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Journal of Virology, October 2008, p. 9318-9328, Vol. 82, No. 19
0022-538X/08/$08.00+0     doi:10.1128/JVI.00583-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Mutations in Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Zinc Fingers Cause Premature Reverse Transcription {triangledown}

James A. Thomas, William J. Bosche, Teresa L. Shatzer, Donald G. Johnson, and Robert J. Gorelick*

AIDS and Cancer Virus Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland 21702

Received 14 March 2008/ Accepted 21 July 2008


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ABSTRACT
 
Human immunodeficiency virus type 1 (HIV-1) requires that its genome be reverse transcribed into double-stranded DNA for productive infection of cells. This process requires not only reverse transcriptase but also the nucleocapsid protein (NC), which functions as a nucleic acid chaperone. Reverse transcription generally begins once the core of the virion enters the cytoplasm of a newly infected cell. However, some groups have reported the presence of low levels of viral DNA (vDNA) within particles prior to infection, the significance and function of which is controversial. We report here that several HIV-1 NC mutants, which we previously identified as being replication defective, contain abnormally high levels of intravirion DNA. These findings were further reinforced by the inability of these NC mutants to perform endogenous reverse transcription (ERT), in contrast to the readily measurable ERT activity in wild-type HIV-1. When either of the NC mutations is combined with a mutation that inactivates the viral protease, we observed a significant reduction in the amount of intravirion DNA. Interestingly, we also observed high levels of intravirion DNA in the context of wild-type NC when we delayed budding by means of a PTAP(–) (Pro-Thr-Ala-Pro) mutation. Premature reverse transcription is most probably occurring before these mutant virions bud from producer cells, but we fail to see any evidence that the NC mutations alter the timing of Pr55Gag processing. Critically, our results also suggest that the presence of intravirion vDNA could serve as a diagnostic for identifying replication-defective HIV-1.


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INTRODUCTION
 
The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) is critical for viral infection and replication. NC is involved in viral genomic RNA (gRNA) packaging (3, 9, 22, 23, 24, 30, 31, 50), particle assembly (13-15, 26, 61, 75), reverse transcription, and integration (11, 16, 17, 27, 65). NC is a nucleic acid chaperone: it enables nucleic acids to form the most thermodynamically stable annealed structures (5, 6, 46, 59, 66). When not bound to nucleic acids, NC is mostly unstructured except for its two zinc fingers of the sequence -Cys-X2-Cys-X4-His-X4-Cys- (4, 44, 51, 52). As expected for a nucleic acid chaperone, HIV-1 NC binds tightly to nucleic acids, in both a sequence-specific and non-sequence-specific manner (44, 46). The zinc fingers are critical for proper NC function, so that alterations that abolish zinc binding (22, 31) or that even slightly alter their structure (20, 29, 63) result in replication defects.

In our previous studies of HIV-1 NC, we observed that there are two mutations to NC, His23Cys (NCH23C) and His44Cys (NCH44C), that result in a replication-defective phenotype, although these viruses are able to infect cells for a single round (11, 29). A detailed examination of the steps of early infection revealed that these mutant viruses are competent for the synthesis of viral DNA (vDNA) at early time points postinfection but that the vDNA produced is less stable and results in far fewer integration events than the wild-type virus (65). A particularly interesting observation was that reverse transcription products appeared much more rapidly in the NCH23C and NCH44C infections than in wild-type virus infections so that even at the earliest time point examined, reverse transcripts were present at their highest levels in the mutants (65). This could be due to accelerated reverse transcription once virion cores enter the cytoplasm, or it could be because a greater proportion of these mutant particles had initiated reverse transcription prior to infection and thus entered the cell preloaded with vDNA, a phenomenon that has recently been reported for several other HIV-1 NC mutants (34). The presence of large amounts of intravirion vDNA is a well-known property of infectious prototype foamy viruses (47).

It has been reported that reverse transcripts are present within wild-type HIV-1 particles at very low levels. Expressed as a ratio of minus-strand strong-stop DNA to gRNA, these are between 1:1,000 and 1:6,000 (48, 68, 74). The significance of this vDNA is unclear: it is possible that this DNA is important for infection, irrelevant for infection, or even inhibits infection.

We investigated HIV-1 NC mutant particles for the presence of intravirion DNA using our system for the detection and quantification of vDNA using real-time PCR (11). We observed that both the NCH23C and NCH44C mutants contained significantly more minus-strand strong-stop vDNA than the wild-type virus did, on a per particle basis. In fact, practically every one of these mutant particles appeared to contain this vDNA species. In addition, with these mutants, reverse transcription products generated after minus-strand strong-stop DNA were also present at higher levels than that measured in wild-type virus. As it is highly unlikely that HIV-1 contains enough deoxynucleotide triphosphates (dNTPs) within a particle to synthesize even minus-strand strong-stop DNA, it is probable that this vDNA was synthesized prior to the time the virus budded from the cell.

In this study, we demonstrated that the high levels of intravirion DNA strictly require reverse transcriptase (RT) possessing both functional polymerase and RNase H activities. In addition, when we combine the NC mutations with a mutation that inactivates the viral protease (PR), we observed a proportional decrease in intravirion DNA for both the NC mutant and wild-type viral particles. Thus, the elevated levels of reverse transcripts present in the mutant particles are dependent on mature RT being present. Collectively, these results provide important insights into the timing and mechanism of reverse transcription. These data also support the hypothesis that the presence of intravirion reverse transcripts is indicative of a replication-defective HIV-1 particle.


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MATERIALS AND METHODS
 
Plasmids and mutagenesis. All mutations were introduced into the proviral plasmid pNL4-3 (wild-type NC [NCWT]) (GenBank accession number AF324493) (2). Construction of the NCH23C, NCH44C, and RTD185K/D186L (RT(–)) mutant plasmids were previously described (29). Production of the p6-PTAP to LIRL (PTAP(–)) mutant (35) as well as the Env(–) version of pNL4-3 (54) have been previously described. The pHCMV-g plasmid, which expresses the vesicular stomatitis virus G protein (VSV-G) (12), was a generous gift from Jane C. Burns (University of California, San Diego).

