The structure of two subgenomic RNAs from human influenza virus A/PR/8/34.

The nucleotide sequences of two subgenomic RNA segments from influenza virus A/PR/8/34 have been determined by cloning viral cDNA into the vector M13mp7. Sequence analysis was facilitated by a re-cloning strategy which takes advantage of both wild-type and amber derivatives of the M13 vector. The RNA species (444 and 480 nucleotides) contain the 5' and 3' termini of segment 1 and therefore derive by simple internal deletions of this segment. However, these species are not exact copies of the terminal regions of the progenitor segment but contain a few base changes. These differences suggest that after these RNAs have arisen, their sequences can drift, presumably reflecting a lower selective pressure than on the standard RNA segments.     

Functional domains of the capsid protein of human immunodeficiency virus type 1.

A series of deletions was introduced into the CA domain of the human immunodeficiency virus type 1 Gag polyprotein to examine its role in virus particle and core formation. The mutations resulted in two phenotypes, indicating the existence of two functionally distinct regions within the CA domain. Deletions within a conserved stretch of 20 amino acids referred to as the major homology region (MHR) and deletions C terminal to this region blocked virus replication and significantly reduced the ability to form viral particles. Deletions N terminal to the MHR also prevented virus replication, but the mutants retained the ability to assemble and release viral particles with the same efficiency as the wild-type virus. The mutant particles contained circular rather than cone-shaped cores, and while they were of a density similar to that of wild-type particles, they were more heterogeneous in size. These results indicate that CA domain sequences N terminal to the MHR are essential for the morphogenesis of the mature cone-shaped core.     

In vivo resolution of circular plasmids containing concatemer junction fragments from minute virus of mice DNA and their subsequent replication as linear molecules.

During replication of their linear, single-stranded DNA genomes, parvoviruses generate a series of concatemeric duplex intermediates. We have cloned, into Escherichia coli plasmids, junction fragments from these palindromic concatemers of minute virus of mice DNA spanning both the right end-to-right end (viral 5' to 5') and left end-to-left end (viral 3' to 3') fusions. When mouse cells were transfected with these circular plasmids and superinfected with minute virus of mice, the viral junctions were resolved and the plasmids replicated as linear chromosomes with vector DNA in their centers and viral DNA at their termini. Resolution did not occur when the concatemer joint was replaced by a different palindromic sequence or when the transfected cells were not superinfected, indicating the presence of latent origins of replication which could only be activated by a viral trans-acting factor(s). Moreover, the products of resolution and replication from the two termini were characteristically different. Analysis of individual terminal fragments showed that viral 5' (right-end) sequences were resolved predominantly into "extended" structures with covalently associated copies of the virally encoded NS-1 polypeptide, while bridges derived from the 3' (left) end resolved into both NS-1-associated extended termini and lower-molecular-weight "turn-around" forms in which the two DNA strands were covalently continuous. This pattern of resolution exactly coincides with that seen at the two termini of replicative-form intermediates in normal virus infections. These results demonstrate that the bridge structures are authentic substrates for resolution and indicate that the frequency with which extended versus turn-around forms of each terminus are generated is an intrinsic property of the telomere.     

Altered utilization of splice sites and 5'' termini in late RNAs produced by leader region mutants of simian virus 40.

We compared the 5' termini and splices of the late 16S and 19S RNAs synthesized by wild-type simian virus 40 and five mutants containing deletions in their late leader region. All mutants produced more unspliced 19S RNA than did wild-type virus, and in two mutants, unspliced 19S RNA constituted more than 60% of the total 19S species. The other three mutants each utilized predominantly a different one of the three spliced species of 19S mRNA. All mutants also produced decreased quantities of 16S mRNA, indicating that they may be defective for splicing both late RNAs. None of the 5' termini of the 16S and 19S RNAs made by the five mutants predominated as in those made by the wild type. Some of the mutant 5' termini were the same as those used by the wild type, whereas others were different. Although present, the major 5'-end positions used by the wild type were frequently not used as major sites by the mutants. In addition, mutants with very similar deletion endpoints synthesized RNAs with different 5' ends. Thus, downstream mutations have a pronounced effect on the location of 5' ends of the late RNAs, and there is no obvious involvement of a measuring function in the placement of 5' ends. For all mutants and wild-type virus, the 5' termini used for 16S and 19S RNAs showed major differences, with some degree of correlation found between the 5' ends and the internal splices of specific mRNA species. A model for the regulation of simian virus 40 late gene expression is presented to explain these findings.     

Infectious circular DNA of human adenovirus type 5: regeneration of viral DNA termini from molecules lacking terminal sequences.

