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Like other positive-strand RNA viruses, plant potyviruses assemble viral replication complexes (VRCs) on modified cellular membranes. Potyviruses encode two membrane proteins, 6K2 and P3. The former is known to play pivotal roles in the formation of membrane-associated VRCs. However, P3 remains to be one of the least characterized potyviral proteins. The P3 cistron codes for P3 as well as P3N-PIPO which results from RNA polymerase slippage. In this study, we show that the P3N-PIPO of Turnip mosaic virus (TuMV) is required for viral cell-to-cell movement but not for viral replication. We demonstrate that the C-terminal region of P3 (P3C) is indispensable for P3 to form cytoplasmic punctate inclusions and target VRCs. We reveal that TuMV mutants that lack P3C are replication-defective. Taken together, these data suggest that the P3 cistron has two distinct functions: P3N-PIPO as a dedicated movement protein and P3 as an essential component of the VRC.

A series of Wheat streak mosaic virus (WSMV)-based expression vectors were developed by engineering a cycle 3 GFP (GFP) cistron between P1 and HC-Pro cistrons with several catalytic/cleavage peptides at the C-terminus of GFP. WSMV-GFP vectors with the Foot-and-mouth disease virus 1D/2A or 2A catalytic peptides cleaved GFP from HC-Pro but expressed GFP inefficiently. WSMV-GFP vectors with homologous NIa-Pro heptapeptide cleavage sites did not release GFP from HC-Pro, but efficiently expressed GFP as dense fluorescent aggregates. However, insertion of one or two spacer amino acids on either side of NIb/CP heptapeptide cleavage site or deletion in HC-Pro cistron improved processing by NIa-Pro. WSMV-GFP vectors were remarkably stable in wheat for seven serial passages and for 120 days postinoculation. Mite transmission efficiencies of WSMV-GFP vectors correlated with the amount of free GFP produced. WSMV-GFP vectors infected the same range of cereal hosts as wild-type virus, and GFP fluorescence was detected in most wheat tissues.

We constructed dicistronic, subgenomic hepatitis C virus (HCV) replicons in which the sequence encoding the human immunodeficiency virus (HIV) tat protein was placed in the upstream cistron, between the HCV 5′NTR and a picornaviral 2A proteinase sequence fused to the selectable marker Neo. Stably transformed Huh7 cells expressing secreted alkaline phosphatase (SEAP) under transcriptional control of the HIV LTR promoter actively secreted SEAP following transfection with these replicon RNAs. Extracellular SEAP activity correlated closely with intracellular HCV RNA levels, as determined by Northern blotting and real-time RT-PCR analysis. These RNAs replicated efficiently despite the absence of core-protein-coding sequence downstream of the HCV IRES. The replication efficiency of replicons derived from the HCV-N strain of HCV was significantly greater than those derived from Con1 in transiently transfected cells. Using this reporter system, we have demonstrated significant differences in the response to interferon α-2b in cell lines containing replicons derived from these two strains of HCV.

Nucleotide (nt) sequence polymorphism within a collection of Wheat streak mosaic virus (WSMV) isolates was examined. An approximately 1267-nt region encompassing the coat protein (CP) cistron and flanking sequences was amplified by reverse transcription-polymerase chain reaction (RT-PCR) for each of 49 isolates not previously sequenced. Consensus sequences were compiled for each isolate based on sequences derived from three clones per RT-PCR product. Among 59 consensus sequences examined, only two were identical. Clades A-C contained divergent isolates from Mexico (Clade A); the Czech Republic, Hungary, and Russia (Clade B); and Iran (Clade C). Fifty-four closely related consensus sequences of isolates from the U.S. (51 sequences), Canada (1 sequence), and Turkey (2 sequences) comprised Clade D. Pair-wise nt divergence between two of the most distantly related sequences (Sidney 81 of Clade D and El Batán 3 of Clade A) was 20%, representing over half of the variable sites (34.1%) in the entire WSMV data set. Maximum pairwise nt divergence within Clade D was 3.6%, yet the proportion of all variable sites within Clade D (21.4%) was similar to that of the Sidney 81-El Batán 3 pair. Patterns of polymorphism within Clade D and the Sidney 81-El Batán 3 pair were remarkably similar with respect to synonymous, nonsynonymous, and noncoding substitutions, as were the proportions of substitutions as a function of nt position within codons. The majority of substitutions within Clade D were synonymous and randomly distributed throughout the coding region examined, whereas nonsynonymous substitutions exhibited a clumped distribution and mostly occurred within the 5'-proximal portion of the CP cistron. Because over half of the polymorphic sites within Clade D were of allele size class 1, the isolates appear to be evolving independently and in a nondeterministic manner, within the constraints of selection. These results indicate that Clade D has undergone substantial and, most likely, recent divergence with the majority of consensus sequence substitutions potentially neutral with respect to fitness. An estimate of evolution rate suggests that the present diversity within the U.S. population arose in about a century, a timeframe corresponding to the establishment of wheat monoculture in the Great Plains.

We report the construction of a poliovirus genome [pPVM-VPg(3F4A)] harboring a double mutation in VPg. This mutant, in which the tyrosine and the threonine at residues 3 and 4 of the VPg region were replaced by phenylalanine and alanine, respectively, is lethal, that is, all RNA synthesis was abolished and no revertants could be isolated. Using the properties of dicistronic polioviruses (with the general genotype PV 5′NTR-3AB-EMCV IRES-PV ORF-3′NTR), we have observed that the defect in RNA synthesis of the VPg(3F4A) mutant could be rescued by providing wild-type protein 3AB from the first open reading frame in trans. We conclude that the 3AB provided by the first cistron of the dicistronic construct was capable of "intragenomic complementation." Intragenomic complementation, however, was inefficient. Thus, the dicistronic RNAs were only quasi-infectious, and even first-passage viruses were found to have reverted to a functioning VPg in the polyprotein. This phenomenon underlines the role of polypeptide 3AB in multiple functions of viral proliferation. First-passage viruses, all of which expressed a small-plaque phenotype, had retained the original dicistronic genotype. At the fourth passage, however, all isolates were monocistronic, and they displayed complex genetic rearrangements revealing interesting information regarding IRES function.

