Leader sequences downstream of the primer binding site are important for efficient replication of simian immunodeficiency virus

Simian immunodeficiency virus (SIV) infection of macaques is remarkably similar to that of human immunodeficiency virus type 1 (HIV-1) in humans, and the SIV-macaque system is a good model for AIDS research. We have constructed an SIV proviral DNA clone that is deleted of 97 nucleotides (nt), i.e., construct SD, at positions (+322 to +418) immediately downstream of the primer binding site (PBS) of SIVmac239. When this construct was transfected into COS-7 cells, the resultant viral progeny were severely impaired with regard to their ability to replicate in C8166 cells. Further deletion analysis showed that a virus termed SD1, containing a deletion of 23 nt (+322 to +344), was able to replicate with wild-type kinetics, while viruses containing deletions of 21 nt (+398 to +418) (construct SD2) or 53 nt (+345 to +397) (construct SD3) displayed diminished capacity in this regard. Both the SD2 and SD3 viruses were also impaired with regard to ability to package viral RNA, while SD1 viruses were not. The SD and SD3 constructs did not revert to increased replication ability in C8166 cells over 6 months in culture. In contrast, long-term passage of the SD2 mutated virus resulted in a restoration of replication capacity, due to the appearance of four separate point mutations. Two of these substitutions were located in leader sequences of viral RNA within the PBS and the dimerization initiation site (DIS), while the other two were located within two distinct Gag proteins, i.e., CA and p6. The biological relevance of three of these point mutations was confirmed by site-directed mutagenesis studies that showed that SD2 viruses containing each of these substitutions had regained a significant degree of viral replication capacity. Thus, leader sequences downstream of the PBS, especially the U5-leader stem and the DIS stem-loop, are important for SIV replication and for packaging of the viral genome.     

Partial Restoration of Replication of Simian Immunodeficiency Virus by Point Mutations in either the Dimerization Initiation Site (DIS) or Gag Region after Deletion Mutagenesis within the DIS

We used the simian immunodeficiency virus (SIV) molecular clone SIVmac239 to generate a deletion construct, termed SD2, in which we eliminated 22 nucleotides at positions +398 to +418 within the putative dimerization initiation site (DIS) stem. This SD2 deletion severely impaired viral replication, due to adverse effects on the packaging of viral genomic RNA, the processing of Gag proteins, and viral protein patterns. However, long-term culture of SD2 in either C8166 or CEMx174 cells resulted in restoration of replication capacity, due to two different sets of three compensatory point mutations, located within both the DIS and Gag regions. In the case of C8166 cells, both a K197R and a E49K mutation were identified within the capsid (CA) protein and the p6 protein of Gag, respectively, while the other point mutation (A423G) was found within the putative DIS loop. In the case of CEMx174 cells, two compensatory mutations were present within the viral nucleocapsid (NC) protein, E18G and Q31K, in addition to the same A423G substitution as observed with C8166 cells. A set of all three mutations was required in each case for restoration of replication capacity, and either set of mutations could be substituted for the other in both the C8166 and CEMx174 cell lines.     

Novel, live attenuated simian immunodeficiency virus constructs containing major deletions in leader RNA sequences

We have constructed a series of simian immunodeficiency virus (SIV) mutants containing deletions within a 97-nucleotide (nt) region of the leader sequence. Deletions in this region markedly decreased the replication capacity in tissue culture, i.e., in both the C8166 and CEMx174 cell lines, as well as in rhesus macaque peripheral blood mononuclear cells. In addition, these deletions adversely affected the packaging of viral genomic RNA into virions, the processing of Gag precursor proteins, and patterns of viral proteins in virions, as assessed by biochemical labeling and polyacrylamide gel electrophoresis. Different levels of attenuation were achieved by varying the size and position of deletions within this 97-nt region, and among a series of constructs that were generated, it was possible to rank in vitro virulence relative to that of wild-type virus. In all of these cases, the most severe impact on viral replication was observed when the deletions that were made were located at the 3' rather than 5' end of the leader region. The potential of viral reversion over protracted periods was investigated by repeated viral passage in CEMx174 cells. The results showed that several of these constructs showed no signs of reversion after more than 6 months in tissue culture. Thus, a series of novel, attenuated SIV constructs have been developed that are significantly impaired in replication capacity yet retain all viral genes. One of these viruses, termed SD4, may be appropriate for study with rhesus macaques, in order to determine whether reversions will occur in vivo and to further study this virus as a candidate for attenuated vaccination.     