The RTE478Q active site mutant of RNase H (RNase H(–)) (19) was generated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions, changing nucleotide (nt) 3981 from G to C, nt 3983 from G to A, and nt 3986 from A to G. These three point mutations introduced a new MunI restriction endonuclease site that was used for screening purposes. The change at nt 3983 introduced the Glu478Gln (E478Q) change in RT, and the changes at nt 3981 and 3986 are silent, maintaining the correct amino acids at positions Thr477 and Leu479 in RT. Upon introduction of these changes into pNL4-3, the AgeI-PflmI fragment (1,818 bp) containing the mutant RT gene was cloned into pNL4-3 Env(–) to generate the NCWTRNase H(–)Env(–) proviral clone. The NCH23CRNase H(–)Env(–) and NCH44CRNase H(–)Env(–) proviral plasmids were constructed similarly.

The NCH23CRT(–)Env(–) and NCH44CRT(–)Env(–) proviral plasmids were constructed by ligating each of the 11,923-bp SbfI-EcoRI fragments from the NCH23CEnv(–) and NCH44CEnv(–) proviral plasmids with the corresponding 2,900-bp fragment containing the RT(–) mutation.

The PRD25A active site mutant (PR(–)), changing nt 2326 from A to C, was previously described (56). To generate the NCWTPR(–)Env(–), NCH23CPR(–)Env(–), and NCH44CPR(–)Env(–) proviral plasmids, the corresponding NCxEnv(–) proviral plasmids were cut with ApaI and SbfI and the 13,990-bp fragments were each ligated with a (833-bp) ApaI and SbfI fragment containing the PR(–) mutation. All mutations were verified by sequencing.

The plasmids used for quantitation of vDNA have been described previously (11). The RNA standard used for gag RNA quantification has been described previously (65). The DNA standard used for quantification of VSV-G DNA was pHCMV-g.

Cell lines and virus production. The 293T cells (28) and HeLa cells were maintained as described previously (29). Pseudotyped viruses were generated by cotransfection of 2 µg of pHCMV-g DNA with 4 µg of each proviral plasmid per 100-mm dish using the calcium phosphate transfection method (32, 38). The pHCMV-g plasmid was monitored by real-time PCR throughout this study as an irrelevant DNA to assess DNA contamination and the effectiveness of the DNase I plus subtilisin treatments (see below). At 48 and 72 h after transfection, the medium incubated with cells for the previous 24 h was removed from the cultures and clarified by centrifugation at 5,000 x g for 10 min. The resulting supernatant was filtered through a 0.22-µm filter. The supernatants were pooled from both days and processed as described below. For assessment of the ratios of HIV-1 genome to p24CA (CA for capsid protein), significantly more virus was required for the protein analysis. Therefore, 150-cm2 flasks with 293T cells were cotransfected with 50 µg of proviral plasmid and 25 µg of the pHCMV-g plasmid by the CaPO4 coprecipitation method (32, 38) and harvested 48 h posttransfection. To demonstrate that the wild-type pseudotyped virus was amplified by reinfection, one sample was cultivated in the presence of 10 µM of the reverse transcription inhibitor tenofovir (9-R-2-phosphonomethoxypropyl adenine [PMPA]) (Gilead Sciences, Inc., Foster City, CA).

Preparation of virus. To accurately quantitate intravirion vDNA, it is critical that transfection plasmid be removed from the virus preparations. This was performed using a DNase I plus subtilisin digestion procedure. Although DNase I is typically used by many laboratories, the additional subtilisin digestion step ensures that any residual DNase is removed, and it also removes nonviral membrane vesicles present in virus preparations that can interfere with bulk analyses (see Results). Initially, pooled clarified supernatants were treated with 10 U/ml DNase I (Sigma-Aldrich, St. Louis, MO) and 4 mM MgCl2 for 1 to 1.5 h at 37°C. This reaction was stopped by adding EDTA to a final concentration of 10 mM. The virus was then pelleted through a 20% (wt/vol) sucrose pad in phosphate-buffered saline (PBS) without Ca2+ or Mg2+ at 100,000 xg for 1 h at 4°C in a SW-32 rotor (Beckman-Coulter, Fullerton, CA). The viral pellet was resuspended in 300 µl PBS without Ca2+ or Mg2+, mixed with 300 µl of 2x subtilisin buffer (final concentration = 1 mg/ml subtilisin [Sigma-Aldrich], 20 mM Tris, 1 mM CaCl2 [pH 8.0]), and incubated at 37°C overnight. The reaction was stopped by adding phenylmethylsulfonyl fluoride (Sigma-Aldrich) to a final concentration of 250 µg/ml, and then virus was pelleted through a 20% (wt/vol) sucrose pad at 400,000 x g for 2 h at 4°C in a Beckman SW-60 rotor. The pellet was resuspended in 151 µl PBS without Ca2+ or Mg2+. Fifty microliters of this solution was removed for vDNA and gRNA quantitation (see below), while the remainder was used for endogenous reverse transcription (ERT) assays (below).

Virus was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 4 to 20% Novex Tris-glycine gels before and after subtilisin treatment as described above (Fig. 1B and D). The gels were stained with Coomassie brilliant blue. The levels of p24CA in the samples were determined by comparison of band intensities with those from a standard curve using known amounts of p24CA. Densitometric analyses were performed using either TotalLab software from BioSystematica (Mountain Hall, United Kingdom) or Quantity One software from Bio-Rad Laboratories (Hercules, CA).


