Characterization of the block in replication of nucleocapsid protein zinc finger mutants from moloney murine leukemia virus - PubMed (original) (raw)

Characterization of the block in replication of nucleocapsid protein zinc finger mutants from moloney murine leukemia virus

R J Gorelick et al. J Virol. 1999 Oct.

Abstract

Mutagenesis studies have shown that retroviral nucleocapsid (NC) protein Zn(2+) fingers (-Cys-X(2)-Cys-X(4)-His-X(4)-Cys- [CCHC]) perform multiple functions in the virus life cycle. Moloney murine leukemia virus mutants His 34-->Cys (CCCC) and Cys 39-->His (CCHH) were able to package their genomes normally but were replication defective. Thermal dissociation experiments showed that the CCHH mutant was not defective in genomic RNA dimer structure. Primer tRNA placement on the viral genome and the ability of the tRNA to function in reverse transcription initiation in vitro also appear normal. Some "full-length" DNA copies of the viral genome were synthesized in mutant virus-infected cells. The CCCC and CCHH mutants produced these DNA copies at greatly reduced levels. Circle junction fragments, amplified from two-long-terminal-repeat viral DNA (vDNA) by PCR, were cloned and characterized. Remarkably, it was discovered that vDNA isolated from cells infected with mutant virions had a wide variety of abnormalities at the site at which the two ends of the linear precursor had been ligated to form the circle (i.e., the junction between the 5' end of U3 and the 3' end of U5). In some molecules, bases were missing from regions corresponding to the U3 and U5 linear vDNA termini; in others, the viral sequences extended either beyond the U5 sequences into the primer-binding site and 5' leader or beyond the U3 sequences into the polypurine tract into the env coding region. Still other molecules contained nonviral sequences between the linear vDNA termini. Such defective genomes would certainly be unsuitable substrates for integration. Thus, strict conservation of the CCHC structure in NC is required for infection events prior to and possibly including integration.

PubMed Disclaimer

Figures

FIG. 1

FIG. 1

Ligation of the linear vDNA to form the 2-LTR circularized species. The linear form of the vDNA is diagrammed with the 5′-LTR (position and orientations described are with respect to plus-strand sequences) in dark gray and the 3′-LTR in light gray. The 5′ end shows the U3, R, U5 region of the LTR as well as the neighboring PBS. The 3′ end shows the ppt region and the subsequent 3′ LTR. The positions of the PCR primers used to amplify the 2-LTR circularized vDNA species are indicated. The 2-LTR species in the middle portion of the figure shows the expected vDNA species that results after ligation of the linear vDNA form in the cell. PCR amplification with primers 4658-367 and 4658-368 with subsequent cloning into the TA cloning site of the pRC2.1 vector gives the expected form shown at the bottom of the panel. Points of reference from the Mo-MuLV genome (GenBank accession no. J02255 [30]) are indicated in the lower portion of the diagram. The diagram shows one of two possible orientations in the pCR2.1 vector. The positions of the M13-reverse and T7 primers, which were used for sequence analysis of the 2-LTR circularized, vDNA, PCR product inserts, are indicated.

FIG. 2

FIG. 2

Melting analyses of Mo-MuLV dimeric RNAs isolated from wild-type and CCHH and PR− mutant virions. RNAs extracted from the respective virions were resuspended and heated for 10 min at the temperatures indicated in the figure. The positions of the monomer and dimer RNA species are indicated at the left. A comparison of the melting temperatures of the wild type (WT), the CCHH mutant, and the PR− mutant RNA dimers is presented. Wild-type virus contains a “mature” RNA dimer, and PR− virus contains an “immature” RNA dimer (15). The melting analysis of the CCHH mutant RNA was compared with that of the wild-type RNA and the PR− RNA in separate experiments, but in the interest of saving space, only one CCHH analysis is presented. The melting analyses were identical for the CCHH mutant in both experiments.

FIG. 3

FIG. 3

PBS occupancy determination in genomic RNAs isolated from wild-type (WT) and CCHH mutant virions. (A) Primer tagging was performed to determine the levels of primer tRNA at the PBS. Undiluted and 1:10-diluted vRNAs were examined as indicated. Heated (samples heated to 100°C for 5 min and immediately chilled on ice prior to primer tagging) and unheated samples were tested, as indicated. (B) Nondenaturing Northern blot analysis of CCHH mutant and wild-type vRNA were tested undiluted and at a 1:10 dilution (RNA from 7.5 and 0.75 ml of culture supernatant, respectively) is indicated.

FIG. 4

FIG. 4

Agarose gel analysis of 2-LTR circularized vDNA PCR products. Hirt supernatants, isolated from 293T cells infected with mutant and wild-type viruses, were amplified with primers specific for 2-LTR circularized vDNA (see Fig. 1). The RT activities (in cpm per milliliter) of the undiluted inocula (corrected for background) are as follows: (−)-Control, 0; CCCC, 451,760; CCHH, 748,370; SSHC, 637,170; Y28S, 630,670; W35S, 564,340; and wild type, 551,430. The location of the expected 178-bp band is shown on the right. Viruses incubated with the cells are indicated at the top, and the dilutions tested by PCR are indicated at the bottom. The lane labeled (−)-Control contains PCR products from the Hirt supernatant from cells incubated with transfected cell culture supernatant that did not contain any virus. The AZT/ddC lane contains PCR products from the Hirt supernatant from cells incubated with wild-type virus in the presence of the RT-inhibiting compounds AZT and ddC. The No DNA lane contains PCR products generated in the absence of added Hirt supernatant.