The role of the 385 nucleotide 5′ noncoding region (NCR) in the translation of the pestivirus genome was investigated. In vitro translation of an RNA transcript containing the 5′ NCR of the bovine viral diarrhea virus (BVDV) genome followed by the coding sequence of the first gene product (p20) of the BVDV large open reading frame resulted in the synthesis of a 20-kDa polypeptide. Results from hybrid-arrest translation studies identified a region involving a predicted RNA stem—loop structure spanning nucleotides 154–261 within the 5′ NCR that was important for p20 synthesis. An additional inhibitory oligonucleotide was complementary to the sequence at the base of this stem-loop and encompassed the initiating AUG at nucleotide 386. Antisense oligonucleotides both upstream and downstream of those that were inhibitory had no effect on p20 translation. RNA from a dicistronic expression vector in which the BVDV 5′ NCR was inserted between two reporter genes, CAT and LUC, showed strong expression of the second (LUC) cistron upon in vitro translation. This expression was dramatically reduced in an analogous construct in which nucleotides 173–236 of the 5′ NCR were deleted. Similar results were obtained when RNA from these same vectors was evaluated for expression after transfection into BHK cells. These results suggest that the BVDV 5′ NCR contains an internal ribosome entry site for translation initiation. This translational mechanism is similar to that shown for hepatitis C virus, further demonstrating the close relationship between viruses of these two genera within the family Flaviviridae.

Translation of tomato black ring virus (TBRV) RNA-1 in a rabbit reticulocyte lysate leads to the synthesis of a 250Kpolyprotein which cleaves itself into smaller proteins of 50, 60, 120, and 190K. Polypeptides synthesized from synthetic transcripts corresponding to different regions of TBRV RNA-1 are processed only when they encode the 23K protein delimited earlier by sequence homology with the cowpea mosaic virus 24K protease. The proteolytic activity of this protein is completely lost by mutating residues C170 (to I) or L188 (to H), residues which align with conserved residues of the viral serine-like proteases. The 120K protein is generated by cleavage of the dipeptide K/A localized in front of the VPg but is not further cleaved in vitro at the K/S site (at the C terminus of the VPg) or between the protease and polymerase domains. However, both the protein VPgProPol (120K) and the protein ProPol (117K) produced in vitro from synthetic transcripts can cleave in trans the RNA-2-encoded 150K polyprotein, but they cannot cleave in trans polypeptides containing a cleavage site expressed from RNA-1 transcripts in which the protease cistron is absent or modified.

We report the complete nucleotide sequence of the single-stranded RNA phage PP7 from Pseudomonas aeruginosa .There are three open reading frames which code for apparent protein homologues of the single-stranded RNA coliphages, i.e., maturation protein, coat protein, and replicase. A fourth overlapping reading frame exists that probably encodes a lysis protein, similar to what has been found in the group A coliphages such as MS2. The genetic map of PP7 is colinear with group A coliphages and we accordingly classify the phage as a levivirus. There is, generally speaking, no significant nucleotide sequence identity between PP7 and the coliphages except for a few regions where homologous parts of proteins are encoded, most notable in the replicase gene. In these regions the nucleotide sequence similarity between PP7 and MS2 is no greater than between PP7 and the group B coliphages such as Q β . Surprisingly, Q β and MS2 are no closer to each other than they are to PP7. Several regulatory RNA secondary structure features that are present in the coliphages were identified also in PP7 RNA although the sequences involved cannot be aligned. Among these are the coat protein binding helix at the start of the replicase gene, structures at the 5′ and 3′ terminus of the RNA, a replicase binding site, and the structure of the coat protein cistron start. Some of these features resemble MS2 type coliphages but others the Q β type. These findings suggest that PP7 is related to the coliphages but branched off before the coliphages diverged into separate groups.

Since internal initiation of translation of cardiovirus RNAs requires the approximately 450-nt segment of the viral genome immediately upstream of the authentic initiation codon for viral polyprotein synthesis, the question arises as to whether the immediately adjacent sequences, the start of the polyprotein coding region, also influence the efficiency of internal initiation. Therefore, a variety of constructs derived from encephalomyocarditis virus and Theiler's murine encephalomyelitis virus retaining various lengths of viral coding sequence were translated in rabbit reticulocyte lysates. Efficient internal initiation showed no requirement specifically for viral coding sequences, but on the other hand certain motifs, notably G-rich sequences, located immediately downstream of the initiation codon were highly inhibitory. These results suggest a possible explanation for the evolution of cardiovirus polyproteins lacking N-terminal myristylation signals and are also pertinent to the design of constructs in which the cardiovirus internal ribosome entry signal is used to drive the expression of a reporter cistron.

Grapevine fanleaf nepovirus (GFLV) has a bipartite plus-sense RNA genome. Its structural and functional proteins originate from polyprotein maturation by at least one virus-encoded proteinase. Here we describe the cloning of the 24-kDa proteinase cistron located between the virus-linked protein (VPg) and the RNA-dependent RNA polymerase cistron in GFLV RNA1 (nucleotides 3966 to 4622). Proteinase expressed from this clone is able to cleave GFLV polyprotein P2 in order to produce the coat protein and a 66-kDa protein which is further processed to the 38-kDa presumed movement protein. The GFLV 24-kDa proteinase sequence contains sequence similarities with other nepovirus and comovirus proteinases, particularly at the level of the conserved domains corresponding to the hypothetical catalytic triad and to the substrate-binding pocket (amino acids 192 to 200). Site-directed mutagenesis of residues His43, Glu87, and Leu197 abolished proteinase activity. Inactivation of the enzyme is also observed if the catalytic residue Cys179 was substituted by isoleucine, but replacement by a serine at the same position produced a mutant with an activity identical to that of native proteinase. All our data show that GFLV cysteine proteinase presents structure similarities to the proteinases of cowpea mosaic virus and potyviruses but is most closely related to trypsin.

RNA editing of the human parainfluenza virus type 3 (HPIV3) phosphoprotein (P) gene was found to occur for the accession of an alternate discontinuous cistron. Editing occurred within a purine-rich sequence (AAUUAAAAAAGGGGG) found at the mRNA nucleotides 791–805. This sequence resembles an HPIV3 consensus transcription termination sequence and is located at the 5′-end of the putative D protein coding sequences. Editing at an alternate site (AAUUGGAAAGGAAAGG), mRNA nucleotides 1121–1136, for accession of a conserved V cistron, which is present in a number of paramyxovirus P genes, was not found to occur in HPIV3. In contrast with many other paramyxoviruses, editing was indiscriminate with the insertion of 1–12 additional G residues not present in the gene template. RNA editing was found to occur in both in vivo (HPIV3 infected cells) and in vitro (purified nucleocapsid complexes) synthesized mRNAs. Further, the in vitro prepared mRNA was edited regardless of whether the nucleocapsid complexes were transcribed in the presence or absence of uninfected human lung carcinoma (HLC) cell lysates. These results support the notion that RNA editing appears to be exclusively a function of viral proteins.