Figure 1
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  		FIG. 1. Quantitation of intravirion HIV-1 DNA. (A) Representative experiment showing the efficacy of our DNase I plus subtilisin treatment protocol in reducing contamination from transfection-derived virus. Results are presented as a ratio of the amount of each indicated DNA species per ml to the amount of gRNA per ml (gRNA quantities before [pre] and after [post] treatment with DNase I plus subtilisin were 3.6 x 108 and 9.4 x 107 per ml, respectively). (B) Coomassie brilliant blue-stained SDS-PAGE fractionation of an NCWT virus preparation before and after the DNase I plus subtilisin digestion procedure. Sixty milliliters of viral supernatant (viral sup) was split into two equal parts; half was pelleted through a 20% sucrose pad and resuspended in loading buffer (without subtilisin [– subtilisin]), while the other half was digested with DNase I plus subtilisin, then pelleted through a 20% sucrose pad, and resuspended in loading buffer (with subtilisin [+ subtilisin] indicated at the top of the gel). We ran the amount of extract equivalent to either 15 ml or 1.5 ml of the original supernatant as indicated at the top of the gel. The remaining lanes were loaded with the indicated quantities of p24CA protein standard (STD). The positions of molecular mass standards (in kilodaltons) are indicated to the left of the gel. (C) Relative amounts of intravirion DNA (from two independent experiments) for the wild-type virus (NCWT) and NCH23C and NCH44C mutant viruses (error bars indicate the standard deviations). These amounts were calculated by dividing the DNA target quantity (R-U5, U3-U5, Gag, R-5'UTR, or VSV-G) per ml by the quantity of gRNA per ml, both measured after treatment with DNase I plus subtilisin. The average numbers of copies of gRNA per ml from the two experiments were 1.4 x 109 for NCWT, 1.2 x 105 for NCH23C, and 6.1 x 104 for NCH44C. (D) Relative expression of VSV-G-pseudotyped viruses containing either the NC mutations or NCWT from a representative experiment after the DNase I plus subtilisin treatment. The starting volume equivalents (eq.) of cell culture supernatants are indicated at the top of the gel for each species. Immediately under the gel is shown the amount of p24CA (ng/ml) of culture fluid determined using p24CA standards indicated in the gel image. The quantity of gRNA/ml in the cell culture supernatant as determined by real-time RT-PCR is shown, then the ratio of gRNA to ng p24CA. The sample labeled WTPMPA is a wild-type provirus/pHCMV-g cotransfection performed in the presence of PMPA, a chain terminator reverse transcription inhibitor. Note that the amount of wild-type virus is greater in panel D than in panel B, since substantially more DNA was used for the transfections in panel D.

ERT analysis. The endogenous reverse transcription assay was adapted from the protocol reported by the Freed laboratory (41) with the major difference being the use of our real-time PCR analyses (11) to assess and quantify vDNA synthesis rather than to measure incorporation of radiolabeled nucleotides by quantification of autoradiographs. The virus preparations treated with DNase I plus subtilisin described above were divided into two tubes containing 50 µl each; one tube was heated to 68°C for 20 min to inactivate reverse transcriptase (67), and the other was kept on ice. Triton X-100 was added to all samples at a final concentration of 0.01% (vol/vol), and the samples were then incubated for 10 min at room temperature. ERT buffer (final composition after addition to virus sample, 50 mM Tris-HCl [pH 8.0], 2 mM MgCl2, 10 mM dithiothreitol, 25 µM [each] dNTPs) was then added to each tube, and the reaction mixtures were incubated at 37°C for 20 h. The reactions were stopped by adding an equal volume of 2x lysing buffer (2x lysing buffer is 100 mM Tris-HCl [pH 7.4], 20 mM EDTA, 2% [wt/vol] SDS, 200 mM NaCl, 100 µg/ml yeast tRNA, 20 µg/ml proteinase K [Invitrogen, Carlsbad, CA]), and the reaction mixtures were incubated for 3 to 5 h at 37°C. Samples were then extracted twice with phenol-chloroform-isoamyl alcohol (Invitrogen) and then ethanol precipitated overnight.

Quantitation of viral RNA and DNA. The ethanol precipitates from the ERT assays or lysed viral pellets were collected by centrifugation and resuspended in 50 µl of diethylpyrocarbonate-treated water. Half of the sample was used for gag RNA quantitation, while the other half was used for DNA quantitation. To quantitate gag targets in gRNA, samples were treated with 10 U of RQ1 RNase-free DNase (Promega, Madison, WI) for 1 h at 37°C in a volume of 100 µl according to the manufacturer's instructions. The reaction was stopped by adding 100 µl of 4 M guanidine isothiocyanate, and the nucleic acids were ethanol precipitated together with 1 µl of 25 mg/ml yeast tRNA (a precipitation aid). The RNA precipitate was resuspended in diethylpyrocarbonate-treated water with 0.1 mM dithiothreitol (Promega) and 1 U/µl RNaseOUT (Invitrogen). The quantities of gRNA were measured by real-time RT-PCR as described previously (65). To quantitate DNA, samples were diluted in buffer (10 mM Tris-HCl [pH 8.0], 0.1 mM EDTA) and then used in the real-time PCR assays described previously (11). The quantities of VSV-G DNA were determined using the following primers and probe that produce an amplicon of 79 bp: VSV-G For primer (5'-TGAATCCAGGCTTCCCTCCTC; Invitrogen), VSV-G Rev primer (5'-GGAGTCACCTGGACAATCACTGC; Invitrogen), P-VSVg-01 probe (5'-FAM-TGGATATGCAACTGTGACGGATGCCG-TAMRA [FAM is 6-carboxyfluorescein and TAMRA is 6-carboxytetramethylrhodamine]; BioSource Invitrogen). The reaction conditions were described previously (11), and the thermocycling conditions were 95°C for 10 min, followed by 45 cycles, with 1 cycle consisting of 15 seconds at 95°C and 30 seconds at 60°C. Real-time PCR was performed using either an ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA) or a Stratagene MX 3000 QPCR system.

35S metabolic labeling. 293T cells in 150-cm2 flasks were transfected with 40 µg of either sheared salmon sperm DNA per flask (negative control), NCH23C (29), NCH44C (29), PR(–) (56), or NCWT (pNL4-3 [2]) with TransIT 293 (Mirus Bio, Madison, WI) according to the manufacturer's instructions. These transfections were performed in the absence of pHCMV-g, thus preventing reinfection of the wild-type sample, which would complicate interpretation of results. After 48 h, cells were dislodged from the flask surfaces with 10 ml of PBS without Ca2+ or Mg2+ and transferred to 50-ml tubes. Cells were then processed and labeled essentially as described by Rudner et al. (60), except the cells were pulsed with [35S]Cys and [35S]Met for 30 min instead of 6 h and chased for the indicated times with complete medium that did not contain radiolabel. One flask was used for each sample at each time point. Cell and viral lysates were prepared, and 35S-radiolabeled Gag proteins were collected by immunoprecipitation using the goat anti-p24CA and rabbit anti-p7NC antibodies as described previously (60); the p6 antiserum was not used. Immunoprecipitated proteins were fractionated on 4 to 20% Novex, Tris-glycine gels (Invitrogen); the gels were dried and exposed to a Kodak phosphorimager screen. The radiolabeled images were collected using a Personal Molecular Imager FX phosphorimager (Bio-Rad Laboratories).


