Equine Infectious Anemia Virus Gag p9 Function in
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病菌学杂志 2005年第14期
Department of Molecular Genetics and Biochemistry, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
ABSTRACT
We have previously reported that serial truncation of the Gag p9 protein of equine infectious anemia virus (EIAV) revealed a progressive loss in replication phenotypes in transfected cells, such that a proviral mutant (E32) expressing the N-terminal 31 amino acids of p9 produced infectious virus particles similarly to parental provirus, while a proviral mutant (K30) with two fewer amino acids produced replication-defective virus particles, despite containing apparently normal levels of processed Gag and Pol proteins (C. Chen, F. Li, and R. C. Montelaro, J. Virol. 75:9762-9760, 2001). Based on these observations, we sought in the current study to identify the precise defect in K30 virion infection of permissive equine dermal (ED) cells. The results of these experiments clearly demonstrated that K30 virions entered target ED cells and produced early (minus-strand strong-stop) and late (Gag) viral DNA products as efficiently as did the replication-competent E32 mutant and parental EIAVUK viruses. However, in contrast to the replication-competent E32 mutant and parental viruses, infection with K30 mutant virus failed to produce detectable two-long-terminal-repeat DNA circles, stable integrated provirus, virus-specific Gag mRNA expression, or intracellular viral protein expression. Taken together, these data demonstrate that the K30 mutant is defective in the ability to produce sufficient nuclear viral DNA to establish a productive infection in ED cells. Thus, these observations indicate for the first time that the EIAV Gag p9 protein performs a critical role in viral DNA production and processing to provirus during EIAV infection, in addition to its previously defined role in viral budding mediated by the p9 L domain.
INTRODUCTION
The functions of retroviral Gag proteins in virus-infected cells to accomplish various steps in virion assembly and budding have been the subject of intense investigation leading to an increasingly intricate model of highly specific Gag protein interactions with other virion protein and RNA components and with host cell proteins (1, 13, 25, 40, 41, 43, 45). However, there are also increasingly more data suggesting important functional roles for retroviral Gag proteins during virus infection of target cells postentry. For example, the matrix (MA), capsid (CA), and nucleocapsid (NC) proteins have all been implicated in reverse transcription of the retrovirus genomic RNA to produce viral DNA (16, 19, 22, 35, 36, 53, 59). The integrase (IN) and MA proteins are believed to be critical components of the preintegration complex (PIC) that translocates the viral DNA to the cell nucleus, where it is integrated into the host chromosome (2, 12, 46, 50, 60, 62). In addition to the CA, MA, and NC Gag proteins common to all retroviruses, the lentiviruses characteristically contain an additional Gag protein (human immunodeficiency virus type 1 [HIV-1] p6 and equine infectious anemia virus [EIAV] p9) that has been shown to provide critical late functions in virion assembly and budding via highly specific interactions with various endocytic proteins in infected cells (11, 17, 27, 30, 39, 51, 52, 55, 58). A specific role for HIV-1 p6 or EIAV p9 in virus infection has to date not been definitively established.
We previously reported that serial truncations of the p9 protein in the context of the EIAVUK provirus revealed a progressive loss of replication competence in transfected cells with increased reduction in p9 length (11). The results of these studies demonstrate that EIAV proviral mutant viruses containing at least the N-terminal 31 amino acids of p9 had replication levels similar to those of the parental EIAVUK virus, indicating that the first 31 amino acids can supply all of the necessary functions for productive infection of equine dermal (ED) cells. In contrast, proviral mutants containing larger p9 truncations were found to be replication defective in ED cells. Further functional characterization of the various replication-defective p9 truncation proviral constructs revealed that p9 mutants lacking a functional L domain (19YPDL22) were greatly suppressed in virion production, the expected phenotype for an L-domain-negative mutant. Interestingly, intermediate p9 truncation proviral mutants containing the L domain but less than 31 amino acids were found to produce virus particles from transfected COS-7 cells at levels similar to those for transfections with the parental EIAVUK provirus DNA, and the mutant p9 virions appeared to be normal for Gag and Pol incorporation and processing. Based on these observations, we hypothesized that the replication-defective nature of these p9 truncation mutants might be due to defects in virion infectivity.
In the current study, we examine this hypothesis by comparing at each step of virus infection the functional competence of the replication-defective mutant K30 expressing the N-terminal 29 amino acids of p9, the replication-competent mutant E32 expressing the N-terminal 31 amino acids of p9, and the parental EIAVUK provirus expressing the full-length p9 protein containing 51 amino acids. The results of these studies revealed that the defect in replication by the K30 mutant is associated with an apparent block in the production of nuclear viral DNA from linear DNA reverse transcripts. Thus, these observations demonstrate for the first time a critical role for the EIAV p9 protein in the early stages of viral infection that lead to the generation of stable integrated provirus necessary for establishing productive infection of target cells.
MATERIALS AND METHODS
Cells. The ED cell line permissive for EIAV replication was obtained from the American Type Culture Collection (ATCC CCL-57) and grown in minimal essential medium supplemented with 10% fetal bovine serum from HyClone Laboratories (Ogden, UT), 2 mM glutamine, nonessential amino acids, 100 U of penicillin, and 100 μg of streptomycin per ml (Gibco BRL). The EIAV-nonpermissive human 293T cells (ATCC CRL-11268), murine NIH 3T3 cells (ATCC CRL-1658), and simian COS-7 cells (ATCC CRL-1651) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U of penicillin, and 100 μg of streptomycin per ml. All cell lines were maintained at 37°C in a humidified incubator at 5% CO2.