Compensatory Point Mutations in the Human Immunodeficiency Virus Type 1 Gag Region That Are Distal from Deletion Mutations in the Dimerization Initiation Site Can Restore Viral Replication

The dimerization initiation site (DIS), downstream of the long terminal repeat within the human immunodeficiency virus type 1 (HIV-1) genome, can form a stem-loop structure (SL1) that has been shown to be involved in the packaging of viral RNA. In order to further determine the role of this region in the virus life cycle, we deleted the 16 nucleotides (nt) at positions +238 to +253 within SL1 to generate a construct termed BH10-LD3 and showed that this virus was impaired in viral RNA packaging, viral gene expression, and viral replication. Long-term culture of these mutated viruses in MT-2 cells, i.e., 18 passages, yielded revertant viruses that possessed infectivities similar to that of the wild type. Cloning and sequencing showed that these viruses retained the original 16-nt deletion but possessed two additional point mutations, which were located within the p2 and NC regions of the Gag coding region, respectively, and which were therefore named MP2 and MNC. Site-directed mutagenesis studies revealed that both of these point mutations were necessary to compensate for the 16-nt deletion in BH10-LD3. A construct with both the 16-nt deletion and the MP2 mutation, i.e., LD3-MP2, produced approximately five times more viral protein than BH10-LD3, while the MNC mutation, i.e., construct LD3-MNC, reversed the defects in viral RNA packaging. We also deleted nt +261 to +274 within the 3′ end of SL1 and showed that the diminished infectivity of the mutated virus, termed BH10-LD4, could also be restored by the MP2 and MNC point mutations. Therefore, compensatory mutations within the p2 and NC proteins, distal from deletions within the DIS region of the HIV genome, can restore HIV replication, viral gene expression, and viral RNA packaging to control levels.     

Effects of a single amino acid substitution within the p2 region of human immunodeficiency virus type 1 on packaging of spliced viral RNA

Human immunodeficiency virus type 1 encapsidates two copies of viral genomic RNA in the form of a dimer. The dimerization process initiates via a 6-nucleotide palindrome that constitutes the loop of a viral RNA stem-loop structure (i.e., stem loop 1 [SL1], also termed the dimerization initiation site [DIS]) located within the 5' untranslated region of the viral genome. We have now shown that deletion of the entire DIS sequence virtually eliminated viral replication but that this impairment was overcome by four second-site mutations located within the matrix (MA), capsid (CA), p2, and nucleocapsid (NC) regions of Gag. Interestingly, defective viral RNA dimerization caused by the DeltaDIS deletion was not significantly corrected by these compensatory mutations, which did, however, allow the mutated viruses to package wild-type levels of this DIS-deleted viral RNA while excluding spliced viral RNA from encapsidation. Further studies demonstrated that the compensatory mutation T12I located within p2, termed MP2, sufficed to prevent spliced viral RNA from being packaged into the DeltaDIS virus. Consistently, the DeltaDIS-MP2 virus displayed significantly higher levels of infectiousness than did the DeltaDIS virus. The importance of position T12 in p2 was further demonstrated by the identification of four point mutations,T12D, T12E, T12G, and T12P, that resulted in encapsidation of spliced viral RNA at significant levels. Taken together, our data demonstrate that selective packaging of viral genomic RNA is influenced by the MP2 mutation and that this represents a major mechanism for rescue of viruses containing the DeltaDIS deletion.     

Mutations in the 5' nontranslated region of bovine viral diarrhea virus result in altered growth characteristics

The 5' nontranslated region (NTR) of pestiviruses functions as an internal ribosome entry site (IRES) that mediates cap-independent translation of the viral polyprotein and probably contains additional cis-acting RNA signals involved in crucial processes of the viral life cycle. Computer modeling suggests that the 5'-terminal 75 nucleotides preceding the IRES element form two stable hairpins, Ia and Ib. Spontaneous and engineered mutations located in the genomic region comprising Ia and Ib were characterized by using infectious cDNA clones of bovine viral diarrhea virus. Spontaneous 5' NTR mutations carrying between 9 and 26 A residues within the loop region of Ib had no detectable influence on specific infectivity and virus growth properties. After tissue culture passages, multiple insertions and deletions of A residues occurred rapidly. In contrast, an engineered mutant carrying 5 A residues within the Ib loop was genetically stable during 10 tissue culture passages. This virus was used as starting material to generate a number of additional mutants. The analyses show that (i) deletion of the entire Ib loop region resulted in almost complete loss of infectivity that was rapidly restored during passages in cell culture by insertions of variable numbers of A residues; (ii) mutations within the 5'-terminal 4 nucleotides of the genomic RNA severely impaired virus replication; passaging of the supernatants obtained after transfection resulted in the emergence of efficiently replicating mutants that had regained the conserved 5'-terminal sequence; (iii) provided the conserved sequence motif 5'-GUAU was retained at the 5' end of the genomic RNA, substitutions and deletions of various parts of hairpin Ia or deletion of all of Ia and part of Ib were found to support replication, but to a lower degree than the parent virus. Restriction of specific infectivity and virus growth of the 5' NTR mutants correlated with reduced amounts of accumulated viral RNAs.     