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RESULTS
 
Efficacy of DNase I plus subtilisin treatment. Because we observed high initial quantities of vDNA shortly after infection of HOS cells with the NCH23C and NCH44C mutant viruses (65), we decided to examine the levels of vDNA contained within newly produced mutant NC and wild-type NC virions. As these two NC mutations produce viruses that are replication defective (29), mutant virions must be generated by transfection. We cotransfected 293T cells with the proviral plasmids and a VSV-G expression plasmid in an equal molar ratio to generate pseudotyped virus particles. This was done for two reasons: to study virus particles prepared in a manner identical to that in our previous reports (11, 65) and to monitor the quantities of VSV-G DNA in our virus preparations for the effectiveness of the DNase I plus subtilisin treatment (see below). Transfection-generated virus is typically highly contaminated with the plasmid DNAs used in the transfection, and this plasmid DNA will obviously interfere with accurate quantitation of vDNA. Therefore, we treated virus stocks with DNase I and then with subtilisin, pelleting the virus through a 20% (wt/vol) sucrose pad after each step. This stringent protocol very effectively removes the vast majority of contaminating plasmid DNA from our virus preparations that would occur in free form and DNA that might be contained within contaminating microvesicles (10, 56, 57) or the reported tubulovesicular structures that arise upon transfection of pHCMV-g (58). For each virus preparation that was treated with DNase I plus subtilisin, we isolated the total nucleic acid contents and then quantitated viral RNA and vDNA using the TaqMan primers and probes that we used for our previous study of early infection events (11, 65). The species detected include minus-strand strong-stop DNA (R-U5), minus-strand transfer product (U3-U5), late minus-strand synthesis product (Gag), and plus-strand transfer product (R-5' untranslated region [5'UTR]).

The efficacy of the DNase I plus subtilisin procedure is illustrated in Fig. 1A, which depicts the quantities of vDNA measured in a typical experiment before and after treatment of wild-type virus with DNase I plus subtilisin. In untreated samples, the amount of DNA is very high (>10 copies of each vDNA species per gRNA; VSV-G DNA at ~1 copy per HIV-1 gRNA). Because virions contain two genomes, any more than two copies of vDNA per virion is undoubtedly the result of contamination by the transfection plasmid DNA. This conclusion is further supported by VSV-G DNA being present at such high levels. After DNase I plus subtilisin treatment, we observed a decrease of between 104- and 105-fold for reverse transcription sequence targets, while quantities of VSV-G DNA decreased 106-fold (Fig. 1A). The dramatic decrease in all DNA quantities indicates that our protocol was effective at removing carryover plasmid DNA. Remarkably, after removing this plasmid contamination, the quantities of minus-strand strong-stop (R-U5) DNA per wild-type virion (accounting for two genomes per virion) is around 1 in 400, while the reverse transcription products formed after the minus-strand strong-stop DNA are present in even lower amounts from 1 in 1,700 virions for minus-strand transfer (U3-U5) to 1 in 8,300 vDNA molecules per virion for plus-strand transfer (R-5'UTR) products. Because proviral plasmid carryover would be detected by all the primers used, the fact that each subsequent species is present at progressively lower amounts is consistent with this signal being authentic. In addition, the reduction in the relative amount of VSV-G DNA in virions from 2 in 1 to 1 in 1,700,000 indicates that the residual level of contaminating plasmid, while still detectable, is trivial.

As we previously mentioned, depending on the cell type used, virus preparations can contain high levels of microvesicles (10). These membrane enveloped structures can contain cellular and viral proteins as well as cytoplasmic RNAs and tend to copurify with virus particles unless specific methods are used to remove them (i.e., subtilisin digestion [57] or CD45 depletion [18]). Subtilisin digestion and the subsequent density-dependent pelleting step typically remove the majority of these artifactual proteins (56) (Fig. 1B). Although we see a significant reduction in total protein in each preparation after subtilisin digestion, the reduction in p24CA levels are less than twofold (Fig. 1B). The band migrating at ~55 kDa in the untreated samples (no-subtilisin lanes in Fig. 1B) is not Pr55Gag, as confirmed by Western blotting (data not shown), and it does not survive subtilisin digestion (compare the two leftmost lanes in Fig. 1B). Curiously, in these same samples, we typically see an approximately 10-fold decrease in the amount of HIV-1 gRNA before and after the DNase I plus subtilisin treatment. The difference in the amount of p24CA and gRNA decrease may be due to some viral RNA being present in microvesicles (i.e., viral RNA as a portion of the cellular mRNA present [10]), while the majority of p24CA is present in virus particles. In summary, these observations highlight the importance of stringently purifying virus preparations for these types of analyses and also demonstrate the effectiveness of our protocol for investigating intravirion DNA in virions generated from transfections.

NCH23C and NCH44C mutations result in high levels of intravirion DNA. Pseudotyped virus particles containing either the NCH23C or NCH44C mutation were examined for the presence of intravirion DNA, and the average results from two independent experiments are shown in Fig. 1C. Only quantities after DNase I plus subtilisin treatment, relative to gRNA are shown, but the quantities before DNase I plus subtilisin treatment showed high levels of plasmid carryover (i.e., all present at similar levels of ~10 copies per virus particle; data not shown). Remarkably, based on the amount of gRNA measured, almost every one of these NC mutant virions contains a copy of minus-strand strong-stop DNA. This is approximately 1,000-fold more R-U5 copies than what was observed with wild-type particles (Fig. 1C). The reverse transcription intermediates synthesized after the minus-strand strong-stop DNA are present in progressively lower amounts as expected, so that when theses quantities are compared to quantities of R-U5, the relative amounts are similar for the mutant and wild-type NC HIV-1. This indicates that reverse transcription processivity (or progression) is similar for the mutant and wild-type viruses. We transfected equal amounts of mutant and wild-type NC proviral plasmid DNA along with the pHCMV-g plasmid into 293T cells to produce the viruses; when VSV-G DNA was measured, we found that these quantities were independent of the number of virus particles produced so that the approximate numbers of copies were similar for the different samples (the numbers of copies per ml before/after DNase I plus subtilisin treatment were 5,360/92 for NCWT, 3,800/30 for NCH23C, and 3,770/43 for NCH44C). Thus, these functionally conservative zinc finger mutations (they preserve Zn2+ binding) (37, 43) cause a tremendous increase in intravirion DNA corresponding to reverse transcripts.