The six major conserved genes of figwort mosaic virus (FMV), a caulimovirus, appear in tandem array on an RNA transcript that spans the entire viral genome. Gene VI, the only cistron that appears as a separate subgenomic RNA, has been reported to transactivate the expression of downstream genes of the full-length transcript. This transcript has a long 5′-leader of about 600 nucleotides followed by a small nonconserved region (gene VII), a smaller intergenic region (57 nucleotides), and the major conserved genes in a closely spaced array. In our present experiments we have constructed expression units containing the promoter for the full-length transcript followed by the 5′ leader region, gene VII, and a reporter gene. These have been tested for expression with and without gene VI as a separate plasmid by electroporation into plant protoplasts. A series of these expression units containing truncated versions of the 5′ leader region placed upstream of a reporter gene (CAT) showed that gene VI transactivation occurred only when gene VII sequences were present in cis between the leader region and the reporter gene. In addition, a more complete version of the FMV genome containing the reporter gene further downstream (in viral gene IV) showed CAT expression only when gene VII sequences were present in an upstream position. A similar construct failed to express CAT activity when gene VII was absent.

The region of the genomic RNA-1 from grapevine fanleaf virus isolate F13 (GFLV-F13), containing the proteinase cistron and flanking sequences (nucleotides 3894 to 4789) of the GFLV polyprotein, was modified by PCR mutagenesis to create a start codon and cloned in a transcription vector. The transcripts from the resulting clone (pVP7) produced, upon translation in rabbit reticulocyte lysate, a 37.8-kDa protein which was subsequently cleaved to a stable 28-kDa product. Autocleavage was maximal at pH 7.0–8.5 and at 30°. Inhibition of the activity was greater than 80% when translation was performed in the wheat germ system. In rabbit reticulocyte lysate, inhibition was also obtained with PMSF, EDTA, E-64, Ca 2+ , Zn +2 , and Co +2 . The pVP7 translation product acts in cis, in the case of its autocleavage, or in trans in the processing of the viral 122-kDa polyprotein from GFLV RNA-2 into a 66-kDa protein and the 56-kDa coat protein. The carboxy extremity of the complete pVP7 translation product, encoded by nucleotides 4633 to 4789 of RNA-1, was not required for the proteinase activity, at least in trans.

Previous to this study, the Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) was known to express only one spliced RNA (spliced IE1 or IE0). We have conducted an analysis of RNA expressed during infection of Spodoptera frugiperda cells with AcMNPV and have identified a set of five additional spliced RNAs expressed late in infection. A reverse transcription-polymerase chain reaction analysis was used to confirm the identification of the LS (late, spliced) RNAs. S1 nuclease and primer extension analyses were used to map the transcription initiation sites of LS RNAs. LS1 and LS2 initiated at positions -138 and -117, respectively (relative to the IE0 +1 transcription start site). Both LS1 and LS2 contain an additional cistron potentially encoding a small, highly basic polypeptide. LS3 (-79), LS4 (-22), and LS5 (+51/52) RNAs encode only the predicted downstream IE0 ORF. Although several baculovirus late gene consensus transcription initiation sites (ATAAG) were identified within this region, only LS5 initiated at one of these conserved motifs. An S1 nuclease analysis was done to determine whether unspliced precursors of LS RNAs could be detected. Early in infection, a greater proportion of IE0 RNA was detected in the spliced form; however, during the late phase of infection a significantly greater amount of unspliced precursor forms of LS RNAs was observed.

The nucleotide sequence of the RNA 3 of the peanut stunt virus strain J (PSV-J) was determined and compared with those of the cucumber mosaic virus strain Y (CMV-Y, subgroup I) and the tomato aspermy virus strain C (TAV-C) at both the nucleotide and protein levels. The RNA 3 of PSV-J consists of 2186 nucleotides and has two large open reading frames (ORFs). By analogy to other tripartite plant viruses, it is presumed that the first ORF (867 nucleotides) codes for the 3a protein and the second ORF (654 nucleotides) is the cistron of the coat protein which is expressed from subgenomic RNA 4 of 1010 nt residues. At the nucleotide level, PSV-J RNA 3 shows 60.6% homology to RNA 3 of CMV-Y and 66.3% to that of TAV-C, much lower than the value between the two CMV subgroups. For the 3a proteins there is 65.4% homology between PSV-J and CMV-Y and 70.2% homology between PSV-J and TAV-C. Although it has been known that PSV shows a distant serological relationship to CMV, the coat protein of PSV-J shows only 50% homology with that of CMV-Y.

Transfection of barley protoplasts with brome mosaic virus (BMV) RNAs 1 + 2 in the absence of RNA-3 yielded a molar ratio for (+):(-)-strand progeny at 24 hr postinoculation near unity, whereas over 100-fold more (+)- than (-)-strand progeny accumulated in its presence. The presence of RNA-3 enhanced total (+)-strand RNA production 205-fold and that of RNAs 1 + 2 by 29-fold. In contrast, total (-)-strand RNA accumulation decreased by 68% and that for (-)RNAs 1 + 2 by 79% in the presence of RNA-3. Transfections containing an RNA-3 mutant (Gsgi----U RNA-3) that is incapable of yielding RNA-4 as a result of a single nucleotide substitution at the subgenomic RNA initiation site yielded only 66% of the (+):(-) asymmetry seen in the presence of wild-type RNA-3. Only 1.8-fold excess of (+)-over (-)-strand production was obtained for transfections that included delta SGP RNA-3, a deletion that includes the subgenomic promoter core and extends 43 nt into the RNA-4 sequence. Transfections containing RNA-3 mutants bearing frameshifts or deletions in the coat protein cistron yielded levels of asymmetry similar to those seen for Gsgi----U RNA-3. These findings implicate the subgenomic promoter and other sequences in the intercistronic region of RNA-3 as the primary determinants of asymmetric replication, although the coat protein may be an additional factor enhancing the accumulation of (+)-strand RNA.

Maize chlorotic mottle virus (MCMV) is a 30-nm icosahedral plant virus composed of a single 25-kDa capsid protein component and a 4.4-kb single-stranded, positive-sense genomic RNA. Northern blot hybridization analysis detected a single 3′-terminal 1.1-kb subgenomic RNA in infected plants. Virion RNA directs the synthesis of several polypeptides in a rabbit reticulocyte lysate in vitro translation system of which only the 25-kDa polypeptide is immunoprecipitated by MCMV capsid protein antiserum. The 1.1-kb subgenomic RNA is a highly efficient messenger RNA for capsid protein synthesis. Positive polarity in vitro transcripts from 3′-proximal MCMV cDNA clones direct the synthesis of the capsid protein in in vitro translation experiments. These data suggest that the MCMV capsid protein is expressed from a subgenomic RNA in vivo , and that the 25-kDa capsid protein is encoded by the 3′-proximal open reading frame in the MCMV genome.

The P1 cistron encodes the first and most variable part of the polyprotein of potyviruses. A site tolerant to a pentapeptide insertion at the N-terminus of Potato virus A P1 (Genome Res. 12, 584–594) was used to express heterologous proteins (insertions up to 783 nucleotides) with or without flanking new proteolytic sites. Aequorea victoria green fluorescent protein (GFP) accumulated to high levels when proteolytically released from P1 and showed strong fluorescence in leaves systemically infected with vector virus. Deletions in GFP and adjacent viral sequences emerged 2–4 weeks after infection, revealing putative recombination hot spots. The inserts in P1 diminished infectivity host-specifically, reduced virus accumulation in protoplasts and systemically infected leaves, alleviated symptoms and reduced accumulation of mRNA and HCpro in cis in a virus-free system. This heterologous protein expression site is the first within a protein-encoding cistron and the third in the genome of potyviruses.

