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. 2001 Mar;75(6):2776-85.
doi: 10.1128/JVI.75.6.2776-2785.2001.

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

Y Guan  1 , J B Whitney, C Liang, M A Wainberg
Affiliations

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

Y Guan et al. J Virol. 2001 Mar.

Abstract

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.

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Figures

FIG. 1
FIG. 1
Illustration of the deletion constructs used in this study. Secondary structures of the U5-leader stem and the putative DIS stem-loop of SIVmac239 leader RNA are shown. The positions of deletion constructs are relative to the transcription initiation site and are shown next to the RNA structure. These positions are also indicated in the diagram of secondary structure. Both the primer binding site (PBS) and the DIS palindrome sequences are highlighted.
FIG. 2
FIG. 2
Replication capacity of mutated viruses in C8166 cells. (A) Equivalent amounts of virus from COS-7-transfected cells were used to infect C8166 cells based on levels of p27 antigen (10 ng per 106 cells). Viral replication was monitored by RT assay of culture fluids. Mock transfection denotes exposure of cells to heat-inactivated wild-type (WT) virus as a negative control. (B) Replication capacity of mutated viruses during long-term tissue culture in C8166 cells.
FIG. 3
FIG. 3
Replication capacity of mutated viruses in CEMx174 cells. (A) Equivalent amounts of virus from COS-7-transfected cells were used to infect CEMx174 cells based on levels of p27 antigen (10 ng per 106 cells). Viral replication was monitored by RT assay of culture fluids. Mock infection denotes exposure of cells to heat-inactivated wild-type (WT) virus as a negative control. (B) Replication capacity of mutated viruses during long-term tissue culture.
FIG. 4
FIG. 4
Replication capacity of mutated virus during the second passage in CEMx174 cells. CEMx174 cells were infected with equivalent amounts of virus from the peak time of RT production after initial infection based on levels of p27 antigen (10 ng per 106 cells). Viral replication was monitored by RT assay of culture fluids. Mock infection denotes exposure of cells to heat-inactivated wild-type (WT) virus as a negative control.
FIG. 5
FIG. 5
Infectiousness of the wild type (WT) and various mutated viruses. The results shown are the averages of three independent experiments. Each of the SD, SD1c, SD3, and SD4 viruses was shown to be poorly infectious, with RT values being below the threshold sensitivity of the assay (dashed line). Mock infection represents a negative control in which cells were exposed to heat-inactivated wild-type virus. (A) TCID50s of the wild type and various mutated viruses were determined by infection of CEMx174 cells as described in Materials and Methods. (B) Infectivity was tested by sMAGI assays as described previously (5). Numbers of blue-stained cells were scored and plotted.
FIG. 6
FIG. 6
Replication capacity of the wild type (WT) and mutated viruses in monkey PBMCs. Equivalent amounts of virus were used to infect rhesus macaque PBMCs based on levels of p27 antigen as described in Materials and Methods. Viral replication was monitored by SIV p27 antigen ELISA of culture fluids. Mock infection denotes exposure of cells to heat-inactivated wild-type virus as a negative control. The dashed line representing 0.01 ng of p27 per ml illustrates the threshold sensitivity of the assay. (A) Growth curves in PBMCs obtained from monkey 1. (B) Growth curves in PBMCs harvested 6 months later from the same animal. (C) Growth curves in PBMCs from monkey 2.
FIG. 7
FIG. 7
Replication capacity of mutated virus derived from monkey PBMCs. Viruses equivalent to 200 pg of p27 antigen derived from infected PBMCs of monkey 1 were used to infect 105 PBMCs of the same monkey or 105 CEMx174 cells. Viral replication was monitored by SIV p27 antigen enzyme-linked immunosorbent assay of culture fluids. Mock infection denotes exposure of cells to heat-inactivated wild-type (WT) virus as a negative control. The dashed line representing 0.01 ng of p27 per ml illustrates the threshold sensitivity of the assay. (A) Growth curves of second-passage virus in monkey PBMCs. (B) Growth curves of second-passage virus in CEMx174 cells.
FIG. 8
FIG. 8
Viral RNA packaging in the wild type (WT) and mutated viruses. Equivalent amounts of virus derived from transfected COS-7 cells, based on levels of p27 antigen, were used to prepare viral RNA, which was then used as a template for quantitative RT-PCR to detect the full-length viral RNA genome in an 18-cycle PCR. Relative amounts of a 114-bp DNA product were quantified by molecular imaging, with wild-type values arbitrarily set at 1.0. Reactions run with RNA template, digested by DNase-free RNase, served as a negative control for each sample to exclude any potential DNA contamination. Relative amounts of viral RNA that were packaged were determined on the basis of four different experiments.
FIG. 9
FIG. 9
Processing of SIV Gag precursor proteins. 293T cells transfected with wild-type (WT) or mutated SIV constructs were radiolabeled and viral proteins in the cell lysates were then immunoprecipitated with MAbs against SIV p27 as described in Materials and Methods. The positions of viral Gag proteins are shown on the right side of the gel (A). Mo, mock transfection control. The percentage of each viral protein relative to all viral proteins detected was calculated with the NIH Image program. The results are illustrated as well by a bar graph (B) showing the different percentages of each band associated with each of the constructs studied, as well as by a line chart (C), showing a steady change in band representation from wild-type virus to the mutated constructs.
FIG. 10
FIG. 10
Protein band patterns in the wild type (WT) and mutated SIV. 35S-labeled viral progeny that had been released over 1 h from 293T cells were purified at 24 h after transfection; protein patterns are shown (A). Mo, mock transfection control. Seven of the protein bands were quantified by densitometry with the NIH Image program. For each virus, the percentage of each band versus all seven bands was calculated. The results are illustrated as well by a bar graph (B) showing the different percentages of each band associated with each of the constructs studied, as well as by a line chart (C) showing a steady change in band representation from wild-type virus to the mutated constructs.
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