A series of plasmids containing the entire human adenovirus genome with viral DNA termini joined 'head to tail' has been isolated. Several plasmids were able to generate infectious virus following transfection of human cells in spite of having small deletions and rearrangements at the junctions of termini. One plasmid has lost 2 bp of DNA from one end of the viral genome and 11 bp from the other end yet produced viruses with complete wild-type sequences at both ends of the genome. We propose a model for replication of viral DNA off circular templates in which regeneration of terminal information involves translocation of primer and polymerase during initiation of DNA replication. The model suggests a novel mechanism for extension of the 5' ends of linear DNA molecules which could be applicable to chromosomal telomeres.     

Bipartite 3'-cis-acting signal for replication in yeast 23 S RNA virus and its repair

23 S RNA narnavirus is a persistent positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome is small (2.9 kb) and only encodes its RNA-dependent RNA polymerase. Recently, we have succeeded in generating 23 S RNA virus from an expression vector containing the entire viral cDNA sequence. Using this in vivo launching system, we analyzed the 3'-cis-acting signals for replication. The 3'-non-coding region of 23 S RNA contains two cis-elements. One is a stretch of 4 Cs at the 3' end, and the other is a mismatched pair in a stem-loop structure that partially overlaps the terminal 4 Cs. In the latter element, the loop or stem sequence is not important but the stem structure with the mismatch pair is essential. The mismatched bases should be purines. Any combination of purines at the mismatch pair bestowed capability of replication on the RNA, whereas converting it to a single bulge at either side of the stem abolished the activity. The terminal and penultimate Cs at the 3' end could be eliminated or modified to other nucleotides in the launching plasmid without affecting virus generation. However, the viruses generated regained or restored these Cs at the 3' terminus. Considering the importance of the viral 3' ends in RNA replication, these results suggest that this 3' end repair may contribute to the persistence of 23 S RNA virus in yeast by maintaining the genomic RNA termini intact. We discuss possible mechanisms for this 3' end repair in vivo.     

Selection of functional 5' cis-acting elements promoting efficient sindbis virus genome replication

The 5' portion of the Sindbis virus (SIN) genome RNA is multifunctional. Besides initiating translation of the nonstructural polyprotein, RNA elements in the 5' 200 bases of the SIN genome RNA, or its complement at the 3' end of the negative-strand intermediate, play key roles in the synthesis of both negative- and positive-strand RNAs. We used here a combination of genetic and biochemical approaches to further dissect the functions of this sequence. Replacement of the SIN 5' end in defective-interfering (DI) and genome RNAs with sequences from a distantly related alphavirus, Semliki Forest virus (SFV), resulted in nonviable chimeras. The addition of five nucleotides from the 5' terminus of SIN restored negative-strand RNA synthesis in DI genomes but not their replication in vivo. Pseudorevertants of various SFV-SIN chimeras were isolated, and suppressor mutations were mapped to AU-rich sequences added to the 5' end of the original SFV 5' sequence or its "deleted" versions. Early pseudorevertants had heterogeneous 5' termini that were inefficient for replication relative to the parental SIN 5' sequence. In contrast, passaging of these pseudorevertant viral populations in BHK cells under competitive conditions yielded evolved, more homogeneous 5'-terminal sequences that were highly efficient for negative-strand synthesis and replication. These 5'-terminal sequences always began with 5'-AU, followed by one or more AU repeats or short stretches of oligo(A). Further analysis demonstrated a positive correlation between the number of repeat units and replication efficiency. Interestingly, some 5' modifications restored high-level viral replication in BHK-21 cells, but these viruses were impaired for replication in the cells of mosquito origin. These studies provide new information on sequence determinants required for SIN RNA replication and suggest new strategies for restricting cell tropism and optimizing the packaging of alphavirus vectors.     

Construction and properties of a recombinant herpes simplex virus 1 lacking both S-component origins of DNA synthesis.

The herpes simplex virus 1 (HSV-1) genome contains three origins of DNA synthesis (Ori) utilized by viral DNA synthesis proteins. One sequence (OriI) maps in the L component, whereas two sequences (OriS) map in the S component. We report the construction of a recombinant virus, R7711, from which both OriS sequences have been deleted, and show that the OriS sequences are not essential for the replication of HSV-1 in cultured cells. In addition to the deletions of OriS in R7711, the alpha 47 gene and the 5' untranscribed and transcribed noncoding regions of the U(S)11 gene were deleted, one of the alpha 4 promoter-regulatory regions was replaced with the simian virus 40 promoter, and the alpha 22 promoter was substituted with the alpha 27 promoter. The total amount of viral DNA synthesized in Vero cells infected with the OriS-negative (OriS-) virus was approximately that seen in cells infected with the OriS-positive virus. However, cells infected with the OriS- virus accumulated viral DNA more slowly than those infected with the wild-type virus during the first few hours after the onset of DNA synthesis. In single-step growth experiments, the yield of OriS- progeny virus was reduced at most fourfold. Although a single OriS (R. Longnecker and B. Roizman, J. Virol. 58:583-591, 1986) and the single OriL (M. Polvino-Bodnar, P. K. Orberg, and P. A. Schaffer, J. Virol. 61:3528-3535, 1987) have been shown to be dispensable, this is the first indication that both copies of OriS are dispensable and that one copy of an Ori sequence may suffice for the replication of HSV-1.     