In this report, we present the quantities of vDNA normalized to the quantity of gRNA present in each sample. This was performed since RT-PCR is far more sensitive than measuring quantities of p24CA, which allowed us to investigate smaller-scale virus preparations for our experiments. For all of these viruses (NCWT, NCH23C, and NCH44C), we see similar ratios of gRNA to p24CA (Fig. 1D). The yield of VSV-G-pseudotyped NCWT virus is much greater than yields of either NCH23C or NCH44C VSV-G-pseudotyped virus (~1,000-fold), because wild-type virus is amplified during the transfection procedure by reinfection. VSV-G-pseudotyped virus can infect 293T cells, but while NCWT is replication competent, the NC mutant viruses are not. Therefore, wild-type virus is produced both from the transfection and subsequent infection events, while virus containing the NC mutants is limited to that produced mainly from the transfection. Amplification by infection in the wild-type case can be blocked by culturing the transfected cells in the presence of reverse transcription inhibitor PMPA (Fig. 1D). When mutant and wild-type NC proviral plasmids are transfected without pHCMV-g, we observe similar yields of virus (see Fig. 4) (29).


Figure 4
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  		FIG. 4. Processing of Pr55Gag. A pulse-chase metabolic labeling experiment is presented where p24 or p7 antiserum was used to immunoprecipitate radiolabeled Gag proteins from 0 to 6 h after labeling. 293T cells were transfected with mutant or wild-type NC plasmids in the absence of pHCMV-g. Gag species present in the cell lysates (A) and viral lysates (B). The positions of Pr55Gag, p24CA, and p7NC are indicated by the arrows to the right of the gels. The positions of molecular mass markers (in kilodaltons) are indicated to the left of the gels. (–)-Control, negative control.

A functional RT is required for intravirion DNA. The high levels of intravirion DNA were unexpected in HIV-1, so we determined the impact of eliminating either the polymerase or RNase H activities of RT on the presence of intravirion DNA. To this end, we incorporated the RTD185K/D186L (inactivates polymerase [RT(–)] [36]) or RTE478Q (inactivates RNase H [RNase H(–)] [19]) active site mutations individually into the NCWT, NCH23C, and NCH44C proviruses. Results from a representative experiment are shown in Fig. 2A to C. With NCWT virus, we see very little difference between the quantities of vDNA when the polymerase activity is inactivated. In contrast to this, we see that the high levels of intravirion DNA in the two NC mutants is greatly reduced when RT lacks polymerase activity.


Figure 2
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  		FIG. 2. Requirement for a fully functional RT for intravirion DNA. The relative amounts of intravirion DNA from a representative experiment, expressed as a ratio of the amount of each indicated DNA species per ml to the amount of gRNA per ml, are shown for wild-type and mutant NC HIV-1. (A) NCWT, NCWTRT(–), and NCWTRNase H(–); (B) NCH23C, NCH23CRT(–), NCH23CRNase H(–); (C) NCH44C, NCH44CRT(–), NCH44CRNase H(–). Error bars indicate the standard deviations for duplicate PCR wells. The quantities of gRNA per ml were 6.0 x 109 for NCWT, 3.4 x 107 for NCWTRT(–), 2.9 x 106 for NCWTRNase H(–), 3.5 x 106 for NCH23C, 7.2 x 105 for NCH23CRT(–), 4.3 x 106 for NCH23CRNase H(–), 1.8 x 106 for NCH44C, 1.5 x 105 for NCH44CRT(–), and, 4.4 x 105 for NCH44CRNase H(–).

In addition, when we incorporated the RNase H(–) mutation into the various NC mutant proviruses, we observe that quantities of minus-strand strong-stop DNA are similar to those in virus containing a functional RNase H activity, but all subsequent DNA species are at levels similar to those of viruses lacking functional polymerase activity (Fig. 2B and C). This result is consistent with the mechanism of reverse transcription, where RNase H digests the gRNA after the polymerase reverse transcribes it into DNA. If the RNase H activity is removed, the gRNA complementary to the minus-strand strong-stop DNA remains annealed and minus-strand transfer cannot occur (64, 69). Thus, in these experiments, we established that the high levels of intravirion DNA were absolutely dependent on a fully functional RT enzyme (both the polymerase and RNase H activities are necessary).

Again, it should be noted that the quantities of VSV-G DNA appear to differ substantially between the different mutant and wild-type RT viruses (most obviously with NCWT, NCWT RT(–), and NCWT RNase H (–) [Fig. 2A]), but this is because the numbers of virions produced is much less with the RT(–)- and RNase H(–)-containing viruses, as these are replication defective (as discussed above).

ERT activity of mutant and wild-type viruses. Because the synthesis of vDNA by RT results in degradation of the gRNA template, we were interested in determining whether these mutant virions exhibited ERT activity. The ERT assay requires that RT reverse transcribe the gRNA within the confines of the virion membrane, in contrast to exogenous-template RT assays that employ an uncomplicated poly(A) template and oligo(dT) primer. Reverse transcription in the ERT setting is much more stringent, as the gRNA is highly structured (71). If the NCH23C and NCH44C mutants actually contain such high levels of intravirion DNA, one would expect to see little or no ERT activity because during reverse transcription the RNA template (genome) is degraded by the RNase H activity of RT (see above). To investigate this issue, we adapted an ERT assay (41) for measuring vDNA products. The main advantages for using quantitative PCR analysis rather than incorporation of radiolabeled nucleotides are that the formation of reverse transcription products can be reliably quantitated and the progression of reverse transcription can be ascertained. Figure 3A shows ERT results along with several control reactions for NCWT virus, which are expressed as a ratio of the quantity of vDNA measured at the end of the ERT reaction divided by the quantity of gRNA present (in the input virus) before the 20-h ERT incubation was initiated. An increase of 2 orders of magnitude in the quantities of minus-strand strong-stop DNA was observed with NCWT upon addition of detergent and dNTPs (data set with dNTPs) over quantities present in "untreated" virions (detergent without dNTPs), to the point that practically every genome gave rise to the R-U5 product. We see an increase of 3 orders of magnitude in the quantities of minus-strand transfer product (U3-U5) and an increase of 2 orders of magnitude in the quantities of both late minus-strand synthesis product (Gag) and plus-strand transfer product (R-5'UTR; Fig. 3A). Because heating virus samples (68°C for 20 min) results in inactivation of reverse transcription, we see no change in the quantities of products, whether or not dNTPs were added with this control. Thus, our ERT system is highly active and processive enough for quantities of plus-strand DNA transfer (late reverse transcription) products to reach approximately 1% of input genomes. However, it is important to note that because our PCR assays detect small vDNA targets, the presence of plus-strand transfer intermediates does not necessarily indicate that full-length double-stranded vDNA was synthesized.