Large deletion mutations involving the first pyrimidine-rich tract of the 5' nontranslated RNA of human hepatitis A virus define two adjacent domains associated with distinct replication phenotypes.

The 5' nontranslated RNA (5'NTR) of the HM175 strain of human hepatitis A virus contains several pyrimidine-rich regions, the largest and most 5' of which (pY1) is an almost pure polypyrimidine tract located between nucleotides (nt) 99 and 138, which includes five tandem repeats of the sequence motif (U)UUCC(C). Previous modeling of the RNA secondary structure suggested that this region was likely to be single-stranded, but repetitive RNase V1 cleavage sites within these (U)UUCC(C) motifs indicated that pY1 possesses an ordered structure. To assess the role of this domain in replication of the virus, a series of large deletion mutations were created which involved the pY1 domain of an infectious cDNA clone. Deletion of 44 nt between nt 96 and 139, including the entire pY1 domain, did not reduce the capacity of the virus to replicate in BS-C-1 or FRhK-4 cells, as assessed by the size of replication foci in radioimmunofocus assays or by virus yields under one-step growth conditions. In contrast, viable virus could not be recovered from transfected RNAs in which the deletion was extended in a 5' direction by an additional 3 nt (delta 93-134), most likely because of the destabilization of a predicted stem-loop structure upstream of pY1. Deletion mutations extending in a 3' fashion to nt 140, 141, or 144 resulted in moderately (delta 96-140 and delta 96-141) or strongly (delta 99-144, delta 116-144, and delta 131-144) temperature-sensitive replication phenotypes. Although deletion of the pY1 domain did not by itself affect the replication phenotype of virus, the additional deletion of sequence elements within the pY1 domain (nt 99 to 130) substantially enhanced the temperature-sensitive phenotype of delta 131-144 virus. These data suggest that the (U)UUCC(C) motifs within the pY1 domain are conserved among wild-type viruses in order to serve a function required during infection in vivo but not in cell culture. In contrast, the single-stranded region located immediately downstream of pY1 (nt 140 to 144) is essential for efficient replication in cultured cells at physiological temperature. Viruses with deletion mutations involving nt 140 to 144 and viruses with large pY1 deletions but normal replication phenotypes in cell culture may have attenuation properties which could be exploited for vaccine development.     

Blocking rolling circle replication with a UV lesion creates a deletion hotspot

UV light irradiation increases genetic instability by causing mutations and deletions. The mechanism of UV-induced rearrangements was investigated making use of deletion-prone plasmids. Chimeric plasmids carrying pBR322 and M13 replication origins undergo deletions that join the M13 replication origin to a random nucleotide. A restriction fragment was UV irradiated, introduced into such a hybrid plasmid and deletions formed at the M13 origin were analysed. In most of the deletant molecules, the M13 replication nick site was linked to a nucleotide in the irradiated fragment, showing that UV lesions are deletion hotspots. These deletions were independent of the UvrABC excision repair proteins, suggesting that the deletogenic structure is the lesion itself and not a repair intermediate. They were not found in the absence of M13 replication, indicating that they result from the encounter of the M13 replication fork with the UV lesion. Furthermore, UV-induced deletions occurred independently of pBR322 replication. We conclude that, in contrast to pBR322 replication forks, M13 replication forks blocked by UV lesions are deletion prone. We propose that the deletion-prone properties of a UV-arrested polymerase depend on the associated helicase.     

