAs each possessing a leader (65 to 90 nucleotides [nt] in length, depending on the viral species) identical to and derived from the genomic leader. One widely supported model for leader acquisition states that a template switch takes place during the generation of negative-strand antileader-containing templates used subsequently for subgenomic mRNA synthesis. In this process, the switch is largely driven by canonical heptameric donor sequences at intergenic sites on the genome that match an acceptor sequence at the 3' end of the genomic leader. With experimentally placed 22-nt-long donor sequences within a bovine coronavirus defective interfering (DI) RNA we have shown that matching sites occurring anywhere within a 65-nt-wide 5'-proximal genomic acceptor hot spot (nt 33 through 97) can be used for production of templates for subgenomic mRNA synthesis from the DI RNA. Here we report that with the same experimental approach, template switches can be induced in trans from an internal site in the DI RNA to the negative-strand antigenome of the helper virus. For these, a 3'-proximal 89-nt acceptor hot spot on the viral antigenome (nt 35 through 123), largely complementary to that described above, was found. Molecules resulting from these switches were not templates for subgenomic mRNA synthesis but, rather, ambisense chimeras potentially exceeding the viral genome in length. The results suggest the existence of a coronavirus 5'-proximal partially double-stranded template switch-facilitating structure of discrete width that contains both the viral genome and antigenome.     

Electron microscopy of the segmented RNA genome ofLa Crosse virus: absence of circular molecules.

The three species of single-stranded RNA present in La Crosse virus were examined in the electron microscope. Because large amounts of contaminating cellular DNA are copurified with the virus despite extensive attempts to purify the virus, it was necessary to use procedures that eliminated the bulk of this DNA before the viral RNA was analyzed. When this was done, the modal lengths of La Crosse virus RNA were 0.4, 2.0, and 3.1 mum. These lengths correspond well to their known molecular weights of 0.4 x 106, 1.8 x 106, and 2.9 x 106. Under the denaturing conditions used to permit complete spreading of these single-stranded RNA molecules, no single-stranded circular molecules are observed. Therefore, the circular nucleocapsids present in La Crosse virus and some other bunyaviruses do not appear to be due to convalent linkage of the ends of the RNA genome.     

A 3'' co-terminus of two early herpes simplex virus type 1 mRNAs.

A 3' co-terminus of two early herpes simplex virus type 1 mRNAs has been identified using the nuclease -S1 mapping procedure with cloned virus DNA probes. These mRNAs (5.0 kb and 1.2 kb), located within the genome region 0.56-0.60, are unspliced and are transcribed rightwards on the prototype genome orientation. The position of their 3' ends has been located on the virus DNA sequence and lies downstream from the polyadenylation signal 5'-AATAAA-3'. This hexanucleotide sequence also was present in the complementary DNA strand and was shown to be the polyadenylation signal for a leftwards-transcribed late mRNA. The abundance within the cytoplasm of the 5.0 kb and 1.2 kb mRNAs was investigated. Results indicated that these mRNAs were regulated in concert. It is suggested that sequences at the 3' co-terminus may be involved in their regulation.     

Detailed analysis of the mRNAs mapping in the short unique region of herpes simplex virus type 1.

We have analysed the mRNAs which map within the short unique (US) region of the herpes simplex virus type 1 (HSV-1) genome. US has a total length of 12979 base pairs (1) and is extensively transcribed with approximately 94% of the total sequence present in cytoplasmic mRNAs and 79% of the total sequence considered to be protein coding. There are several examples of overlapping functions and multiple use of DNA sequence within this region. US contains 12 genes (1) which are expressed as 13 mRNAs. Two of these mRNAs are thought to arise from the same gene since they differ only slightly in the positions of their 5' ends and probably specify the same polypeptide. 11 of the 13 mRNAs are arranged into four nested families with unique 5' ends and common 3' co-termini. The other two mRNAs have unique 5' and 3' ends.     

Self-ligating RNA sequences on the antigenome of human hepatitis delta virus.

A 179-base fragment of RNA from the 1,679-base antigenome of hepatitis delta virus can not only self-cleave but, when the ends of the resultant fragments are brought into apposition by base pairing to another RNA, also self-ligate. Thus, processing events needed for genome replication in vivo may be strictly RNA mediated.     

La Crosse virus infection of mammalian cells induces mRNA instability.

La Crosse virus infection of BHK cells leads to a dramatic shutoff of not only host protein synthesis but also viral protein synthesis later in infection. This shutoff can be accounted for by the loss of the cytoplasmic cellular and viral mRNAs. The induction of mRNA instability requires extensive virus replication, since when cycloheximide is added early in infection the preexisting viral and cellular mRNAs do not decrease upon incubation of the cultures. Pretreatment of the cultures with actinomycin D does not affect the ability of La Crosse virus infection to induce mRNA instability, and examination of the rRNAs shows no evidence of specific degradation due to activation of the interferon-associated latent RNase. The induction of mRNA instability therefore does not appear to operate through an interferon pathway. Viral mRNA synthesis, on the other hand, is not turned off during infection, and the cap-dependent endonuclease involved in viral mRNA initiation may be responsible for the mRNA instability.     

Mutations in the 5' trailer region of a respiratory syncytial virus minigenome which limit RNA replication to one step

The 3' termini of the genomic and antigenomic RNAs of human respiratory syncytial virus (RSV) are identical at 10 of the first 11 nucleotide positions and 21 of the first 26 positions. These conserved 3'-terminal sequences are thought to contain the genomic and antigenomic promoters. Furthermore, the complement of each conserved sequence (i.e., the 5' end of the RNA it encodes) might contain an encapsidation signal. Using an RSV minigenome system, we individually mutated each of the last seven nucleotides in the 5' trailer region of the genome. We analyzed effects of these mutations on encapsidation of the T7 polymerase-transcribed negative-sense genome, its ability to function as a template for RSV-driven synthesis of positive-sense antigenome and mRNA, and the ability of this antigenome to be encapsidated and to function as template for the synthesis of more genome. As a technical complication, mutations in the last five nucleotides of the trailer region were found to affect the efficiency of the adjoining T7 promoter over more than a 10-fold range, even though three nonviral G residues had been included between the core promoter and the trailer to maximize the efficiency of promoter activity. This was controlled in all experiments by monitoring the levels of total and encapsidated genome. The efficiency of encapsidation of the T7 polymerase-transcribed genome was not affected by any of the trailer mutations. Furthermore, neither the efficiency of positive-sense RNA synthesis from the genome nor the efficiency of encapsidation of the encoded antigenome was affected by the mutations. However, nucleotide substitution at positions 2, 3, 6, or 7 relative to the 5' end of the trailer blocked the production of progeny genome, whereas substitution at positions 1 and 5 allowed a low level of genome production and substitutions at position 4 were tolerated. Position 4 is the only one of the seven positions examined that is not conserved between the 3' ends of genomic and antigenomic RNA. The mutations that blocked the synthesis of progeny genome thus limited RNA replication to one step, namely, the synthesis and encapsidation of antigenome. Restoration of terminal complementarity for one of the trailer mutants by making a compensatory mutation in the leader region did not restore synthesis of genomic RNA, confirming that its loss was not due to reduced terminal complementarity. Interestingly, this leader mutation appeared to prevent antigenome synthesis with only a slight effect on mRNA synthesis, apparently providing a dissociation between these two synthetic activities. Genomes in which the terminal 24 or 325 nucleotides of the trailer have been deleted were competent for encapsidation and the synthesis of mRNA and antigenomic RNA, further confirming that terminal complementarity was not required for these functions.