Figure 3
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  		FIG. 3. ERT activities of mutant and wild-type NC viruses. (A) dNTP requirement for ERT activity using wild-type, VSV-G-pseudotyped virus. These results are expressed as a ratio of the amount of each indicated DNA species per ml (measured at the end of the ERT reaction) to the amount of gRNA per ml (measured before the ERT incubation commenced). Bars labeled "untreated" represent viral samples that were put into ERT buffer without dNTPs. Bars labeled "+ dNTPs" indicate that the viral samples were incubated in ERT buffer containing dNTPs. Bars labeled "heated" and "heated + dNTPs" were analogous samples that were heat inactivated prior to the ERT reaction. Values are the averages of two independent experiments, with error bars indicating the standard deviations. The average quantity of gRNA used was 3.5 x 1010 genomes per ml. (B) Relative amounts of DNA targets in parallel samples measured after ERT using virus samples either heat inactivated prior to ERT (heated/+ dNTPs) or not (+ dNTPs). Values are the averages of two independent experiments, with error bars representing standard deviations. Results are expressed as a ratio of the quantity of each DNA species per ml after the ERT to the quantity of gRNA per ml used to start the ERT reaction. Average quantities of genomes per ml were 3.2 x 109 for NCWT, 2.3 x 106 for NCH23C, and 1.1 x 106 for NCH44C.

Next we examined the ERT activity for both NC mutants, and these results are presented in Fig. 3B. Alternating bars in Fig. 3B report vDNA quantities per gRNA in heat-treated samples (white bars) or just after ERT (black bars). As expected, neither NC mutant exhibited increases in quantities of vDNAs after ERT compared to heat-treated controls for any vDNA species. If anything, the relative levels of vDNA were slightly lower after ERT. These results correlate with the high levels of intravirion DNA observed in the NC mutants (everything that could have been reverse transcribed has already been consumed) and also indicate that these virions cannot synthesize additional late products. It should also be noted that the efficiency of premature reverse transcription (comparing relative amounts of late versus early reverse transcripts) is greater in the NC mutants than the efficiency of ERT for the wild-type virus (Fig. 3B).

Pr55Gag proteolytic processing. The characterization of viruses containing an RT that was defective in either polymerase or RNase H activity demonstrated that the intravirion DNA detected was truly synthesized by viral enzymes (Fig. 2), but it is unlikely that any significant reverse transcription can occur within extracellular virions (see Discussion). Conceptually, it is intriguing to consider the possibility that these NC mutations cause reverse transcription to begin prior to particles budding from producer cells. One possible mechanism for this would be that the NC mutations cause alterations to the structure of Pr55Gag or Pr170Gag-Pol so that PR is activated too early. More rapid proteolytic processing would be predicted to result in the earlier appearance of mature RT, which could give rise to earlier reverse transcription of the viral genome. In addition, with earlier PR activity, the budding particles may liberate p9NC or p7NC earlier, which could assist in the reverse transcription process via their nucleic acid chaperone function (5, 6, 46, 59, 66). Alternatively, these NC mutants may allow reverse transcription to proceed without the premature processing of Pr55Gag or Pr170Gag-Pol. It has been reported that Pr170Gag-Pol from wild-type virus is very active in ERT assays (39) (see below).

We investigated potential changes in Pr55Gag processing kinetics by performing a pulse-chase metabolic labeling experiment (Fig. 4). 35S-radiolabeled HIV-1 Gag proteins were immunoprecipitated with a mixture of p24CA and p7NC antisera and then fractionated by SDS-PAGE. Cells transfected with mutant and wild-type NC proviral plasmids produced Pr55Gag in abundance, the presence of the NCH23C or NCH44C mutation did not appreciably change the level of Pr55Gag synthesis, and over the chase period, Pr55Gag decreased slightly for all samples (Fig. 4A). In lysates from cells transfected with the NCWT, NCH23C, or NCH44C clone, one sees low levels of p24CA at 1 h, with increasing amounts at 3 and 6 h. In addition, for NCWT, p7NC is evident at 3 and 6 h. Examination of viral lysates for Gag (Fig. 4B) shows somewhat less p24CA and p7NC in virions containing NC mutations compared to NCWT virions. This result indicates that the NC mutations do not appreciably accelerate proteolytic processing of Pr55Gag and thus is probably not the main reason for the premature reverse transcription phenotype. It should be noted that the viral protein levels in Fig. 4 are similar for the mutant and wild-type NC samples; these transfections were performed in the absence of pHCMV-g.

Intravirion DNA production in a PTAP mutant. Interestingly, if the balance between budding and reverse transcription is sensitive enough for these minor alterations in NC to cause such extensive premature reverse transcription, we postulated that if the budding process were delayed, we might observe high levels of intravirion DNA even in the presence of wild-type NC. We approached this possibility by measuring the amounts of intravirion DNA in otherwise wild-type virus particles containing a mutation in the PTAP (Pro-Thr-Ala-Pro) sequence of p6 (35). This mutant exhibits slower budding kinetics from the plasma membrane compared to wild-type virus (55). In addition, the phenotype of this PTAP mutant is similar to those of the NC mutants: mutant PTAP virus can initiate a single cycle of infection (albeit inefficiently), but this infection does not lead to a spreading infection (55).

We transfected both 293T and HeLa cells with NCWT or PTAP(–) proviral plasmids (without pHCMV-g) and examined the amount of minus-strand strong-stop DNA present in particles (Fig. 5). These viruses were generated from transfections using Mirus Bio's TransIT 293 or HeLa Monster instead of the CaPO4/DNA coprecipitation method previously used (similar levels of intravirion DNA were observed with NCWT virus by either method [compare NCWT in Fig. 1 and 5]). Interestingly, we observed that quantities of R-U5 were 100-fold higher within PTAP(–) virions than in wild-type virions. We performed these experiments using the two different cell types because this mutant has been studied more extensively in HeLa cells (35, 55), but in our experiments the amount of intravirion DNA was independent of cell type. Thus, it would seem that slowing the release rate of particles from the plasma membrane can result in premature reverse transcription (increased intravirion DNA).