Deletion Mutagenesis within the Dimerization Initiation Site of Human Immunodeficiency Virus Type 1 Results in Delayed Processing of the p2 Peptide from Precursor Proteins

Previous work has shown that deletions of genomic segments at nucleotide (nt) positions +238 to +253, i.e., construct BH10-LD3, or nt positions +261 to +274, i.e., construct BH10-LD4, within the human immunodeficiency virus type 1 (HIV-1) dimerization initiation site (DIS) destroyed DIS secondary structure and dramatically reduced viral replication capacity. Surprisingly, two point mutations located within the viral peptide 2 (p2) and nucleocapsid (NC) protein termed MP2 and MNC, respectively, were able to compensate for this defect. Since the MP2 mutation involves an amino acid substitution near the cleavage site between p2 and NC, we investigated the effects of the above-mentioned deletions on the processing of Gag proteins. Immunoprecipitation assays performed with monoclonal antibodies against viral capsid (CA) (p24) protein showed that p2 was cleaved from CA with less efficiency in viruses that contained the LD3 and LD4 deletions than in wild-type viruses. The presence of the two compensatory mutations, MP2 and MNC, increased the efficiency of the cleavage of p2 from CA, but neither mutation alone had this effect or was sufficient to compensate for the observed impairment in infectiousness. A virus that contained both of the above-mentioned deletions within the DIS was also impaired in regard to processing and infectiousness, and it could likewise be compensated by the MP2 and MNC point mutations. These results suggest that the DIS region of HIV-1 RNA plays an important role in the processing of Gag proteins.     

The replication termination signal terB of the Escherichia coli chromosome is a deletion hot spot.

Hybrids composed of phage M13, plasmid pBR322 and the termination signal of Escherichia coli chromosome replication terB were used to show that arrest of DNA synthesis creates a very efficient deletion hot spot. Up to 80% of deletions occurring in these hybrids had one deletion end-point at terB provided that (i) terB was oriented to arrest M13 and pBR322 leading strand synthesis; and (ii) the host cells contained the Tus protein necessary for arresting DNA synthesis at terB. The position of terB and the flanking sequences had little effect on deletion hot spot activity. About 90% of the deletions at terB ended 5-6 nucleotides in front of the major replication arrest site. We propose two models to account for deletion formation and speculate that many genome rearrangements may be due to the pausing of DNA replication.     

Defined mutations in the 5'' nontranslated sequence of Sindbis virus RNA.

We have constructed 24 deletion mutants which contain deletions of from 1 to 15 nucleotides in the 5' nontranslated region of Sindbis virus RNA and tested the effect of these mutations on virus replication. The results showed that the first 44 nucleotides, which are capable of forming a hairpin structure, are important for virus replication, as all deletions tested in this region were either lethal or resulted in virus that grew poorly in comparison to the parental virus. Many of these deletions had different effects in mosquito cells than in chicken cells, suggesting that cellular factors, presumably proteins, bind to this region. This domain may function in at least two processes in viral replication. It seems likely that in the minus strand, this sequence element is bound by the viral replicase and promotes RNA replication. In the plus strand, this element may modulate initiation of translation of the nonstructural proteins. The results suggest that the hairpin structure itself is important. All deletions within it had deleterious effects on virus replication, and in particular, deletion of one of the G residues at nucleotide 7 or 8 or of one of the C residues at nucleotide 36 or 37 which are theoretically base-paired with these G's resulted in temperature-sensitive viruses that behaved very similarly. In contrast, large deletions between the 44-nucleotide hairpin and the translation start site at nucleotides 60 to 62 resulted in virus that grew as well as or better than the parental virus in both chicken and mosquito cells. The A residue at position 5 of the HRSP strain used was examined in more detail. Deletion of this A was lethal, whereas substitution by G resulted in a virus that grew poorly, despite the fact that G is present at position 5 in the AR339 parent of HRSP. U at position 5 resulted in a virus that grew less well than the A5 strain but better than the G5 mutant.     

Deletion analysis of the cloned replication origin region from bacteriophage M13.

A cloned 270-nucleotide fragment from the origin region of the M13 duplex replicative form DNA confers an M13-dependent replication mechanism upon the plasmid vector pBR322. This M13 insert permits M13 helper-dependent replication of the hybrid plasmid in polA cells which are unable to replicate the pBR322 replicon alone. Using in vitro techniques, we have constructed several plasmids containing deletions in the M13 DNa insert. The endpoints of these deletions have been determined by DNA sequence analysis and correlated with the transformation and replication properties of each plasmid. Characterization of these deletion plasmids allows the following conclusions. (i) The initiation site for M13 viral strand replication is required for helper-dependent propagation of the chimeric plasmid. (ii) A DNA sequence in the M13 insert, localized between 89 and 129 nucleotides from the viral strand initiation site, is necessary for efficient transformation of polA cells. A chimeric plasmid containing the viral strand initiation site, but lacking this additional 40 nucleotide M13 sequence, transforms helper-infected cells at a frequency approximately 10(4)-fold less than that of plasmids containing this additional DNA segment. (iii) The entire M13 complementary strand origin can be deleted without affecting M13-dependent transformation by the hybrid plasmids. We propose a model in which replication of one strand of duplex chimera initiates by nicking at the gene II protein nicking site in the viral strand of the M13 insert, followed by asymmetric single-strand synthesis. Initiation of the complementary strand possibly occurs within plasmid sequences.     