Virus stocks. The parental pathogenic proviral molecular clone EIAVUK has been described in detail by Cook et al. (15). The replication-competent pEIAVUK p9-E32 truncation mutant containing an introduced termination codon at residue 32 to express the N-terminal 31 amino acids of p9 and the replication-defective pEIAVUK p9-K30 truncation mutant with an introduced termination codon at residue 30 to express the N-terminal 29 amino acids of p9 have been described in the work of Chen et al. (11). For simplicity, the two p9 truncation mutants are designated in the current report as E32 and K30, respectively. Stocks of the parental EIAVUK, E32 mutant, and K30 mutant viruses were generated by transfection of the respective proviral DNAs into COS-7 cells or 293T cells with PolyFect reagent (Qiagen, Valencia, CA). Culture supernatants from transfected cells were harvested at 2 to 3 days posttransfection, clarified by low-speed centrifugation, aliquoted, and frozen at –80°C. Each virus stock was characterized by standard micro-reverse transcriptase (RT) assay (11) to quantify virus production.
Assays for virus infectivity. Virus infections were performed with approximately 4 x 105 cells in six-well plates. Equal amounts of EIAV p9 mutants or parental virus corresponding to 28,000-cpm reverse transcriptase activity were used to infect ED cells in the presence of 8 μg/ml of Polybrene for 2 h at 37°C, as previously described (49). Unabsorbed virus was then washed with phosphate-buffered saline (PBS) three times, and the cells were cultured in fresh medium. At specific time intervals after infection, supernatants were harvested, clarified, and frozen at –80°C. At the conclusion of the experiment, the stored samples were analyzed in parallel for reverse transcription activity, as described previously (11). A portion of each infected cell culture was passaged at each time point, and another portion of cells was collected by trypsinization, washed with PBS, and subjected to DNA isolation. Total cellular DNA was isolated using the DNeasy tissue kit (Qiagen, Valencia, CA). Chromosomal DNA was isolated using a high-molecular-weight DNA purification kit (Qiagen, Valencia, CA). Infections were performed in at least three independent experiments, and RT values were determined in duplicate samples in each assay.
Quantitative real-time PCR analysis of EIAV-specific viral DNA species. Quantitative real-time PCR assays were developed to determine the progression of virus infection by monitoring the formation of intracellular EIAV-specific early cytoplasmic RT products (minus-strand strong-stop DNA), late RT products (gag DNA), nuclear unintegrated nonlinear DNA (two-long-terminal-repeat [2-LTR] circle DNA), and integrated provirus (high-molecular-weight chromosomal DNA). The concentrations of DNA samples extracted from the infected cells were determined by measuring optical density at 260 nm, and 250 ng of either total cellular DNA or high-molecular-weight DNA was used for quantitative PCR amplification. The primer-probe sets (purchased from MWG Biotech Inc., High Point, NC) used to detect each sequence were as follows: early RT product (minus-strand strong-stop DNA), R-forward (5'-CAGATTCTGCGGTCTGAG-3'), U5-reverse (5'-CGAACAGACAAACTAGAGAC-3'), and R-U5 probe [5'-(6-carboxyfluorescein)-CCTTCTCTGCTGGGCTGAAAAGG-(6-carboxytetramethylrhodamine)-3']; late RT product (gag DNA), EIAV-F (5'-GGAGCCTTGAAAGGAGGGCCACTA-3') and EIAV-R (5'-TTGTTGTGCTGACTCTTCTGTTGTATCGGGAA-3) (the probe for late RT product was as described elsewhere [14]); nuclear unintegrated DNA (2-LTR circle DNA), 2-LTR circle forward (5'-CCCTGTCTCTAGTTTGTCTG-3), 2-LTR circle reverse (5'-CAACATGAGCTAGTCCTTTG-3'), and 2-LTR probe [5'-(6-carboxyfluorescein)-AAGAAAGTTGCTGATGCTCTCATAACCTTG-(6-car-boxytetramethylrhodamine)-3'].
For integrated provirus (high-molecular-weight chromosomal DNA), the isolated high-molecular-weight DNA was subjected to quantitative real-time PCR using the gag primer-probe set as described above.
Each DNA sample was normalized by calculating cell numbers using primers and probe specific for equine glyceraldehyde-3-phosphate dehydrogenase (38).
Different standard curves were created for calculating the number of EIAV early RT products, late RT products, 2-LTR circle DNA, and integrated viral DNA with high-molecular-weight DNA isolated from the nucleus. The amplicon being measured was run in duplicate, ranging from 100 to 107 copies plus a negative control lacking template. Standard curves were generated by dilution of molecular clone pEIAVUK and p2LTR, and 250 ng of uninfected cellular DNA was added to normalize the DNA samples. The results of the different dilution series were analyzed by single linear regression with the threshold cycle entered as the dependent variable and log10 dilution as the independent variable. DNA samples contained 1x EZ buffer, 350 μM of each deoxynucleoside triphosphate, 2.5 mM manganese acetate, 200 nM forward primer, 200 nM reverse primer, 125 nM probe primer, and 5 U of rTth (Applied Biosystems, Foster City, CA) in a total reaction volume of 50 μl. After initial incubations at 50°C for 2 min and 95°C for 10 min, 40 rounds of amplification were carried out for 15 s at 95°C, followed by 1 min at 60°C. Reactions were carried out in the ABI Prism 7700 sequence detection system and analyzed with ABI Prism SDS software (Applied Biosystems).