Figure 5
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  		FIG. 5. The PTAP(–) mutant shows high levels of intravirion DNA. The ratio of intravirion minus-strand strong-stop DNA to gRNA was determined for wild-type virus and the PTAP(–) mutant after transfection of 293T or HeLa cells with proviral plasmids (virions were not pseudotyped with VSV-G). Error bars represent the standard deviations of duplicate PCR wells. The quantities of gRNA measured follow: 8.2 x 107 gRNA/ml for wild-type virus produced from 293T cells, 5.6 x 107 gRNA/ml for PTAP(–) virus produced from 293T cells, 3.6 x 107 gRNA/ml for wild-type virus produced from HeLa cells, and 1.1 x 106 gRNA/ml for PTAP(–) virus produced from HeLa cells.

Effect of Gag and Gag-Pol processing on intravirion DNA. It is known that PR is active as part of Pr170Gag-Pol and that PR activity is also evident in budding particles (40). Therefore, it may be possible that the amount of RT released from Pr170Gag-Pol during budding is necessary for the extraordinarily large amounts of intravirion DNA. To investigate this, we individually combined the two NC mutations with a PRD25A active site mutation (42). As expected, the combination NC-PR(–) mutants showed no processing of the Pr55Gag proteins in Western blots (data not shown). Figure 6A shows the copy numbers of R-U5 per viral gRNA from virions with the various combinations of NC and PR. With a defective PR, there was a 100-fold decrease in the amount of minus-strand strong-stop DNA present in NC mutant particles compared to those with a functional PR, but the amounts of vDNA are still higher in the NC mutant virions than in wild-type virions in the PR(–) background. The last observation indicates that blocking processing of Pr55Gag or Pr170Gag-Pol proteins does not entirely eliminate the production of intravirion DNA resulting from the NC mutations. Thus, these NC mutants permit more extensive reverse transcription than the wild type does, even in the presence of unprocessed RT.


Figure 6
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  		FIG. 6. Impact of inactivating PR on intravirion DNA and ERT activity. Mutant and wild-type HIV-1 virions were generated by cotransfection of pHCMV-g and the corresponding proviral plasmids. (A) The ratio of intravirion minus-strand strong-stop (R-U5) DNA relative to gRNA was determined for wild-type and NC mutants without and with the PR(–) mutation. Results are the averages of two experiments, and error bars represent the standard deviations. The average amounts of gRNA per ml were 3.8 x 109 for NCWT, 7.7 x 107 for NCWTPR(–), 5.5 x 106 for NCH23C, 1.4 x 107 for NCH23CPR(–), 1.8 x 106 for NCH44C, and 2.6 x 106 for NCH44CPR(–). (B) ERT activity of the wild type and NC mutants without and with the PR(–) mutation. The values are the ratios of minus-strand strong-stop DNA per ml measured after ERT to gRNA per ml before ERT in parallel samples either heat inactivated prior to ERT (heated/+ dNTPs) or not (+ dNTPs). The results are the average of two experiments, with error bars representing the standard deviations. The quantities of gRNA per ml were 2.4 x 109 for NCWT, 4.4 x 107 for NCWTPR(–), 1.6 x 105 for NCH23C, 9.1 x 106 for NCH23CPR(–), 2.0 x 105 for NCH44C, and 3.6 x 105 for NCH44CPR(–).

We also examined the ERT activity of PR(–) viruses. Figure 6B compares the amount of minus-strand strong-stop DNA in heat-inactivated virions and virions after undergoing ERT. Intriguingly, we see that PR(–) virus particles do exhibit ERT activity. As expected, in NCWT PR(–) virions, ERT activity is lower than in wild-type virions in which RT is fully processed. However, for the NC mutants, we could now observe some ERT activity when the NC mutation was combined with the PR(–) mutation (Fig. 6B). Interestingly, it has been reported that Pr170Gag-Pol shows high activity in ERT reactions (39); thus, the quantity of intravirion DNA present in the NC mutants (without a PR(–) mutation) does appear to require the presence of some proteolytically processed RT. This result also corroborates the observation made above (Fig. 6A) that premature reverse transcription is less extensive in NC mutants with a defective PR.


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DISCUSSION
 
The NCH23C and NCH44C mutant viruses are replication defective; however, they are still able to enter cells and reverse transcribe their genomes (11, 29, 65). We previously reported that after infection with these particular mutants, the principal defect appears to be at the integration step (65). Curiously, despite an extensive body of work showing the importance of NC for reverse transcription in vitro, we do not observe any serious defects in vivo, although we do see that reverse transcription proceeds faster for the NC mutants than for wild-type virus (65). Faster reverse transcription may be due to an increase in the rate of reverse transcription during infection or premature reverse transcription resulting in nascent virions containing abnormally high levels of vDNA. In this report, we examined cell-free virus particles to ascertain whether premature reverse transcription did in fact occur.

We developed a stringent method to remove contaminating plasmid or extravirion DNA and quantitated the amount of various reverse transcription intermediates in virus particles. Using these methods, we observed that both the NCH23C and NCH44C virions contained significantly higher levels of vDNA than wild-type virions did (Fig. 1C). In fact, it appears that every virus particle contains minus-strand strong-stop DNA, which is a 1,000-fold increase over the amount observed in wild-type virus particles. These results were further substantiated by the observation that an active RT is required (Fig. 2) and that these mutants exhibit no ERT activity (Fig. 3B). One question that may be posed is why we typically see such high levels of gRNA in these mutant NC particles (on a per particle basis, similar to wild-type levels) when the quantity of vDNA is so high. It is important to keep in mind that the vDNA species present at the highest level is minus-strand strong-stop (R-U5) DNA but that we see less vDNA representing subsequent reverse transcription steps in virions. We quantitate gRNA by amplifying a gag sequence present only in unspliced RNA (65), but this RNA target is consumed only during late minus-strand synthesis. Because the later vDNA products are present at ~10-fold-lower levels than genomes are, we measure near-wild-type levels of gRNA in the NC mutants. It should also be pointed out that reverse transcription is more extensive upon infection of cells (65) compared to either premature reverse transcription (in the NC mutants) or efficient ERT (with wild-type virus) demonstrated in this work, suggesting the involvement of additional cellular factors.