This study demonstrates that sequences homologous to those of the non-retro RNA virus (Potato virus Y; PVY) are integrated into the genome of several grapevine varieties. The integrated PVY-coat-protein-like cistron is expressed in the grapevine as indicated by Southern and Western blot analyses as well as by RNase protection assay. In addition, genome-walking studies showed that one PVY-like sequence is flanked by 41-bp direct repeats and is embedded in authentic grapevine sequences, flanked by inverted repeats. Rearranged PVY-like sequences were also found in tobacco. It is suggested that nonhomologous recombination of a potyviral RNA with RNA of a retrotransposable element took place at some point in evolution. The initial integration locus was probably within a grapevine gene homologous to a pentatricopeptide repeat-carrying protein, and was later transposed to other locations. The current location is reminiscent of a MITE-type retroelement, indicating transposition history. Because grapevine cultivars are propagated asexually, without going through a meiotic phase, the chance for DNA recombination is minimal and the foreign integrated sequence may be better conserved, enabling it to be expressed correctly in the recipient genome.

The complete nucleotide sequence of the 5′-untranslated region (5′-UTR) of the genome of three ECHO virus type 25 strains (the JV4 reference strain and two wild isolates) was determined. The 5′-UTR of the two isolates shared 85 and 82% nucleotide identity with the reference strain. The overall folding of the predicted secondary structure of the ECHO virus 5′-UTRs showed significant conservation with that of the poliovirus. Most significant differences were observed in specific domains of the predicted IRES of the wild isolates. The efficiency with which ECHO virus type 25 5′-UTRs promoted internal initiation of translation was examined in anin vitrotranslation system. At low doses of input RNA, all three 5′-UTRs were similarly efficient in promoting internal ribosome entry and behaved similarly to the poliovirus IRES in that downstream cistron translation was markedly stimulated by the addition of HeLa cell extracts to standard reticulocyte lysates. At high RNA doses, the JV4 5′-UTR was much more efficient than the other two ECHO virus 5′-UTRs. This difference was much less marked when reticulocyte lysates were supplemented with 20% HeLa S-10 extracts, which suggests that the two less efficient 5′-UTRs were characterized by a reduced binding capacity to at least one factor present in HeLa cells. In MRC5 cell S-10 extract, internal initiation of translation was stimulated from the IRES of all three ECHO strains, the M1262 IRES being the least stimulated.

A short upstream open reading frame (uORF2) in the human cytomegalovirus (CMV) gpUL4 (gp48) transcript leader is conserved among CMV strains and inhibits translation of a downstream cistron. Remarkably, this inhibitory effect depends on the amino acid coding information of uORF2, at least in transient transfection assays in diploid human fibroblasts. Using retroviral vectors, we now report that the gp48 leader inhibits downstream translation in multiple additional cell types, even when expressed from a stably integrated gene, and on a transcript containing an additional kilobase of complex leader sequences. The magnitude of inhibition can be augmented approximately 3- to 10-fold by replacing the context of nucleotides flanking the wild-type initiation codon of uORF2 with an optimal context, suggesting that leaky scanning past the wild-type AUG codon accounts for translation of the downstream cistron. Using an in vivo mutagenesis protocol that relies on reverse transcriptase infidelity, we isolated mutants in which the inhibitory effect of the gp48 leader was inactivated as a result of alterations in the coding information of uORF2. These studies demonstrate that, independent of the cell type or expression system used, CMV gp48 uORF2 is a potent translational inhibitory element.

The expression of plum pox potyvirus (PPV) genomic RNA takes place through translation of its unique long and functional open reading frame (ORF) into a large polyprotein that undergoes extensive proteolytic processing. In this paper we show that the AUG recognized as the initiation codon of the PPV ORF by in vitro translation systems is the one found at nucleotide position 147, in spite of the presence at position 36 of an in-phase AUG that marks the start of the ORF. Deletion of a substantial part of the PPV 5′ nontranslated region (5'-NTR), from nucleotide 19 to 101, does not impair the in vitro translation of PPV synthetic transcripts. By introduction of mutations that disrupt either of these two AUGs into a full-length PPV cDNA clone, it is shown that, while alteration of the first AUG does not have any effect on virus viability, growth, or symptom induction, destruction of the second renders the viral RNA noninfectious. This result indicates that the AUG employed in vivo is also the second. The hypothesis that this AUG could be recognized through a ribosomal internal entry mechanism has been tested in vitro using various bicistronic transcripts in which the PPV 5′-NTR was internally placed. The second cistron of these bicistronic RNAs was translated, but only at low levels, indicating that the PPV 5′-NTR is not able to drive in vitro an efficient internal entry of the ribosomes and suggesting that PPV RNA translation might proceed through a conventional leaky scanning mechanism.

The complete nucleotide sequences of the matrix (M) and fusion (F) genes of simian virus 41 (SV41) were determined. Deduced amino acid sequences confirmed the close relationship of SV41 with human parainfluenza type 2 virus (PIV2). Analyses of noncoding regions between the F and the hemagglutinin-neuraminidase (HN) genes suggested the absence of the small hydrophobic gene, which is present between the F and the HN genes of simian virus 5 and mumps virus. It was striking that there was no apparent consensus gene end sequence between the M and the F genes and that the M gene was transcribed exclusively as a dicistron with the F gene. The number of monocistronic transcripts of the F gene was approximately half that of the dicistronic transcripts. However, the F protein of SV41 seemed to be efficiently translated, since viral multiplication and fusion from within were as efficient as in PIV2. These results suggest that the lack of a consensus gene end sequence resulted in the readthrough of viral RNA polymerases between the M and the F genes and that the initiation of F gene transcription could occur by newly entered polymerases independently of the polymerases that started the upstream M gene transcription.

A nonaphid transmissible (NAT) variant of tobacco vein mottling virus (TVMV) was used to test the hypothesis that the viral coat protein (CP) plays a role in determining aphid transmissibility. Comparison of the nucleotide sequences in the coat protein cistron of an aphid transmissible isolate (TVMV-AT) with that of TVMV-NAT revealed a single nucleotide difference (G----A) at position 8445; this alters a single amino acid residue (G----E) at position 2747. A cDNA fragment representing the CP region of TVMV-NAT was substituted into the CP region of a full-length cDNA clone of TVMV-AT, and transcribed RNA was inoculated to tobacco plants. Aphids were unable to transmit the resultant hybrid virus which had the TVMV-NAT coat protein, although the concentration and infectivity of the hybrid virus in the source plants were similar to those of TVMV-AT. This is the first direct demonstration that a CP mutation affects aphid transmissibility of a potyvirus.

