Single-stranded regions on unintegrated avian retrovirus DNA.

Using chromatography on benzoylated naphthoylated DEAE-cellulose, we found that greater than 99.5% of the unintegrated linear viral DNA species detected in quail embryo cells infected with Rous sarcoma virus contained single-stranded regions, even at 16 h after infection. These regions were distributed across the genome and, on average, were primarily of plus-strand DNA. Within most of the linear viral DNA species, the minus strand was interpreted as being of genome size with two copies of the large terminal redundancy, LTR. In contrast, the plus strands in the linear viral DNA species were exclusively subgenomic.     

Synthesis of murine leukemia viral DNA in vitro: evidence for plus-strand DNA synthesis at both ends of the genome

We studied the synthesis of B-tropic murine leukemia viral DNA in vitro by detergent-disrupted virions. The reaction products (detected by the Southern transfer technique) included full-length, infectious, double-stranded DNA and several subgenomic fragments. Restriction endonuclease analysis and hybridization and specific probes revealed two classes of subgenomic fragments: some were derived from the right end of the genome, and some were derived from the left end. Most of the fragments harbored one long terminal repeat copy at their ends, suggesting that they were initiated correctly. S1 nuclease and restriction endonuclease treatments of these fragments indicated that a single-stranded gap was present near the first initiation site of plus strong-stop DNA. The treatments also suggested the presence of a second initiation site flanked by a single-stranded gap 0.9 kilobase pairs from the right end of the genome. Our data clearly show that plus-strand DNA is synthesized at both ends of the genome, by using plus strong stop as the first initiation site and additional initiation sites.     

Synthesis of plus strands of retroviral DNA in cells infected with avian sarcoma virus and mouse mammary tumor virus

The vast majority of plus strands synthesized in quail cells acutely infected with avian sarcoma virus were subgenomic in size, generally less than 3 kilobases (kb). A series of discrete species could be identified after agarose gel electrophoresis by annealing with various complementary DNAs, indicating specificity in the initiation and termination of plus strands. The first plus strand to appear (within 2 h postinfection) was similar in length to the long redundancy at the ends of linear DNA (0.35 kb), and it annealed with complementary DNAs specific for the 3' and 5' termini of viral RNA (Varmus et al., J. Mol. Biol. 120:50-82, 1978). Several subgenomic plus-strand fragments (0.94, 1.38, 2.3, and 3.4 kb) annealed with these reagents. At least the 0.94- and 1.38-kb strands were located at the same end of linear DNA as the 0.35-kb strand, indicating that multiple specific sites for initiation were employed to generate strands which overlapped on the structural map. We were unable to detect RNA liked to plus strands isolated as early as 2.5 h postinfection; thus, the primers must be short (fewer than 50 to 100 nucleotides), rapidly removed, or not composed of RNA. To determine whether multiple priming events are a general property of retroviral DNA synthesis in vivo, we also examined plus strands of mouse mammary tumor virus DNA in chronically infected rat cells after induction of RNA and subsequent DNA synthesis with dexamethasone. In this case, multiple, discrete subgenomic DNA plus strands were not found when the same methods applied to avian sarcoma virus DNA were used; instead, the plus strands present in the linear DNA of mouse mammary tumor virus fell mainly into two classes: (i) strands of ca. 1.3 kb which appeared early in synthesis and were similar in size and genetic content to the terminally repeated sequence in linear DNA; and (ii) plus strands of the same length as linear DNA. A heterogeneous population of other strands diminished with time, was not found in completed molecules, and was probably composed of strands undergoing elongation. These two retroviruses thus appear to differ with respect to both the number of priming sites used for the synthesis of plus strands and the abundance of full-length plus strands. On the other hand the major subgenomic plus strand of mouse mammary tumor virus DNA (1.3 kb) is probably the functional homolog of a major subgenomic plus strand of avian sarcoma virus DNA (0.35 kb). The significance of this plus strand species is discussed in the context of current models which hold that it is used as a template for the completion of the minus strand, thereby generating the long terminal redundancy.     

A domain of the hepadnavirus capsid protein is specifically required for DNA maturation and virus assembly

Mutations introduced into the capsid gene of duck hepatitis B virus (DHBV) were tested for their effects on viral DNA synthesis and assembly of enveloped viruses. Four classes of mutant phenotypes were observed among a series of deletions of covering the 3' end of the capsid open reading frame. Class I mutant capsids were able to support normal single-stranded and relaxed circular viral DNA synthesis; class II mutant capsids supported normal single-stranded DNA synthesis but not relaxed circular DNA synthesis; class III mutant capsids resembled class II capsids, but viral DNA synthesis was inhibited 5- to 10-fold; and class IV capsids were severely restricted in their ability to support viral DNA synthesis. Class I capsids were assembled into enveloped virions, but class II, III, and IV capsids were not. Viral DNA synthesized inside class II capsids was normal with respect to minus-strand DNA initiation, plus-strand DNA initiation, and circularization of the DNA, but plus strands failed to be elongated to mature 3-kb DNA. The results suggest that a function of the capsid protein specifically required for viral DNA maturation is also required for assembly of nucleocapsids into envelopes. Thus, class II mutants appear to be defective in the appearance of the "packaging signal" for virus assembly (J. Summers and W. Mason, Cell 29:403-415, 1982).     

Coronavirus minus-strand RNA synthesis and effect of cycloheximide on coronavirus RNA synthesis.