The efficiency of amplification of the target sequences generated by the infection of ED cells was compared to that of the plasmid used as the standard for estimated calculation. The ratio of the slope of the sample dilution to the slope of the plasmid standard curve reached 1.00 (data not shown), demonstrating that the amplification efficiency of viral DNA extracted from cells was identical to that of the standards under the experimental conditions. The dilution series reproducibly yielded satisfactory efficacies, with slopes averaging –3.247 (early RT products), –3.036 (late RT products and integrated provirus), –3.305 (2-LTR circles), and –3.161 (glyceraldehyde-3-phosphate dehydrogenase) and correlation coefficients of 0.984, 0.991, 0.988, and 0.998 for each target sequence, respectively. These results indicate that all the genes being studied displayed similar amplification efficiencies in the real-time PCR. Uninfected ED cells and EIAV-nonpermissive COS-7 cells incubated with EIAVUK virus under standard infection conditions were used as negative controls. Viral DNA could not be detected in either negative cell line using the described quantitative real-time PCR (data not shown), confirming the specificity of the amplification for EIAV DNA species.
RT-PCR assays of EIAV mRNA levels. Expression of EIAV proviral DNA transcription in infected ED cells was measured by analysis of EIAV gag-specific mRNA by RT-PCR. ED cells infected with parental EIAVUK or p9 mutant were collected at specific time intervals, and the mRNA fractions were isolated using the mRNA Capture kit (Roche, Mannheim, Germany) according to the manufacturer's instructions. The primers used for the gag gene amplification were FS31 (5'-CTAAGAGCGAAATATGAAAAGAAG-3') and RS25 (5'-TTGAGTCTGAGGAGTCTCTTGTAC-3'). PCR products were resolved alongside a DNA marker on a 0.85% agarose gel and stained with an ethidium bromide solution. ?-Actin was used as an internal control as described previously (32).
EIAV protein expression assays. For detection of viral protein expression, ED cells infected with the parental EIAVUK, the E32 mutant, or K30 mutant virus were collected at 6 days postinfection and lysed in mammalian protein extraction reagent (Pierce, Rockford, IL). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses were performed as described previously (32). A reference horse immune serum (Lady) (44) was used as the primary antibody (1:2,500), and horseradish peroxidase-conjugated anti-horse immunoglobulin G (1:20,000) was used as the secondary antibody. Assay procedures for monitoring Gag polyprotein processing in progeny virions produced in COS-7 cells transfected with proviral DNA plasmids have been described previously (11).
RESULTS
Effect of EIAV p9 truncations on EIAV replication in infected ED cells. We previously observed that a serial truncation of EIAV p9 at its C terminus revealed a critical minimum length of p9 required for replication competence in transfected ED cells that are permissive for EIAV replication (11). For instance, the p9 E32 mutant that expresses 31 amino acids of the p9 N terminus was replication competent, while the p9 K30 mutant expressing two fewer amino acids was replication defective. To confirm the reported phenotypes for the p9 mutant constructs in transfection experiments, we infected ED cells with equivalent amounts of K30, E32, and parental EIAVUK virus stocks, respectively. Viral production in cell-free supernatants of infected ED cells was monitored by measuring extracellular RT activity, as shown in Fig. 1A. Consistent with our previous results, we observed that the virus production in E32 mutant-infected cells was similar to that observed with the parental EIAVUK virus infection; virus production by E32 infection was only about 20% less than that obtained with parental EIAVUK infection. In contrast, there was no detectable virus production in cells infected with the K30 mutant during the 30-day observation period. Thus, these results confirm the replication-competent phenotype of the E32 p9 mutant and the replication-defective phenotype of the K30 p9 truncation mutant and validate the use of these mutants in studies to define the defective step in K30 replication compared to E32 and parental EIAVUK.
Effects of p9 truncations on Gag polyprotein incorporation and processing in virions. Since the p9 gene sequences overlap with the viral protease gene, we next sought to examine if the replication deficiency observed in the K30 mutant resulted from defective Gag polyprotein processing in virions from transfected cells. Thus, we compared the extent of Gag protein processing in virions produced from COS-7 cells transfected with the K30, E32, and EIAVUK proviral plasmids, respectively (Fig. 1B). Virus particles were then pelleted from the supernatant of each transfected cell culture by centrifugation, and the virion content was normalized by RT content. Equal quantities of each virion preparation were then fractionated by SDS-PAGE, and the Gag protein composition was visualized by Western blot analysis using a reference equine immune serum. As shown in Fig. 1B, the replication-defective K30 and the replication-competent E32 and EIAVUK virions contained a similar predominant content of mature p26 capsid protein, indicating efficient processing of the precursor Gag polyprotein in each virion preparation. The immunoblots in Fig. 1B also revealed similar minor levels of intermediate Gag polyprotein cleavage products (p47 and p40) and negligible uncleaved Gag polyprotein (p55), confirming the relatively equal Gag processing in replication-competent and -defective virus particles. Thus, these data on virion Gag protein composition indicate that the defective nature of the K30 virions evidently cannot be attributed to a defect in Gag polyprotein incorporation or processing.
Characterization of early and late RT DNA production. To identify which step in the virus life cycle was blocked in the K30 mutant, we examined the sequence of virus replication postentry using a quantitative TaqMan-based real-time PCR assay with specific primer and probe combinations, as described in Materials and Methods. Early reverse transcripts and late reverse transcripts were analyzed after infection of ED cells with p9 mutants or the parental EIAVUK virus stocks. At specific time intervals after infection, cells were harvested by trypsinization. A fraction of the cells was cultured continuously for sampling at later time points, while another two fractions were used for total cellular DNA and high-molecular-weight DNA isolation, respectively.