FIG. 5

FIG. 5

Frequency distribution of the sizes of cloned 2-LTR circularized vDNA PCR products from cells incubated with mutant and wild-type viruses. Individual clones of the PCR products obtained in the experiment in Fig. 4 were analyzed for the sizes of the inserts. Results are reported as the deviation from the expected size of 178 bp (Fig. 1). The frequencies were compiled in 20-nt windows from the expected size. Frequencies are reported as a percentage of the total number of samples examined. Distributions are shown for PCR product inserts obtained from cells incubated with the wild-type (A), CCCC (B), CCHH (C), Y28S (D), W35S (E), and SSHC (F) viruses.

FIG. 6

FIG. 6

Schematic of alignments of PCR product inserts. The cloned inserts of products from the PCR of Hirt supernatants isolated from cells incubated with mutant and wild-type (WT) viruses were sequenced. The individual clone designations are indicated on the right, and the mutant examined is indicated on the left. The thick gray lines in the lower portion of each panel represent nucleotides that are present in each of the clones. The dashed regions depict the nucleotides that are missing from the vDNA product insert. The black regions depict insertions of extraneous (nonviral or noncontiguous viral) DNA sequences with the size of the fragment indicated inside the black region. (A) Alignment of full-length or truncated inserts that are isogenic with the expected sequence for authentic 2-LTR circularized vDNA. The diagram at the top is the same as that shown in Fig. 1. (B) Alignment of clones containing extraneous DNA inserts between the ends of the linear vDNA termini. The diagram at the top shows the arrangement of the termini and fragments. (C and D) Alignments of clones that continue through the PBS (C) or the ppt (D) are depicted. The diagrams at the top of each panel show the arrangement of the consensus viral sequences.

FIG. 6

FIG. 6

Schematic of alignments of PCR product inserts. The cloned inserts of products from the PCR of Hirt supernatants isolated from cells incubated with mutant and wild-type (WT) viruses were sequenced. The individual clone designations are indicated on the right, and the mutant examined is indicated on the left. The thick gray lines in the lower portion of each panel represent nucleotides that are present in each of the clones. The dashed regions depict the nucleotides that are missing from the vDNA product insert. The black regions depict insertions of extraneous (nonviral or noncontiguous viral) DNA sequences with the size of the fragment indicated inside the black region. (A) Alignment of full-length or truncated inserts that are isogenic with the expected sequence for authentic 2-LTR circularized vDNA. The diagram at the top is the same as that shown in Fig. 1. (B) Alignment of clones containing extraneous DNA inserts between the ends of the linear vDNA termini. The diagram at the top shows the arrangement of the termini and fragments. (C and D) Alignments of clones that continue through the PBS (C) or the ppt (D) are depicted. The diagrams at the top of each panel show the arrangement of the consensus viral sequences.

FIG. 7

FIG. 7

Mechanisms for vDNA terminal truncations. (A) The path on the left shows how the termini become truncated after complete synthesis of the vDNA by unabated reverse transcription. Exonuclease degradation of the double-stranded vDNA (ds vDNA) would account for the missing nucleotides at the vDNA termini. (B) The scheme on the right shows how incomplete synthesis of vDNA could occur, due to the inability of NC to completely melt the double-stranded vDNA complementary regions, U3|R|U5 and PBS, thus blocking RT from completing synthesis to the proper ends. Nucleases would then degrade single-stranded vDNA overhangs (ss vDNA).

Similar articles

Cited by

References

    1. Allain B, Lapadat-Tapolsky M, Berlioz C, Darlix J-L. Transactivation of the minus-strand DNA transfer by nucleocapsid protein during reverse transcription of the retroviral genome. EMBO J. 1994;13:973–981. - PMC - PubMed
    1. Berg J M. Potential metal-binding domains in nucleic acid binding proteins. Science. 1986;232:485–487. - PubMed
    1. Berkowitz R, Fisher J, Goff S P. RNA packaging. Curr Top Microbiol Immunol. 1996;214:177–218. - PubMed
    1. Bushman F D, Craigie R. Activities of human immunodeficiency virus (HIV) integration protein in vitro: specific cleavage and integration of HIV DNA. Proc Natl Acad Sci USA. 1991;88:1339–1343. - PMC - PubMed
    1. Casas-Finet J R, Urbaneja M A, Nower P N, Gorelick R J, Bosche W J, Kane B P, Johnson D, Henderson L E. Functional properties of point-mutated MoMuLV nucleocapsid (NC) protein p10. FASEB J. 1997;11:A981.

Publication types

MeSH terms

LinkOut - more resources