Limits to the role of palindromy in deletion formation.

We tested the effect of palindromy on deletion formation. This involved a study of reversion of insertion mutations in the pBR322 amp gene at a site where deletions end either in 9-bp direct repeats or in adjoining 4-bp direct repeats. Inserts of palindromic DNAs ranging from 10 to more than 26 bp and related nonpalindromic DNAs were compared. The frequency of deletions (selected as Ampr revertants) was stimulated by palindromy only at lengths greater than 26 bp. The 4-bp direct repeats, one component of which is located in the palindromic insert, were used preferentially as deletion endpoints with palindromes of at least 18 bp but not of 16 or 10 bp. We interpret these results with a model of slippage during DNA replication. Because deletion frequency and deletion endpoint location depend differently on palindrome length, we propose that different factors commit a molecule to undergo deletion and determine exactly where deletion endpoints will be.     

Interference between M13 and oriM13 plasmids is mediated by a replication enhancer sequence near the viral strand origin

The origin of replication for the viral strand of bacteriophage M13 DNA is contained within a 507 base-pair intergenic region of the phage chromosome. The viral strand origin is defined as the specific site at which the M13 gene II protein nicks the duplex replicative form of M13 DNA to initiate rolling-circle synthesis of progeny viral DNA. Using in vitro techniques we have constructed deletion mutations in M13 DNA at the unique AvaI site which is located 45 nucleotides away on the 3' side of the gene II protein nicking site. This deletion analysis has identified a sequence near the viral strand origin that is required for efficient replication of the M13 genome. We refer to this part of the intergenic region as a "replication enhancer" sequence. We have also studied the function of this sequence in chimeric pBR322-M13 plasmids and found that plasmids carrying both the viral strand origin and the replication enhancer sequence interfere with M13 phage replication. Based upon these findings we propose a model for the mechanism of action of the replication enhancer sequence involving binding of the M13 gene II protein.     

Induction of deletion and insertion mutations by 9-aminoacridine. An in vitro model

The ability of 9-aminoacridine to induce mutagenic lesions during DNA replication in vitro was investigated. The ampicillinase gene of pBR322 was replicated in vitro in the presence of 9-aminoacridine. Transfection of the replicated DNA into Escherichia coli gave Amps mutants. Determination of the base changes in 76 of these mutants indicated that the spectrum of mutations induced by 9-aminoacridine was consistent with its action in vivo. Both large (407-base) and small (1- and 2-base) deletions were induced at repetitive sequences. The frequency of deletion mutations depended on the identity of the base deleted and sequences surrounding the deletions. The characteristics of the frameshift mutations induced were consistent with the interactions of 9-aminoacridine with DNA. These results establish that 9-aminoacridine can induce frameshift mutations during the replication process and provide an in vitro model of frameshift induction for mechanistic studies.     

Construction and characterization of replication-competent simian immunodeficiency virus vectors that express gamma interferon.

We report the construction and characterization of several replication-competent simian immunodeficiency virus (SIV) vectors with a deletion in the viral nef gene (SIV(delta nef)) that express gamma interferon (IFN-gamma). The expression of the cytokine gene was controlled either by the simian virus 40 early promoter or by the SIV 5' long terminal repeat regulatory sequences, utilizing the nef gene splice signals. To enhance the expression of IFN-gamma, the two in-frame nef start codons were mutated without altering the Env amino acid sequence (SIV(HyIFN)). Plasmids containing full-length proviral genomes were used to obtain high-titer stocks of each recombinant virus in cell cultures. Expression of IFN-gamma by SIV(HyIFN) reached levels as high as 10(6) U/ml after 11 days in culture. The IFN-gamma gene was unstable and sustained deletions after serial passage of SIV(delta nef) vectors in CEM-X-174 cells. The degree of instability appears to depend on size and orientation of the insert and the expression of IFN-gamma. Only one virus, SIV(HyIFN), expressed detectable levels of IFN-gamma up to the sixth passage. Prospects for the use of IFN-gamma and other lymphokines to enhance the safety and efficacy of live attenuated vaccines are discussed.