The presence of early RT DNA products (minus-strand strong-stop EIAV DNA) was analyzed by specific primers as detailed in Materials and Methods. Thus, ED cells were infected with an equal dose of EIAVUK, E32, and K30 virus stocks based on virion RT content. Total cellular DNA was then isolated at 0, 3.5, 7, 15, 24, and 144 h postinfection. The synthesis of minus-strand strong-stop DNA (early RT), the first major intermediate of reverse transcription, was determined as shown in Fig. 2A. The results revealed that the infection of ED cells with the replication-defective K30 produced levels of early RT DNA products at 3.5 h similar to those observed for the replication-competent E32 and EIAVUK virus infections, about 10 early RT DNA copies per cell. The levels of early RT DNA product increased for all three viruses at 7 h and 15 h postinfection and then markedly declined at 24 h to similar levels of about 7.0 copies per cell. The data in Fig. 2A also revealed a lower level of early RT DNA products detected at 144 h (6 days) in the K30-infected ED cells (0.5 copies per cell), compared to the cells infected with the E32 virus (9.1 copies per cell) or EIAVUK virus (20.8 copies per cell).
In parallel to the preceding assays for early RT DNA products, we assayed the levels of late RT DNA production based on amplification of the EIAV gag gene as detailed in Materials and Methods. The results of these assays are presented in Fig. 2B. These data indicate that about 0.05 copies of late RT DNA per cell was observed at 3.5 h in all three virus infections and that detectable levels of late RT DNA products increased by at least 50-fold at 7 h postinfection for all three viruses. At this time postinfection, the ED cells infected with the K30 or E32 viruses contained about two late RT DNA copies per cell, while the EIAVUK infection yielded about five late RT DNA copies per cell. Interestingly, the levels of late RT DNA products in all of the infections were less than 10% of the level of early RT DNA products observed at 7 h postinfection. In the case of the E32 and EIAVUK infections, the levels of late RT DNA products observed increased at 15 h and were sustained at 144 h postinfection. In contrast, the levels of late RT DNA products observed in the K30-infected cells increased to about four copies per cell at 15 h but then declined to one copy per cell at 24 h and to undetectable levels at 144 h postinfection. The relatively similar levels of early RT DNA products postentry in cells infected with the replication-defective K30 virus compared to cells infected with replication-competent E32 or EIAVUK virus evidently indicate similar efficiencies of virus entry in ED cells, presumably via receptor-mediated uptake.
Taken together, these data demonstrate that the replication-defective K30 virus was able to enter target ED cells with efficiency equal to those of the replication-competent E32 and EIAVUK viruses, as evidenced by the similar amounts of early and late RT DNA products at 3.5 h postinfection, by the increasing early and late RT DNA product levels up to 15 h postinfection, and by the similar early RT DNA product levels at 24 h postinfection. Moreover, the high levels of early and late RT DNA products observed in the E32 and EIAVUK infections compared to the K30 infection at 144 h postinfection are compatible with multiple cycles of infection produced by the replication-competent E32 and EIAVUK viruses, compared to single-cycle infection by the replication-defective K30 virus.
Characterization of nuclear viral DNA. It has been established that translocation of retroviral DNA from the cytoplasm to the nucleus is accurately reflected in the formation of 2-LTR DNA circles within the nucleus of the infected cell (6, 7). Therefore, the efficiency of translocation of viral DNA from cytoplasmic to nuclear compartments of the various infected ED cells was monitored by measuring 2-LTR circular DNA species using primers that span the junction between the 5'-LTR R region and 3'-LTR U5 region as created during formation of the 2-LTR circle. The results of these assays are summarized in Fig. 2C. These data demonstrated detectable levels of 2-LTR DNA circles in the E32- and EIAVUK-infected ED cells in the range of 0.004 to 0.03 copies per cell at 7 to 24 h postinfection, with levels increasing to an average of about 0.08 to 0.11 copies per cell at 144 h postinfection. In marked contrast, no detectable 2-LTR DNA circles were observed in the K30-infected ED cells, even at 144 h postinfection. To exclude the possibility that the observed block of K30 integration was due to insufficient amount of virus input, ED cells were infected with 16-fold-more K30 virus than in the infections described above and analyzed daily for late viral DNA and 2-LTR circle DNA production. As summarized in Fig. 3, even at the higher viral infectious dose, the K30 infection failed to produce detectable 2-LTR DNA circles, despite a 22-fold increase in late DNA transcripts produced at 24 h postinfection in these infected cells compared to the level observed above in cells infected with the lower infectious dose. Thus, these data indicate that, in contrast to the replication-competent E32 and EIAVUK viruses, the replication-defective K30 mutant is deficient in the production of 2-LTR DNA circles from the linear DNA products observed postinfection.