We demonstrated that premature reverse transcription was not caused by more rapid Pr55Gag (and/or Pr170Gag-Pol) processing (Fig. 4). However, proteolytic processing is required, as inactivating PR caused a substantial decrease in the quantities of intravirion DNA in the NC mutants (Fig. 6). Curiously, although quantities decreased, viruses containing the NC mutations still contain significantly higher levels of vDNA compared to the level in wild-type virus. Thus, it would appear that the majority of vDNA synthesized is by the fraction of RT liberated by PR during the budding process; however, it is difficult to determine the level of processed RT present, as even particles harvested rapidly after budding have been shown to contain fully processed viral proteins as shown in Western blots (40).

We would assert that the vDNA observed in particles must be synthesized prior to or during the budding process, while virions are still attached to the cell membrane and thus have access to cellular dNTP pools. Support for this hypothesis comes primarily from our calculations regarding the extent to which reverse transcription can occur in extracellular virus particles. If the average diameter of HIV-1 particles is around 100 nm (70) and assuming that the particle is a sphere, the volume is ~5 x 10–19 liters. Intracellular concentrations of dNTPs depend on the cell type but are typically 3 to 5 µM (49). If dNTP incorporation into viral particles is by passive diffusion, this would mean that no more than one molecule of dNTP is present per virion, which is certainly not enough to synthesize even minus-strand strong-stop DNA. Thus, it is very likely that the intravirion vDNA observed is synthesized only before or during the budding process, when the RT (either processed or as a domain of Gag-Pol) still has access to physiologically significant intracellular dNTP pools. It should also be noted that we found a strong requirement for detergent in our ERT reactions and that ERT in wild-type virions was not as efficient as the premature reverse transcription observed in the mutant NC viruses (Fig. 3B). This later observation argues against premature reverse transcription occurring when virions are exposed to dNTPs present in extracellular fluids (34, 73).

A prediction of our model would be that if budding could be delayed, we may see an increase in intravirion DNA. Our data suggest that whatever fraction of Gag-Pol is processed prior to budding (yielding mature RT) is capable of premature reverse transcription in the context of these NC mutations. Interestingly, in the context of the PTAP(–) mutation with a delayed-budding phenotype (35), premature reverse transcription was observed in the context of wild-type NC (Fig. 5). The relationship between NC and budding has also been demonstrated by the fact that a zinc finger deletion mutant of Rous sarcoma virus exhibited a 10-fold reduction in budding (45). It is unknown whether this mutant would give rise to premature reverse transcription.

There has been a recent report that HIV-1 virions containing a severe zinc finger mutation ({Delta}ZF2) in NC can also cause an increase in the quantity of intravirion DNA, approximately 100-fold higher than the quantity in wild-type HIV-1 virions, although the authors are unable to determine how many particles contain vDNA (34). However, the nature and phenotypes of the NC mutants examined in our report (NCH23C and NCH44C) are significantly different from those of the {Delta}ZF2 mutant: the zinc finger deletion mutant packages very little gRNA (<5% [34]), while the NCH23C and NCH44C mutants package close to wild-type quantities (Fig. 1D) (29). Even more significant is that the {Delta}ZF2 mutant cannot infect a cell (i.e., enter and reverse transcribe its genome) (33), while the two NC mutants investigated in this work can infect cells for a single round (11, 65). However, it is interesting to note that these highly divergent mutations result in this similar phenotype.

In conclusion, there are two key implications of our data regarding (i) the nature of the early infection defect in the NCH23C and NCH44C mutants and (ii) the significance of intravirion DNA in HIV-1.

The early infection defect caused by the NCH23C and NCH44C mutations appears to be primarily a defect in integration (65). However, although we mainly see a reduction in the formation of proviruses, the nature of the defect is likely a result of the uncoupling of the reverse transcription and uncoating processes (for a more extensive discussion, see reference 66). Our results suggest that reverse transcription occurring too early can be detrimental to infection. In agreement with this proposal, certain mutations to the primer activation signal in HIV-1 cause an increase in reverse transcription activity and corresponding replication defects (1, 7, 8). In addition, there are several CA and MA (matrix protein) mutations that stabilize the core and inhibit reverse transcription (21, 25, 41). We do not have direct evidence that premature reverse transcription would cause premature uncoating, although it has been reported that virus particles that carried out natural endogenous reverse transcription exhibited poorly defined cores as seen in electron micrographs (72).

If uncoupling reverse transcription and uncoating results in these replication defects, it is also likely that the presence of intravirion DNA indicates a replication-defective HIV-1 particle. This is in contrast to the highly divergent spumaretrovirus, prototype foamy virus, that maintains its extracellular genetic material in the form of DNA (47). Although the ratio of infectious units per virus particle is very low (~1:1,000), the actual number of infectious particles per virus particle is much higher (1:3 to 1:8) having more to do with the probability that a virion will contact a cell than intrinsic defectiveness (53, 67). Because the number of wild-type virions that contain intravirion DNA are a very small fraction compared to the number of infectious particles, the majority, if not all, infectious particles do not contain DNA. Ultimately, these data support the model in which there is tight regulation/association between the steps of viral assembly, proteolytic processing, budding, maturation, entry, core uncoating, reverse transcription, nuclear transport, and integration so that altering any of these steps would result in a defective virus particle (21, 25, 33, 41, 62, 65, 66).


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ACKNOWLEDGMENTS
 
We thank David Ott of the AIDS Vaccine Program, SAIC-Frederick, Inc., Alan Rein of the HIV-Drug Resistance Program, NCI-Frederick, and Christopher Aiken of Vanderbilt University School of Medicine, Nashville, TN, for critical comments and helpful suggestions. We also thank Gilead Sciences, Inc., Foster City, CA, for the PMPA.

This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-CO-12400.

The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government.


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FOOTNOTES
 
* Corresponding author. Mailing address: AIDS and Cancer Virus Program, SAIC Frederick, Inc., NCI-Frederick, P.O. Box B, Frederick, MD 21702-1201. Phone: (301) 846-5980. Fax: (301) 846-7119. E-mail: gorelick{at}ncifcrf.gov Back

{triangledown} Published ahead of print on 30 July 2008. Back


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Journal of Virology, October 2008, p. 9318-9328, Vol. 82, No. 19
0022-538X/08/$08.00+0     doi:10.1128/JVI.00583-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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