Minus strand RNA synthesis by the positive strand alphaviruses, Sindbis and Semliki Forest viruses, normally occurs early in infection, is coupled to synthesis of viral nonstructural proteins and to formation of viral replication complexes, and terminates and does not occur late in infection. Previously, ts24 of the A complementation group of Sindbis virus RNA-negative mutants was found to possess, among its other temperature sensitive defects, a temperature sensitivity in the normal cessation of minus strand synthesis which enabled minus strands to be synthesized late in infection at 40 degrees in the absence of protein synthesis. Revertants of ts24 (ts24R1, ts24R2) retained the defect in the shutoff of minus strand synthesis, indicating the lesion was not conditionally lethal and could map outside the A cistron. The studies reported here used an infectious clone of Sindbis virus to identify the mutation responsible for this phenotype. Hybrid viruses were prepared from constructs containing restriction fragments of the cDNA of ts24R1 in place of the corresponding fragments in the infectious SIN HR clone and screened for their ability to synthesize minus strands at 40 degrees in the presence of cycloheximide. A unique base change of an A for a C residue at nt 6339, predicting a change from glutamine to lysine at amino acid 195 in nsP4, was found in genomes of ts24, ts24R1, and ts24R2. Other nucleotide changes present at the 5' and 3' termini did not affect minus strand synthesis. The substitution of the parental Sindbis virus sequence that encompassed nt 6339 in an infectious clone of the ts24R1 revertant eliminated the mutant phenotype. We conclude that the ability to continue minus strand synthesis at 40 degrees exhibited by ts24 and its revertants is caused by an alteration in nsP4, which is the alphavirus replicase or an essential component of the replicase. We hypothesize that this domain of nsP4 functions to fix the minus strand as the stable template of alphavirus replication complexes.

Purified preparations of TYMV contain a number of minor nucleoprotein components distinguishable on the basis of their density in CsCl gradients (R. E. F. Matthews, Virology 12 , 521–539, 1960). When analysed by polyacrylamide-gel eletrophoresis, the RNA from each of these various components was found to consist of a characteristic set of molecular-weight species. Full-size RNA (2 × 10 6 daltons) was present only in nucleoproteins B 1 and B 2 . Nucleoproteins B 0 , B 00 , and B 000 contained RNAs ranging in size from approx 1.3 to 0.28 × 10 6 daltons. The 0.28 × 10 6 -dalton RNA species was detectable in all nucleoprotein fractions, but was a significant proportion of the RNAs of B 000 and B 00 . Translation of the RNA from each of the nucleoproteins in the wheat germ cell-free protein synthesizing system yielded essentially similar patterns of polypeptides which ranged in size from 5000 to 70,000 daltons. The major radioactive product migrated on SDS-polyacrylamide gels to the same position as TYMV coat protein. The data suggest that the 0.28 × 10 6 -dalton RNA component is the cistron for coat protein, and that it and other RNAs associated with TYMV infection are encapsidated.

An approach to correlate functions with cistrons in bacteriophage PM2 is presented. Two of the temperature-sensitive mutants obtained differed from the wild type in heat sensitivity and four differed in host range. Comparison of the electrophoretic patterns of DNA from cells infected at the restrictive temperature with those obtained in wild-type infection revealed that viral DNA bands were absent with three additional mutants. Complementation analysis assigned the four host range mutants to cistron I and the three mutants defective in DNA synthesis to cistron IV. Recombination frequencies were used to locate cistrons III and IV on the partial genetic map of PM2.

The nature of DNA synthesis and recombination has been examined in bacteriophage T4D mutants deficient in recombination and repair. The interactions of several of these mutations have been assessed by measuring DNA synthesis, uv sensitivity, and genetic recombination in multiply mutant strains. Certain multiple mutants exhibited a 60-fold reduction in recombination frequency. Gene x - and gene y -deficient strains each exhibited an arrest in DNA synthesis and a concomitant interruption of concatemer formation as measured by alkaline sucrose density gradient centrifugation. The arrest in DNA synthesis was suppressed by the presence of a second mutation in gene 55. Strains carrying x and y mutations failed to complement mutants fds A and fds B, respectively. A gene w mutation was found to suppress the arrest in DNA synthesis caused by mutations in genes 46–47 and in gene 59. In the case of genes 46–47, genetic recombination was not restored, while for gene 59 mutants genetic recombination and uv resistance were partially restored. A gene w mutation did not suppress the DNA arrest phenotype produced by mutations in the x or y genes. The T4B strain 1206 was found to contain a mutation in gene w , along with several uncharacterized mutations.

Electron microscopic length measurement of Eco RI limit digest products of mature P2 DNA reveals four fragments, A, B, C and D, of defined length. Under our standard conditions, the efficiency of plating of P2 on an RI-carrying strain is 7–10%. An exception is the deletion mutant P2 del2 having a plating efficiency of 30–40%. Electron microscopy of Eco RI digests of P2 del2 DNA shows that del2 removes the middle RI-sensitive site. Since the location of del2 is precisely known from previous work, and the two end segments are sometimes observed joined through their cohesive ends, it is possible to locate the three sites (leftmost, middle, and rightmost) at 10.5, 47.5, and 79.9% from the left end of P2 DNA, respectively. The rightmost site is most probably within cistron B. A mutant, P2 del2 sr1 , was isolated which has a plating efficiency of 70–90% on the RI-carrying host. Electron microscopy of P2 del2 sr1 DNA and phage crosses show that mutation sr1 makes the rightmost site resistant to Eco R1. The sr1 mutation, when separated from del2 , gives a plating efficiency of 20–30% on the RI-carrying host. It would seem that the three sites do not contribute equally to the lowering of the efficiency of plating.

Temperature-sensitive B, C, and BC mutants of Simian virus 40 (SV40) map in the late region of the viral genome in Hin fragments K, F, J, and G, a DNA segment of about 1200 nucleotide pairs ( Lai, C.-J., and Nathans, D. (1975) Virology 66, 70–81). To define the B/C region further, mutants of SV40 with deletions in this genomic segment were constructed by enzymatic excision of DNA from the viral genome, followed by cloning in the presence of a complementing ts A mutant of SV40. After localization of deleted genome segments by analysis of endo R fragments and electron microscopic heteroduplex mapping, selected deletion mutants were tested for complementation by ts mutants and were screened for their ability to produce new viral proteins in infected cells. Complementation tests indicated that B/C deletion mutations are in a cistron distinct from that of ts A and ts D mutations and that the junction between the B/C and D genes is within Hin -K. Two of the deletion mutants produced new proteins detectable in infected cells. More detailed analysis of one of these proteins (of molecular weight 25,000) indicated that it precipitated with antiserum against dissociated SV40 capsids, and that all but one of its lysine-containing tryptic peptides cochromatographed with SV40 VP1 tryptic peptides. We conclude that the B/C gene, containing approximately 1200 nucleotide pairs, codes for VPl. Since deletion mutants lacking Hin -E do not complement B mutants, we suggest that the Hin -E DNA segment has a signal required for expression of the B/C gene.