The temporal sequence of coronavirus plus-strand and minus-strand RNA synthesis was determined in 17CL1 cells infected with the A59 strain of mouse hepatitis virus (MHV). MHV-induced fusion was prevented by keeping the pH of the medium below pH 6.8. This had no effect on the MHV replication cycle, but gave 5- to 10-fold-greater titers of infectious virus and delayed the detachment of cells from the monolayer which permitted viral RNA synthesis to be studied conveniently until at least 10 h postinfection. Seven species of poly(A)-containing viral RNAs were synthesized at early and late times after infection, in nonequal but constant ratios. MHV minus-strand RNA synthesis was first detected at about 3 h after infection and was found exclusively in the viral replicative intermediates and was not detected in 60S single-stranded form in infected cells. Early in the replication cycle, from 45 to 65% of the [3H]uridine pulse-labeled RF core of purified MHV replicative intermediates was in minus-strand RNA. The rate of minus-strand synthesis peaked at 5 to 6 h postinfection and then declined to about 20% of the maximum rate. The addition of cycloheximide before 3 h postinfection prevented viral RNA synthesis, whereas the addition of cycloheximide after viral RNA synthesis had begun resulted in the inhibition of viral RNA synthesis. The synthesis of both genome and subgenomic mRNAs and of viral minus strands required continued protein synthesis, and minus-strand RNA synthesis was three- to fourfold more sensitive to inhibition by cycloheximide than was plus-strand synthesis.     

Initiation of Genomic Plus-Strand RNA Synthesis from DNA and RNA Templates by a Viral RNA-Dependent RNA Polymerase

In contrast to the synthesis of minus-strand genomic and plus-strand subgenomic RNAs, the requirements for brome mosaic virus (BMV) genomic plus-strand RNA synthesis in vitro have not been previously reported. Therefore, little is known about the biochemical requirements for directing genomic plus-strand synthesis. Using DNA templates to characterize the requirements for RNA-dependent RNA polymerase template recognition, we found that initiation from the 3' end of a template requires one nucleotide 3' of the initiation nucleotide. The addition of a nontemplated nucleotide at the 3' end of minus-strand BMV RNAs led to initiation of genomic plus-strand RNA in vitro. Genomic plus-strand initiation was specific since cucumber mosaic virus minus-strand RNA templates were unable to direct efficient synthesis under the same conditions. In addition, mutational analysis of the minus-strand template revealed that the -1 nontemplated nucleotide, along with the +1 cytidylate and +2 adenylate, is important for RNA-dependent RNA polymerase interaction. Furthermore, genomic plus-strand RNA synthesis is affected by sequences 5' of the initiation site.     

Genomic plus-strand RNA synthesis by the brome mosaic virus (BMV) RNA replicase requires a sequence that is complementary to the binding site of the BMV helicase-like protein

Initiation of genomic plus-strand RNA synthesis by the brome mosaic virus (BMV) replicase in vitro requires a 26-nucleotide (nt) RNA sequence at the 3' end of the minus-strand RNA and a nontemplated nucleotide 3' of the initiation cytidylate [ Sivakumaran, K. and Kao, C.C. (1999) J. Virol. 64 , 6415–6423]. At the 5' end of this RNA is a 9-nt sequence called the cB box, the complement of the previously defined B box. The cB box can not be functionally replaced by the B box and has specific positional and sequence requirements. The portion of the cB box that is required for RNA synthesis in vitro is well-conserved in species in the Bromoviridae family. An equivalent RNA from Cucumber mosaic virus was unable to direct efficient RNA synthesis by the BMV replicase until the cB box was positioned at the same site relative to the BMV RNA and guanylates were present at positions +6 and +7 from the initiation cytidylate. These results further define the elements required for the recognition and initiation of viral genomic plus-strand RNA synthesis and suggest that a sequence important for minus-strand RNA synthesis is also required for plus-strand RNA synthesis.     

A second origin of DNA plus-strand synthesis is required for optimal human immunodeficiency virus replication.

We recently reported that human immunodeficiency virus type 1 (HIV-1) unintegrated linear DNA displays a discontinuity in its plus strand, precisely defined by a second copy of the polypurine tract (PPT) located near the middle of the genome (P. Charneau and F. Clavel, J. Virol. 65:2415-2421, 1991). This central PPT appears to determine a second initiation site for retrovirus DNA plus-strand synthesis. We show here that mutations replacing purines by pyrimidines in the HIV-1 central PPT, which do not modify the overlapping amino acid sequence, are able to significantly slow down viral growth as they reduce plus-strand origin at the center of the genome. One of these mutations, introducing four pyrimidines, results in a 2-week delay in viral growth in CEM cells and abolishes plus-strand origin at the central PPT. The introduction in this mutant of a wild-type copy of the PPT at a different site creates a new plus-strand origin at that site. This new origin also determines the end of the upstream plus-strand segment, probably as a consequence of limited strand displacement-synthesis. Our findings further demonstrate the role of PPTs as initiation sites for the synthesis of the retroviral DNA plus strand and demonstrate the importance of a second such origin for efficient HIV replication in vitro.     

Similarities and differences between the subgenomic and minus-strand promoters of an RNA plant virus

Promoter regions required for minus-strand and subgenomic RNA synthesis have been mapped for several plus-strand RNA viruses. In general, the two types of promoters do not share structural features even though they are recognized by the same viral polymerase. The minus-strand promoter of Alfalfa mosaic virus (AMV), a plant virus of the family Bromoviridae, consists of a triloop hairpin (hpE) which is attached to a 3' tRNA-like structure (TLS). In contrast, the AMV subgenomic promoter consists of a single triloop hairpin that bears no sequence homology with hpE. Here we show that hpE, when detached from its TLS, can function as a subgenomic promoter in vitro and can replace the authentic subgenomic promoter in the live virus. Thus, the AMV subgenomic and minus-strand promoters are basically the same, but the minus-strand promoter is linked to a 3' TLS to force the polymerase to initiate at the very 3'end.