Integration of EIAV viral DNA in ED cells infected with p9 mutants or wild-type virus. To complete the comparative analysis of provirus production by the three test viruses, we next evaluated the production of integrated proviral DNA in ED cells infected with the K30, E32, or EIAVUK virus by quantifying the amount of EIAV DNA contained in purified high-molecular-weight chromosomal DNA. These data are summarized in Fig. 2D. In the case of the E32 and EIAVUK infections, the first integrated proviral DNA was reproducibly detected at 15 h postinfection at levels of about 0.0064 copies per cell. The level of integrated proviral DNA increased at 144 h postinfection to an average of about 0.17 copies per cell for E32 and 0.33 copies per cell for EIAVUK infections. The level of provirus integration observed in the ED cells infected with the E32 or EIAVUK virus represented about 5% of the total viral DNA contained in the infected cell. This level of integration is very similar to that reported for other lentiviruses (8, 9, 29). In contrast to these replication-competent EIAV stocks, there was no detectable integrated proviral DNA in ED cells infected with the K30 virus, consistent with the absence of precursor 2-LTR DNA circles observed in the preceding assays.
Specificity of viral DNA synthesis in virus-infected cells. Several experiments were performed to confirm the specificity of the viral DNA synthesis observed in the ED cells infected with the p9 mutants or the parental EIAVUK virus. To exclude the possibility that the intracellular DNA detected in virus-infected cells could be the result of contaminating DNA contained in the culture supernatants collected from the transfected cells, we assayed for viral DNA in several EIAV-nonpermissive cells (NIH 3T3, COS-7, and 293T) incubated with the EIAVUK virus preparations, respectively. In parallel, separate cultures of ED cells were incubated with the infectious virus supernatant or with supernatant containing purified proviral plasmid DNA (2 μg) for 2 h and then washed with PBS three times, as described in Materials and Methods. After 24 h, the treated cell cultures were then assayed for intracellular viral DNA using the specific primers and PCR conditions described above to detect EIAV early RT and late RT products. As summarized in Fig. 4, the results of these control experiments demonstrated a lack of detectable viral DNA in any of the nonpermissive cell lines incubated with the parental EIAVUK virus preparations. In addition, there was no detectable DNA in permissive ED cells incubated with proviral DNA. The results conclusively demonstrate the requirement for EIAV entry into target cells, as observed in the permissive ED cells, to yield detectable viral DNA transcripts, confirming the specificity of the DNA species observed by PCR assays in the preceding experiments.
Detection of the viral transcription and viral protein synthesis. To extend the analyses of the replication defect of the K30 mutant, we next evaluated the levels of virus-specific transcription and protein production in the ED cells infected with the K30, E32, and EIAVUK virus constructs. While one might not predict detectable EIAV transcription and protein synthesis in the absence of detectable K30 provirus, we reasoned that the transcription and translation assays might provide a more sensitive assay for K30 infection than the DNA amplifications used to detect various proviral products. Alternatively, these latter assays could serve to confirm the data, indicating a lack of stable provirus production after infection of ED cells by the K30 virus.
To assess the transcription levels in the respective infected cells, the Gag mRNA was quantified by RT-PCR, as described in Materials and Methods. As shown in Fig. 5A, virus-specific RNA could be detected at 7 h postinfection and at all later time points in the lysates of cells infected with the E32 or EIAVUK virus strain. In contrast, there was no detectable EIAV mRNA in the lysates of cells infected by the K30 mutant virus at all of the time points tested. These data demonstrate a lack of detectable transcription of full-length viral RNA in K30 infections, consistent with the concept of a defect in the production of stable provirus in these infected cells.
Finally, virus-specific protein expression produced by each of the virus infections was monitored by examining the intracellular levels of EIAV proteins by Western blotting of cellular lysates from infected cells at 6 days postinfection, using a reference equine immune serum. The resulting immunoblots (Fig. 5B) revealed similar amounts of EIAV proteins in lysates of ED cells infected with the E32 or EIAVUK virus, but no detectable viral proteins in cells infected with the K30 mutant virus. These protein expression data are consistent with the absence of detectable provirus transcription in K30 virus-infected cells, in contrast to the robust transcription and protein synthesis observed in cells infected with either the E32 or EIAVUK virus.
DISCUSSION
We previously reported that a p9 truncation mutant of EIAV, designated E32, expressing the N-terminal 31 amino acids of the p9 protein, was replication competent, whereas the deletion of two additional amino acids from the p9 protein produced a provirus (designated K30) that could produce virus particles in transfected COS-1 cells but was replication defective in permissive ED cells (11). Since proviral DNA transfection produced evidently normal virus particles, we deduced that the p9 protein might perform a critical role in EIAV infectivity leading to integrated provirus that was defective in the K30 mutant provirus. The current study tested this hypothesis by examining the sequential steps of EIAV infection, including early and late reverse transcription; viral DNA nuclear localization; and provirus integration, transcription, and translation. The results of these studies clearly demonstrated that the K30 virus is able to produce similar levels of early and late viral DNA from the infecting virion genomic RNA postentry, compared to the replication-competent E32 and EIAVUK viruses assayed in parallel. In contrast to the replication-competent viruses, however, the K30 infection evidently failed to effectively produce 2-LTR DNA proviral circles, indicative of nuclear localization of late DNA products, resulting in a lack of detectable integrated provirus, EIAV mRNA, and viral proteins in the K30 virus-infected cells. Thus, the current data for the first time reveal a critical role for the EIAV p9 protein in early steps of infection necessary for the production of stable integrated provirus and gene expression required for a productive infection. The function of EIAV p9 in early steps of infection is very similar to that of Moloney murine leukemia virus p12, which is known to be important for both late and early events in the virus life cycle (65, 66).