Some physical and chemical properties of the V strain of tomato aspermy virus (TAV) and the Q strain of cucumber mosaic virus (CMV) have been compared. The size, morphology, sedimentation rate, RNA base ratio, and buoyant density of the two viruses are indistinguishable. Preparations of RNA from both viruses were each resolved into four distinct species by polyacrylamide-gel electrophoresis. TAV-RNA preparations contained species with molecular weights of 1.26 × 10 6 ,1.10 × 10 6 , 0.90 × 10 6 , and 0.43 × 10 6 daltons, and CMV-RNA, species of 1.26 × 10 6 , 1.10 × 10 6 , 0.77 × 10 6 , and 0.34 × 10 6 daltons. Analysis of sodium dodecyl sulphate (SDS)-treated viral proteins by polyacrylamide-gel electrophoresis showed that both viruses have protein subunits of molecular weight 24,500 daltons. The amino acid compositions of proteins from the two viruses, although similar, were distinguishable, and the calculated molecular weights of protein subunits were 26,100 and 26,300 daltons for TAV and CMV, respectively. The two viruses were serologically distinct. On the data presented it is suggested that in preparations of both TAV and CMV three distinct particles are present in each with identical protein shells, but different RNA cores.

Nucleotide sequence determination of the coat protein cistron of the alfalfa mosaic virus (AIMV) temperature-sensitive mutant, Tbts 7 (uv) revealed a small number of point mutations of which only one results in the replacement of an amino acid: the asparagine residue at position 126 is replaced by an aspartate residue. RNA transcribed in vitro from a Tbts 7 cDNA 4 clone directed the production in vitro of a polypeptide which shows the same altered electrophoretic mobility in SDS-polyacrylamide gels as the Tbts 7 coat protein. Nucleotide sequence analysis of the 32-kDa open reading frame revealed some base changes, but none of these lead to changes in the primary structure of the protein. The 5′-terminal sequence of Tbts 7 RNA 3 was analyzed by cDNA cloning. At least three different types of nontranslated leader sequences were found, indicating considerable heterogeneity at the 5′ end of the mutant RNA 3. The results indicated that the low abundance of RNA 3-containing particles in Tbts 7 virus preparations might be due to malfunctioning of the 5′ terminus of Tbts 7 RNA 3 during replication.

A mutant (Tbts7) of alfalfa mosaic virus, the coat protein of which is unable to activate the viral genome (the RNA species 1, 2, and 3, which need some coat protein for infectivity) at 30 degrees , can be rescued at this temperature by adding to the inoculum wild-type RNA 3 (the genome part that contains the coat protein cistron), but not adding wild-type RNA 4 (the subgenomic messenger for the coat protein). Unless RNA 3 of Tbts 7 has a second ts mutation at a site not occurring in RNA 4, it may be concluded from the above finding that RNA 4 does not replicate autonomously.

Temperature-sensitive mutants of simian virus 40 (SV40), representing each complementation class, have been mapped by marker rescue with Endo R fragments of wild-type SV40-DNA. The 41 mutants mapped cover most of the genome, but there is a notable absence of mutants mapping between 0.43 and 0.85 map-units. Mutants which are defective in viral DNA synthesis (tsA mutants) all map in the “early” region of the genome; 12 of 13 such mutants are clustered in about one-fourth of the “early” region between 0.32 and 0.43 map-units. Mutants in complementation classes defective in a late function map in the “late” region and also show clustering: eight of nine B mutants map between 0.94 and 0.06 map-units, all C mutants map between 0.06 and 0.11 map-units, and seven of eight BC mutants map between 0.11 and 0.17 map-units. D mutants, which are thought to be defective in virus uncoating at high temperature, all map between 0.85 and 0.94 map-units, i.e., in a small segment of the “late” region. Therefore, the genomic segment associated with the defect of D mutants likely codes for a virion protein. One C mutant proved to be a double mutant with one mutation in the B region and another in the C region, both mutations being required for the tsC phenotype. On the basis of the mapping data and prior complementation tests we suggest that B, C, and BC mutations are in one cistron, complementation occurring at the protein level.

Mapping of the h and hh∗ host-range mutations in phage λ by two-point crosses with reference J − point mutations, and with λ gal deleted for part of J , locates these mutations in the promoter-distal portion of the J cistron. Analysis of phenotypic h + recombinants, formed in crosses of the type h or hh∗ × J t- , or h + revertants of h, hh∗ , and J def mutants, indicates that such phenotypic h + particles often retain cryptic h determinants. Similar determinants are also present in some common laboratory strains of λ. These h + recombinants and revertants carry a variety of different h markers, since recombination analysis allows several classes of particles carrying cryptic h markers to be distinguished. These genetic data suggest that the extended host-range phenotype in λ is due to multiple rather than single, mutations in the distal region of gene J , although the number of sites involved and their arrangement remain uncertain. The genetic location of the h and hh∗ mutations is confirmed at the physical level by comparing the tryptic peptide maps of the J proteins purified from lysates of cells infected with different h + , h, hh∗, J am , and J 434 phage and from purified λh + virions. Examination of these peptide maps shows there are several methionine-containing peptides altered in the h and hh∗ maps. Some of these altered peptides are derived from the C-terminal 5–10% of the J polypeptide in the region of nonhomology between λ and 434.

High-density, defective DNA ( ϱ CsCl = 1.732 g/cm 3 ) produced in infections of the 15th through 20th undiluted serial passage of herpes simplex virus type 1 (HSV-1), Strain KOS, was purified by preparative CsCl density gradient ultracentrifugation. Purified defective DNA expressed no discernible cytopathic effects when transfected into receptive cell monolayers. Cleavage of defective DNA preparations with EcoRI restriction endonuclease revealed four major and three minor bands upon electrophoresis in agarose gels. Restriction endonuclease Hind III failed to cleave defective DNA. Marker rescue studies revealed that of 13 temperature-sensitive mutants representing 12 complementation groups, only mutants in group B were rescued by defective DNA. These studies demonstrate, therefore, that defective DNA is enriched for at least part of the B cistron and suggest that this cistron is located in the region of the viral genome from which defective DNA is generated.