Vesicular stomatitis virus defective interfering particles can contain extensive genomic sequence rearrangements and base substitutions

We sequenced the 5' and 3' RNA termini of 16 defective interfering (DI) particles of vesicular stomatitis virus (VSV) isolated at intervals from persistent infections and from a series of undiluted lytic passages. All DI RNAs exhibited complementary termini, but sequences internal to these termini were extensively rearranged in a variety of ways. Despite extensive rearrangement, these internal sequences (in addition to the termini) apparently are important for DI particle interference properties. Some of these DI particles are derived from multiple intrastrand and interstrand recombination events, and the generation of each can be explained by current replicase error models. During viral evolution in persistent and acute infections, DI particles with specific termini base substitutions are selected. One DI particle exhibits a remarkable clustering of specific A----G (and complementary U----C) substitutions, apparently as a result of repetitive misincorporations by an error-prone viral polymerase complex.     

Two spatially distinct genetic elements constitute a bipartite DNA replication origin in the minute virus of mice genome.

Mutations were introduced into plasmid pMM984, a full-length infectious clone of the fibrotropic strain of minute virus of mice, to identify cis-acting genetic elements required for the excision and replication of the viral genome. The replicative capacity of these mutants was measured directly, using an in vivo transient DNA replication assay following transfection of plasmids into murine A9 cells and primate COS-7 cells. Experiments with subgenomic constructs indicated that both viral termini must be present on the same DNA molecule for replication to occur and that the viral nonstructural protein NS-1 must be provided in trans. The necessary sequences were located within 1,084 and 807 nucleotides of the 3' and 5' ends of the minute virus of mice genome, respectively. The inhibitory effect of deletions within the 206-bp 5'-terminal palindrome demonstrated that these sequences comprise a cis-acting genetic element that is absolutely essential for the excision and replication of viral DNA. The results further indicated a requirement for a stem-plus-arms T structure as well as for the formation of a simple hairpin. In addition, the removal of one copy of a tandemly arranged 65-bp repeat found 94 nucleotides inboard of the 5'-terminal palindrome inhibited viral DNA replication in cis by 10- and just greater than 100-fold in A9 and COS-7 cells, respectively. The latter results define a novel genetic element within the 65-bp repeated sequence, distinct from the terminal palindrome, that is capable of regulating minute virus of mice DNA replication in a species-specific manner.     

Structure and role of the herpes simplex virus DNA termini in inversion, circularization and generation of virion DNA

The herpes simplex virus genome consists of two components, L and S, which invert relative to each other during viral replication. The a sequence is present at the genomic termini in direct orientation and at the L-S junction in inverted orientation. Previously, we showed that insertion of a fragment spanning the L-S junction into the viral genome causes additional inversions. In this study, we determine the nucleotide sequence of the genomic termini and show that insertion of either the free S terminus or the L terminus causes inversions in the viral genome. We conclude that the a sequence is the inversion-specific sequence, that linear unit-length molecules packaged in virions are generated by cleavage between adjacent copies of the a sequence, that cleavage produces 3' single-base extensions on the genomic termini and that the signal for cleavage is contained within the a sequence.     

Influenza B viruses with site-specific mutations introduced into the HA gene.

We have succeeded in engineering changes into the genome of influenza B virus. First, model RNAs containing the chloramphenicol acetyltransferase gene flanked by the noncoding sequences of the HA or NS genes of influenza B virus were transfected into cells which were previously infected with an influenza B helper virus. Like those of the influenza A viruses, the termini of influenza B virus genes contain cis-acting signals which are sufficient to direct replication, expression, and packaging of the RNA. Next, a full-length copy of the HA gene from influenza B/Maryland/59 virus was cloned. Following transfection of this RNA, we rescued transfectant influenza B viruses which contain a point mutation introduced into the original cDNA. A series of mutants which bear deletions or changes in the 5' noncoding region of the influenza B/Maryland/59 virus HA gene were constructed. We were able to rescue viruses which contained deletions of 10 or 33 nucleotides at the 5' noncoding region of the HA gene. The viability of these viruses implies that this region of the genome is flexible in sequence and length.