The precise role of EIAV p9 in virus infection of target cells remains to be defined. However, based on the premise that 2-LTR circular DNAs (3, 21, 47, 54, 63) are a reliable indicator of transport of retroviral DNA PICs from the cytoplasm to the nucleus, we propose that EIAV p9 performs a critical role in the efficient progression of cytoplasmic linear DNA to nuclear circular DNA. The role for p9 may be in stabilizing or mediating transport of cytoplasmic viral DNA. In this regard, studies with other retroviruses, including HIV-1, have implicated a number of virion proteins as important components of the viral DNA PIC, including the viral integrase (4, 6, 57), matrix (18, 20, 26, 28, 33, 61), capsid (10, 23, 64), and Vpr (5, 31, 42, 56). While the EIAV p9 protein and other lentivirus homologs like HIV-1 p6 have been shown to supply important L-domain functions in viral assembly and budding, this appears to be the first evidence for a role of this lentivirus Gag protein in the early steps of virus infection, as described previously for the Moloney murine leukemia virus p12 (65, 66). Therefore, it will be important to determine if HIV-1 and simian immunodeficiency virus p6 and the homologous Gag proteins of other animal lentiviruses supply infection functions in addition to assembly functions during viral replication.
How might the EIAV p9 protein mediate the progression of the PIC from the cytoplasm to the nucleus following completion of reverse transcription postentry? One possible model is that the EIAV p9 protein is necessary to stabilize the linear viral DNA products to prevent degradation during translocation from the cytoplasm to the nucleus. A second model is that the p9 protein may provide a nuclear localization signal (NLS) to target the PIC to the nucleus. The possibility of p9 providing an NLS is supported by the location of a number of lysine residues in the sequences surrounding the K30 and E32 mutation sites that differentiate replication competence and differ by only two amino acids, 30KKEYNVKEK38. To determine whether the basic lysine residues in this sequence would be predicted to form an NLS sequence, we analyzed the p9 open reading frame using a computer program (PSORT II) to identify the potential for NLS motifs. However, no NLS-like sequence in the p9 protein was predicted by this computer modeling. Also arguing against the role of a p9 NLS function is the previous study from this laboratory (11) demonstrating that the mutation of the lysine residues at positions 31 and 32 to methionine residues resulted in a replication-competent provirus construct, indicating that these two lysines are dispensable for virus replication. As a third model, we postulate that p9 protein association with other viral proteins and specific cellular factors may direct nuclear import of PICs. It has been reported that HIV-1 p6 protein serves as an anchor to incorporate the Vpr proteins into the virion and that the Vpr protein is necessary for viral DNA nuclear localization (34, 37, 48). Since EIAV does not encode an apparent Vpr-like protein, the proposed role of EIAV p9 in the nuclear import of viral DNA might be similar to the function of the accessory protein Vpr of HIV-1 translocation of HIV PICs (24, 31). Experiments are currently in progress to examine the EIAV PIC and to determine the role of EIAV p9 in viral DNA stabilization and nuclear transport.
ACKNOWLEDGMENTS
This work was supported by the grant R01 CA49296 from the National Cancer Institute of the National Institutes of Health.
We thank Shannon Durkin for critical editing of the manuscript.
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ABSTRACT
We have previously reported that serial truncation of the Gag p9 protein of equine infectious anemia virus (EIAV) revealed a progressive loss in replication phenotypes in transfected cells, such that a proviral mutant (E32) expressing the N-terminal 31 amino acids of p9 produced infectious virus particles similarly to parental provirus, while a proviral mutant (K30) with two fewer amino acids produced replication-defective virus particles, despite containing apparently normal levels of processed Gag and Pol proteins (C. Chen, F. Li, and R. C. Montelaro, J. Virol. 75:9762-9760, 2001). Based on these observations, we sought in the current study to identify the precise defect in K30 virion infection of permissive equine dermal (ED) cells. The results of these experiments clearly demonstrated that K30 virions entered target ED cells and produced early (minus-strand strong-stop) and late (Gag) viral DNA products as efficiently as did the replication-competent E32 mutant and parental EIAVUK viruses. However, in contrast to the replication-competent E32 mutant and parental viruses, infection with K30 mutant virus failed to produce detectable two-long-terminal-repeat DNA circles, stable integrated provirus, virus-specific Gag mRNA expression, or intracellular viral protein expression. Taken together, these data demonstrate that the K30 mutant is defective in the ability to produce sufficient nuclear viral DNA to establish a productive infection in ED cells. Thus, these observations indicate for the first time that the EIAV Gag p9 protein performs a critical role in viral DNA production and processing to provirus during EIAV infection, in addition to its previously defined role in viral budding mediated by the p9 L domain.
INTRODUCTION
The functions of retroviral Gag proteins in virus-infected cells to accomplish various steps in virion assembly and budding have been the subject of intense investigation leading to an increasingly intricate model of highly specific Gag protein interactions with other virion protein and RNA components and with host cell proteins (1, 13, 25, 40, 41, 43, 45). However, there are also increasingly more data suggesting important functional roles for retroviral Gag proteins during virus infection of target cells postentry. For example, the matrix (MA), capsid (CA), and nucleocapsid (NC) proteins have all been implicated in reverse transcription of the retrovirus genomic RNA to produce viral DNA (16, 19, 22, 35, 36, 53, 59). The integrase (IN) and MA proteins are believed to be critical components of the preintegration complex (PIC) that translocates the viral DNA to the cell nucleus, where it is integrated into the host chromosome (2, 12, 46, 50, 60, 62). In addition to the CA, MA, and NC Gag proteins common to all retroviruses, the lentiviruses characteristically contain an additional Gag protein (human immunodeficiency virus type 1 [HIV-1] p6 and equine infectious anemia virus [EIAV] p9) that has been shown to provide critical late functions in virion assembly and budding via highly specific interactions with various endocytic proteins in infected cells (11, 17, 27, 30, 39, 51, 52, 55, 58). A specific role for HIV-1 p6 or EIAV p9 in virus infection has to date not been definitively established.