Preparations of the cowpea strain of TMV contain both large and small particles. RNA extracted from these particles has been translated in a cell-free protein synthesizing system derived from wheat germ. The polypeptide products have been characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), by immunoprecipitation followed by SDS-PAGE and also by two-dimensional tryptic peptide mapping of the immunoprecipitated material. Coat-protein was shown to be a major product of in vitro protein synthesis directed by total viral RNA. It was synthesized in a form slightly larger than the long polypeptide of the virus capsid as judged by peptide mapping and incorporation of [35S]methionine, an amino acid not present in B-TMV coat-protein. RNA extracted from a purified preparation of short particles stimulated the wheat-germ system to synthesize coat-protein almost exclusively. It was concluded that short particles carry a cistron for coat-protein. RNA isolated from a preparation of long particles programmed the synthesis of a series of polypeptides ranging in size from 130,000 to 5000 daltons, and including some coat-protein. More extensive purification of the high molecular weight RNA showed that the synthesis of coat-protein was not attributable to this RNA but to residual low molecular weight RNA from short particles. Thus RNA from long particles resembles RNA from Ul-TMV in its inability to program a wheat-germ system to synthesize appreciable amounts of coat-protein. Evidence is given elsewhere that long particle RNA actually contains a cistron for coat-protein. Translation of long RNA could be suppressed by the addition of short RNA to the in vitro system, resulting in a polypeptide profile dominated by coat-protein. Initiation sites in the coat-protein cistron on the short RNA must therefore be preferentially recognized by the wheat-germ protein synthesizing system.

Newly synthesized products of replication and transcription from vesicular stomatitis virus (VSU)-infected HeLa cells have been separated on Renografin gradients and characterized. The replicative products have been identified by the following criteria: (1) sucrose-velocity gradients and methyl-mercury agarose gel analyses show RNA of sizes between 18 S and genome size 42 S. (2) This RNA is predominantly minus stranded by hybridization analyses. (3) A majority of the nascent RNA molecules of less than half genome size from replicative complexes are complementary to the 3′ end of the VSV plus strand, i.e., the L protein cistron. These short nascent RNA species are nuclease resistant and thus are presumed to be contained in ribonucleoprotein complexes.

25 S TMV protein, when added to a mixture of TMV RNA fragments obtained by partial T1 RNase digestion, is able to recognize only a few species of RNA fragments, all of them deriving from an unique sequence of 103 nucleotides that represents a part of the coat protein cistron. This selective encapsidation leads to the formation of two types of stable nucleoprotein complexes. These complexes have sedimentation coefficients of 26 and 34 S, contain about half the amount of RNA than native TMV and are resistant to pancreatic ribonuclease. Added to a mixture of intact TMV RNA and 25 S TMV protein, the specifically encapsidated RNA fragments (SERF) extracted from the 26 and 34 S particles induce a rapid inhibition of the reconstitution process as shown by light scattering measurements. This inhibition due to the rapid encapsidation of SERF by 25 S TMV protein, demonstrates that TMV protein has a great affinity for SERF even in the presence of native TMV RNA. The affinity is specific since other polynucleotides such as poly(A) or tRNA do not cause the same inhibitory effect, i.e., they do not compete with TMV RNA for 25 S TMV protein.

Double-stranded cDNA copies of cucumber green mottle mosaic virus (watermelon strain, CGMMV-W) RNA polyadenylated in vitro were cloned into the pBR322 at the PstI site. The sequence of 1071 nucleotides from the Tend of the genomic RNA was determined using two recombinant plasmids and the genomic RNA. The coat protein cistron was located in residues 176–661 from the 3′ end. The coat protein was composed of 160 amino acid residues with the molecular weight of 17,261. The 3′ noncoding region of the CGMMVW genome was 175 nucleotides long and highly homologous to that of the common strain of TMV. The assembly origin of reconstitution is positioned within the coat protein cistron as predicted previously. In the 5′ flanking region of the coat protein cistron a long open frame, probably of 30K protein, was found. The predicted 30K and the coat protein cistron would overlap each other as is the case of the cowpea strain of TMV.

The 3' terminal 337 bases of BMV (brome mosaic virus) coat protein mRNA (BMV RNA4) are presented. This sequence includes the terminal portion of the coat protein cistron and the complete 300-base 3' noncoding sequence. The 3' noncoding sequence displays significant complementarity to the 5' terminal sequence of BMV RNA3 but not to the 5' terminal sequence of BMV RNA4.

Turnip yellow mosaic virus (TYMV) RNA extracted from virions (“virion” TYMV-RNA) contains two types of molecules of molecular weight 2 × 10 6 : one which resists heat denaturation (“intact” TYMV-RNA) and one which yields smaller fragments upon heat denaturation (TYMV-RNA containing hidden breaks). A small RNA molecule of 0.2 × 10 6 MW is also present in virions. “Virion” TYMV-RNA can therefore be separated into “intact” RNA and smaller molecules by heat denaturation followed by sucrose gradient centrifugation. The RNA from the sucrose gradient fractions were analyzed by formamide PAGE: The small RNA with a molecular weight of 0.2 × 10 6 was separated from “intact” TYMV-RNA. The amount of coat protein synthesized by the RNA fractions when added to the wheat germ cell-free system was quite proportional to the amount of small RNA present. We conclude that coat protein synthesis is not directed by “intact” TYMV-RNA but by the small RNA. In addition, infectivity studies show that “intact” TYMV-RNA is fully infectious and thus it also contains the coat protein cistron.

The nature of the polypeptide products encoded by the 5'-terminal region of the RNA of the potyvirus, tobacco vein mottling virus (TVMV), was investigated. Single-stranded DNA probes complementary to either nucleotides 1100-2100 or 2100-2820 from the 5' terminus of the RNA were prepared by subcloning recombinant plasmids in bacteriophage M13. These were hybridized to TVMV RNA, the DNA:RNA hybrids translated in a reticulocyte lysate cell-free translation system (hybrid-arrested translation), and the products analyzed by electrophoresis on SDS-containing polyacrylamide gels. The hybrids produced altered patterns of polypeptides which indicated that the major product, P75, was translated from the 5' terminus of the RNA. The N-terminal portion (molecular weight approximately 35,000) of P75 did not react with antisera to any of the five known potyviral proteins, suggesting the existence of a previously unidentified cistron at the 5' terminus. Translation of additional RNA sequences produced a polypeptide with a molecular weight of 68,000 which was immunoprecipitable by antiserum to TVMV helper component, establishing the coding region for helper component near the 5' terminus. Limited DNA sequence analysis of the region encoding the C terminus of P75 revealed an open reading frame of 369 nucleotides followed by a pair of UAG Codons. Pulse-chase experiments demonstrated that in the first 10 min of incubation, the only polypeptide initiated was P75, suggesting that products downstream from P75 arose from in vitro fragmentation of TVMV RNA and translation of the RNA fragments.

