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. 2000 Mar;74(6):2694-702.
doi: 10.1128/jvi.74.6.2694-2702.2000.

Replication of Lengthened Moloney Murine Leukemia Virus Genomes Is Impaired at Multiple Stages

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Free PMC article

Replication of Lengthened Moloney Murine Leukemia Virus Genomes Is Impaired at Multiple Stages

N H Shin et al. J Virol. .
Free PMC article

Abstract

It has been assumed that RNA packaging constraints limit the size of retroviral genomes. This notion of a retroviral "headful" was tested by examining the ability of Moloney murine leukemia virus genomes lengthened by 4, 8, or 11 kb to participate in a single replication cycle. Overall, replication of these lengthened genomes was 5- to 10-fold less efficient than that of native-length genomes. When RNA expression and virion formation, RNA packaging, and early stages of replication were compared, long genomes were found to complete each step less efficiently than did normal-length genomes. To test whether short RNAs might facilitate the packaging of lengthy RNAs by heterodimerization, some experiments involved coexpression of a short packageable RNA. However, enhancement of neither long vector RNA packaging nor long vector DNA synthesis was observed in the presence of the short RNA. Most of the proviruses templated by 12 and 16 kb vectors appeared to be full length. Most products of a 19. 2-kb vector contained deletions, but some integrated proviruses were around twice the native genome length. These results demonstrate that lengthy retroviral genomes can be packaged and that genome length is not strictly limited at any individual replication step. These observations also suggest that the lengthy read-through RNAs postulated to be intermediates in retroviral transduction can be packaged directly without further processing.

Figures

FIG. 1
FIG. 1
Structures of proviral clones and other constructs. Virus coding regions are presented as shaded boxes. Restriction sites indicated below pGPP+11 are those used to map reverse transcription products in Fig. 6. The viral sequences in pD1040-2, an RNA probe expression plasmid, are represented by a shaded box. PSV40, simian virus 40 promoter; LTR, long terminal repeat; Ψ+, packaging site; Δ, deletion of packaging site.
FIG. 2
FIG. 2
Replication efficiencies of native-length and lengthened genomes. (A and B) Puromycin-resistant CFU per unit volume of virus-containing culture medium harvested from ET cells transiently transfected with equimolar amounts of vector expression plasmids, normalized to the value obtained for the parental pGPP plasmid. In experiments that included the pΨ+hyg escort plasmid, equimolar pΨ+hyg was added in place of some of the carrier DNA used in the no-pΨ+hyg transfections. (C and D) Puromycin-resistant CFU per unit of virion protein in the culture medium. (E and F) Packaging of viral RNA per unit virion protein in the presence and absence of Ψ+hyg escort RNA. (G and H) Puromycin-resistant CFU per unit of virion RNA in the presence and absence of Ψ+hyg escort RNA. The values in panel B were normalized to the GPP value in panel A. For all other panels, values presented were normalized to values obtained for the parental GPP vector in that panel and were determined from data such as those in Fig. 3, using approaches described in Materials and Methods. In panels E through H, “encapsidated RNA” refers to GPP, GPP derivative, or ΨMLV RNA and does not include Ψ+hyg escort RNA.
FIG. 3
FIG. 3
RNase protection assays to quantify vector RNAs. (A) Assays of RNAs harvested from the culture medium of ET cells cotransfected with pGPP or related plasmid and pΨ+hyg. Lanes: 1, size standards; 2, undigested probe; 3, no sample or recovery marker control; 4, mock-transfected ET medium RNA; 5 to 10, RNA samples purified from the virion-containing culture media from cells transfected with pGPP+4, pGPP+8, pGPP+11, pGPP, pΨMLV, and pNCA (an infectious M-MLV provirus clone), respectively. Migration positions of protected bands diagnostic of each product are indicated at the right. U, undigested probe; G, GPP-type product; E, escort product; RM, recovery marker. (B) Assays of intracellular RNAs harvested from transfected cells. Designations at the top indicate which RNA is analyzed in each lane of panels B and C. (C) Assays of RNAs harvested from the culture medium of ET cells transfected with pGPP or related plasmid in the absence of pΨ+hyg.
FIG. 4
FIG. 4
RNase protection assay of 5′ and 3′ ends of GPP-type RNAs. Lanes: 1, size standards; 2, undigested 3′ (puro) probe; 3, undigested 5′ (gag) probe; 4, probe-alone control; 5, mock-transfected ET medium RNA; 6 to 10, RNA samples from the virion-containing culture media from cells transfected with pGPP, pGPP+4, pGPP+8, pGPP+11, and pNCA (which expresses wild-type MLV), respectively. Migration positions of undigested probes and protected bands diagnostic of each product are indicated at the right.
FIG. 5
FIG. 5
Denaturing Northern blots of vector RNAs. (A) Cellular RNA extracted from ET cells transfected with pGPP and derivative plasmids. Two amounts of RNA were loaded for each sample so that the odd-numbered lanes contain 2.5-fold as much sample as the even-numbered lanes. RNA was from cells expressing GPP (lanes 1 and 2), GPP+4 (lanes 3 and 4), GPP+8 (lanes 5 and 6), and GPP+11 (lanes 7 and 8). (B) Virion RNA harvested from the culture medium of ET cells transfected as above. Lanes: 1, GPP; 2, GPP+4; 3, GPP+8; 4, GPP+11. Numbers at the right indicate the mobilities of RNA size standards of the indicated lengths in kilobases. The identity of the faster-migrating band in lanes 1 and 2 of panel A was not determined.
FIG. 6
FIG. 6
Southern blot analysis of individual proviral products of the GPP+11 vector. (Top) Schematic drawing of a GPP+11 provirus showing the locations of restriction sites used to assess provirus sizes. Because of the template switches during reverse transcription, MLV-based vector DNAs should be 0.6 kb longer than their RNA templates (11). ClaI restriction sites and the size (10.5 kb) of the ClaI restriction fragment predicted for an unrearranged GPP+11 provirus are indicated, as are BstEII site locations and the sizes predicted for BstEII digestion (2.7, 3.2, and 6.3 kb). Drawing is not to scale. For abbreviations, see the legend to Fig. 1. (Bottom) Southern blot analyses of integrated vector proviruses. (A) GPP+11 clonal integrants that contain dystrophin region deletions digested with ClaI. Lanes: 1, size standards; 2, mock-infected cell DNA; 3 to 6, individual clonal integrants. (B) Candidate full-length GPP+11 GM integrant DNA analyzed with BstEII. Lanes: 1, size standards; 2, mock-infected cell DNA; 3, clone 4-8 DNA. (C) Restriction analysis of GPP+11 vector product sizes after a second round of replication. D17 clone DNAs were digested with ClaI. Lanes: 1, marker; 2, mock-infected D17 cellular DNA; 3, second-round product generated by clone 16-6; 4 to 6, second-round products from 4-4, 4-8, and 4-14, respectively; 7, size standards. Mobilities of DNA size standards are indicated at the left. For each panel, arrows indicate provirus-specific products and Δ indicates host-derived products detectable in both mock-infected and transduced cells. First-replication-round products were analyzed in 3T3 cells that express GaLV Env; second-round products were analyzed in D17 cells.

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