We previously reported that serial truncations of the p9 protein in the context of the EIAVUK provirus revealed a progressive loss of replication competence in transfected cells with increased reduction in p9 length (11). The results of these studies demonstrate that EIAV proviral mutant viruses containing at least the N-terminal 31 amino acids of p9 had replication levels similar to those of the parental EIAVUK virus, indicating that the first 31 amino acids can supply all of the necessary functions for productive infection of equine dermal (ED) cells. In contrast, proviral mutants containing larger p9 truncations were found to be replication defective in ED cells. Further functional characterization of the various replication-defective p9 truncation proviral constructs revealed that p9 mutants lacking a functional L domain (19YPDL22) were greatly suppressed in virion production, the expected phenotype for an L-domain-negative mutant. Interestingly, intermediate p9 truncation proviral mutants containing the L domain but less than 31 amino acids were found to produce virus particles from transfected COS-7 cells at levels similar to those for transfections with the parental EIAVUK provirus DNA, and the mutant p9 virions appeared to be normal for Gag and Pol incorporation and processing. Based on these observations, we hypothesized that the replication-defective nature of these p9 truncation mutants might be due to defects in virion infectivity.
In the current study, we examine this hypothesis by comparing at each step of virus infection the functional competence of the replication-defective mutant K30 expressing the N-terminal 29 amino acids of p9, the replication-competent mutant E32 expressing the N-terminal 31 amino acids of p9, and the parental EIAVUK provirus expressing the full-length p9 protein containing 51 amino acids. The results of these studies revealed that the defect in replication by the K30 mutant is associated with an apparent block in the production of nuclear viral DNA from linear DNA reverse transcripts. Thus, these observations demonstrate for the first time a critical role for the EIAV p9 protein in the early stages of viral infection that lead to the generation of stable integrated provirus necessary for establishing productive infection of target cells.
MATERIALS AND METHODS
Cells. The ED cell line permissive for EIAV replication was obtained from the American Type Culture Collection (ATCC CCL-57) and grown in minimal essential medium supplemented with 10% fetal bovine serum from HyClone Laboratories (Ogden, UT), 2 mM glutamine, nonessential amino acids, 100 U of penicillin, and 100 μg of streptomycin per ml (Gibco BRL). The EIAV-nonpermissive human 293T cells (ATCC CRL-11268), murine NIH 3T3 cells (ATCC CRL-1658), and simian COS-7 cells (ATCC CRL-1651) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U of penicillin, and 100 μg of streptomycin per ml. All cell lines were maintained at 37°C in a humidified incubator at 5% CO2.
Virus stocks. The parental pathogenic proviral molecular clone EIAVUK has been described in detail by Cook et al. (15). The replication-competent pEIAVUK p9-E32 truncation mutant containing an introduced termination codon at residue 32 to express the N-terminal 31 amino acids of p9 and the replication-defective pEIAVUK p9-K30 truncation mutant with an introduced termination codon at residue 30 to express the N-terminal 29 amino acids of p9 have been described in the work of Chen et al. (11). For simplicity, the two p9 truncation mutants are designated in the current report as E32 and K30, respectively. Stocks of the parental EIAVUK, E32 mutant, and K30 mutant viruses were generated by transfection of the respective proviral DNAs into COS-7 cells or 293T cells with PolyFect reagent (Qiagen, Valencia, CA). Culture supernatants from transfected cells were harvested at 2 to 3 days posttransfection, clarified by low-speed centrifugation, aliquoted, and frozen at –80°C. Each virus stock was characterized by standard micro-reverse transcriptase (RT) assay (11) to quantify virus production.
Assays for virus infectivity. Virus infections were performed with approximately 4 x 105 cells in six-well plates. Equal amounts of EIAV p9 mutants or parental virus corresponding to 28,000-cpm reverse transcriptase activity were used to infect ED cells in the presence of 8 μg/ml of Polybrene for 2 h at 37°C, as previously described (49). Unabsorbed virus was then washed with phosphate-buffered saline (PBS) three times, and the cells were cultured in fresh medium. At specific time intervals after infection, supernatants were harvested, clarified, and frozen at –80°C. At the conclusion of the experiment, the stored samples were analyzed in parallel for reverse transcription activity, as described previously (11). A portion of each infected cell culture was passaged at each time point, and another portion of cells was collected by trypsinization, washed with PBS, and subjected to DNA isolation. Total cellular DNA was isolated using the DNeasy tissue kit (Qiagen, Valencia, CA). Chromosomal DNA was isolated using a high-molecular-weight DNA purification kit (Qiagen, Valencia, CA). Infections were performed in at least three independent experiments, and RT values were determined in duplicate samples in each assay.