Mutations in 2 of the 36 cistrons known for Bacillus subtilis phage SPOT were unmappable because they showed high frequencies of recombination with each other and with mutations in all other cistrons (Okubo, S., Yanagida, T., Fujta, D. J., and Ohlsson-Wilhelm, B. M., 1972, Biken J. 15 , 81–97). These cistrons (35 and 36) are among the last to be replicated and their replication is specifically inhibited by mutants affected in SPOT cistron 32 (Glassberg, J., Franck, M., and Stewart, C. R., 1977a, J. Virol. 21 , 147–152). We propose that these observations can be explained by the location of these two cistrons within a region of terminal redundancy, and we present the following data in support of this proposal: (1) Mixed infection between sus 35 + and sus 35 − phage yields bursts which include a high proportion of heterozygotes. (2) The sus 35 genetic marker is located on two different restriction fragments. (3) By restriction nuclease mapping, one copy of this marker has been located to the right of the rightmost markers on the known genetic map. (4) Two of the fragments produced by Eco RI ∗ digestion of mature DNA are not found in digests of intracellular DNA, and no new fragment is found, suggesting that these fragments are part of a region of terminal redundancy used for the formation of concatemers. One of these fragments carries the sus 35 marker.

Cross reactivation, multiplicity reactivation, and functional cistron survival have been investigated for bacteriophage T4D X-irradiated in buffer suspension before infection. Rescue is inefficient when compared with identical phenomena after ultraviolet irradiation. Analysis of multiplicity reactivation after X-irradiation supports the inference that two non-plaque-forming phages, have, in mixed infection, a probability approaching zero of producing live progeny. Implications of this work for a study of the recombinogenic effects of X-rays are discussed.

The c III and c III cistrons of lambda play an important role in the lysogenization of a bacterium, but they are not required for the maintenance of the resulting lysogenic strain. As gauged by their ability to complement a mutant superinfecting heteroimmune phage, these cistrons fail to function in an immune cell. This was true whether the wild-type cistron was located in the prophage or in another superinfecting but repressed phage. These c cistrons did function in a lysogenic host so long as they were carried in a genome whose immunity specificity differed from that of the prophage. Although c II + function is necessary to lysogenize a sensitive bacterium, it was not a limiting factor in the formation of a doubly lysogenic cell following superinfection of an already lysogenic bacterium by a homoimmune phage.

Ninety-six conditional lethal mutants of coliphage M13 have been isolated and assigned to complementation groups as part of a program to determine the number of cistrons and their functions in this small DNA virus. Genetic complementation tests show that the 63 temperature sensitive (ts) mutants isolated fall into 5 cistrons and the 33 amber (am) mutants into these same 5 and 1 additional cistron. With the limited evidence on hand it is not possible to conclude whether these comprise all the cistrons in the phage. Temperature shift experiments carried out on ts mutants from cistrons 1 through 5 show that cistron 2 starts to act during the early minutes of M13 infection, while cistrons 1, 3, 4, and 5 start to act only late in the latent period. The functions of all 5 of these cistrons are needed to maintain phage production once it has begun. The phage production stops immediately if any of the late functions are blocked, but only gradually after the early function is blocked. Mutants from the early and late cistrons differ dramatically from each other in their effect on host cells under nonpermissive conditions. Neither am nor ts mutants in the early cistron affect the viability or growth rate of the infected cells, while the mutants in all the late cistrons kill the cells, stopping their growth soon after infection. The wild-type M13 phage does not kill the cells under normal conditions of infection, but does decrease their growth rate. It appears that the host cells are killed when the early cistron is operative and phage production is prevented by a block in any one of the late cistrons.

An in vivo assay for the product of the N gene of bacteriophage lambda is described. Using this assay we show that (1) expression of the N gene starts during the first minute of the vegetative cycle; (2) the N product is functionally unstable between 30° and 35° but stable at 4°; (3) the phage repressor prevents expression of the early genes located to the left of N, even in the presence of N product; (4) the amount of N product synthesized by wild-type phage lambda is in large excess of what is required to obtain maximum expression of the early genes located to the left of N.

The problem raised by the ultraviolet (UV)-target sizes of T4 cistrons determined by the survival of phenotype (SP survival) of irradiated wild-type phage in mixed infection with unirradiated carrier ( Krieg, 1959 ) is investigated. It is noted that the greatly overestimated target sizes obtained by this latter method particularly for macrotarget functions ( Womack and Barricelli, 1965 ) are comparable to the mean target size of the three vulnerable centers determined by multiplicity reactivation ( Barricelli, 1956 ). A suggestion by Harm (1966) that the three vulnerable centers represent early functions required for repairing processes like V-gene reactivation, X-gene reactivation, and possibly other similar processes hereafter collectively designated as “spontaneous reactivation,” is used as a basis for the following interpretation: the greatly overestimated SP-target sizes are assumed to be the effect of a lack of or a reduction of spontaneous reactivation, when an irradiated phage competes with unirradiated phages for phenotypic expression. In order to test this interpretation, a series of SP-survival experiments has been done in which not only the wild-type phage, but also the carrier was irradiated. In each experiment only one UV dose to the carrier (usually smaller than all UV doses given to the wild-type phage) was used while the wild type received several UV doses in order to obtain an SP-survival diagram for irradiated carrier. A UV-dose to the carrier is expected to give a certain dose-dependent probability for spontaneous reactivation of damages in the wild-type cistron of the coinfecting phage. This is a necessary consequence of multiplicity reactivation theory ( Barricelli, 1956 , Barricelli, 1960 ) and the above interpretation ( Harm, 1966 ) implying that, when all phages are equally irradiated, spontaneous reactivation normally occurs in all cistrons except the 3 vulnerable centers. As a result the SP-survival values with irradiated carrier are expected to be significantly higher than one would expect if all damages in the wild-type cistron measured by the SP-survival method were phage lethal damages (instead of partly spontaneously reparable or “sublethal”). In fact it can be expected that a small UV-dose to the carrier may, under certain conditions, increase rather than reduce the SP-survival. All these predictions are confirmed by the experiments. Furthermore it has been possible to outline some of the rules specifying the conditions for spontaneous reactivation of the sublethal wild-type cistron damages, thus obtaining a fairly accurate interpretation of the experimental data. Although the outlined theory is still rudimentary it is already clear that UV-hits (sublethal as well as lethal) in the mutant cistron of the carrier phage are the main factor which makes possible spontaneous reactivation of sublethal damages in the wild-type cistron. This effect does not have to be due to a stimulation of the spontaneous reactivation processes, in a selective manner for the cistron involved. Simply by delaying or preventing the production of the mutant protein, damages in the mutant cistron may buy the time necessary for spontaneous reactivation of the wild-type cistron and production of wild-type protein in time to establish the wild phenotype. This would be possible if the mutant protein is not deprived of all function but is, on the contrary, able, although in a lethal or abnormal fashion, to carry the processes of infection to a stage in which the wild-type protein would no longer be effective. On the other hand, the absence of both proteins may interrupt or suspend the processes at an earlier stage so that the wild phenotype may be expressed as soon as the appropriate protein becomes available.