Quantitative real-time PCR analysis of EIAV-specific viral DNA species. Quantitative real-time PCR assays were developed to determine the progression of virus infection by monitoring the formation of intracellular EIAV-specific early cytoplasmic RT products (minus-strand strong-stop DNA), late RT products (gag DNA), nuclear unintegrated nonlinear DNA (two-long-terminal-repeat [2-LTR] circle DNA), and integrated provirus (high-molecular-weight chromosomal DNA). The concentrations of DNA samples extracted from the infected cells were determined by measuring optical density at 260 nm, and 250 ng of either total cellular DNA or high-molecular-weight DNA was used for quantitative PCR amplification. The primer-probe sets (purchased from MWG Biotech Inc., High Point, NC) used to detect each sequence were as follows: early RT product (minus-strand strong-stop DNA), R-forward (5'-CAGATTCTGCGGTCTGAG-3'), U5-reverse (5'-CGAACAGACAAACTAGAGAC-3'), and R-U5 probe [5'-(6-carboxyfluorescein)-CCTTCTCTGCTGGGCTGAAAAGG-(6-carboxytetramethylrhodamine)-3']; late RT product (gag DNA), EIAV-F (5'-GGAGCCTTGAAAGGAGGGCCACTA-3') and EIAV-R (5'-TTGTTGTGCTGACTCTTCTGTTGTATCGGGAA-3) (the probe for late RT product was as described elsewhere [14]); nuclear unintegrated DNA (2-LTR circle DNA), 2-LTR circle forward (5'-CCCTGTCTCTAGTTTGTCTG-3), 2-LTR circle reverse (5'-CAACATGAGCTAGTCCTTTG-3'), and 2-LTR probe [5'-(6-carboxyfluorescein)-AAGAAAGTTGCTGATGCTCTCATAACCTTG-(6-car-boxytetramethylrhodamine)-3'].
For integrated provirus (high-molecular-weight chromosomal DNA), the isolated high-molecular-weight DNA was subjected to quantitative real-time PCR using the gag primer-probe set as described above.
Each DNA sample was normalized by calculating cell numbers using primers and probe specific for equine glyceraldehyde-3-phosphate dehydrogenase (38).
Different standard curves were created for calculating the number of EIAV early RT products, late RT products, 2-LTR circle DNA, and integrated viral DNA with high-molecular-weight DNA isolated from the nucleus. The amplicon being measured was run in duplicate, ranging from 100 to 107 copies plus a negative control lacking template. Standard curves were generated by dilution of molecular clone pEIAVUK and p2LTR, and 250 ng of uninfected cellular DNA was added to normalize the DNA samples. The results of the different dilution series were analyzed by single linear regression with the threshold cycle entered as the dependent variable and log10 dilution as the independent variable. DNA samples contained 1x EZ buffer, 350 μM of each deoxynucleoside triphosphate, 2.5 mM manganese acetate, 200 nM forward primer, 200 nM reverse primer, 125 nM probe primer, and 5 U of rTth (Applied Biosystems, Foster City, CA) in a total reaction volume of 50 μl. After initial incubations at 50°C for 2 min and 95°C for 10 min, 40 rounds of amplification were carried out for 15 s at 95°C, followed by 1 min at 60°C. Reactions were carried out in the ABI Prism 7700 sequence detection system and analyzed with ABI Prism SDS software (Applied Biosystems).
The efficiency of amplification of the target sequences generated by the infection of ED cells was compared to that of the plasmid used as the standard for estimated calculation. The ratio of the slope of the sample dilution to the slope of the plasmid standard curve reached 1.00 (data not shown), demonstrating that the amplification efficiency of viral DNA extracted from cells was identical to that of the standards under the experimental conditions. The dilution series reproducibly yielded satisfactory efficacies, with slopes averaging –3.247 (early RT products), –3.036 (late RT products and integrated provirus), –3.305 (2-LTR circles), and –3.161 (glyceraldehyde-3-phosphate dehydrogenase) and correlation coefficients of 0.984, 0.991, 0.988, and 0.998 for each target sequence, respectively. These results indicate that all the genes being studied displayed similar amplification efficiencies in the real-time PCR. Uninfected ED cells and EIAV-nonpermissive COS-7 cells incubated with EIAVUK virus under standard infection conditions were used as negative controls. Viral DNA could not be detected in either negative cell line using the described quantitative real-time PCR (data not shown), confirming the specificity of the amplification for EIAV DNA species.
RT-PCR assays of EIAV mRNA levels. Expression of EIAV proviral DNA transcription in infected ED cells was measured by analysis of EIAV gag-specific mRNA by RT-PCR. ED cells infected with parental EIAVUK or p9 mutant were collected at specific time intervals, and the mRNA fractions were isolated using the mRNA Capture kit (Roche, Mannheim, Germany) according to the manufacturer's instructions. The primers used for the gag gene amplification were FS31 (5'-CTAAGAGCGAAATATGAAAAGAAG-3') and RS25 (5'-TTGAGTCTGAGGAGTCTCTTGTAC-3'). PCR products were resolved alongside a DNA marker on a 0.85% agarose gel and stained with an ethidium bromide solution. ?-Actin was used as an internal control as described previously (32).
EIAV protein expression assays. For detection of viral protein expression, ED cells infected with the parental EIAVUK, the E32 mutant, or K30 mutant virus were collected at 6 days postinfection and lysed in mammalian protein extraction reagent (Pierce, Rockford, IL). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses were performed as described previously (32). A reference horse immune serum (Lady) (44) was used as the primary antibody (1:2,500), and horseradish peroxidase-conjugated anti-horse immunoglobulin G (1:20,000) was used as the secondary antibody. Assay procedures for monitoring Gag polyprotein processing in progeny virions produced in COS-7 cells transfected with proviral DNA plasmids have been described previously (11).
RESULTS
Effect of EIAV p9 truncations on EIAV replication in infected ED cells. We previously observed that a serial truncation of EIAV p9 at its C terminus revealed a critical minimum length of p9 required for replication competence in transfected ED cells that are permissive for EIAV replication (11). For instance, the p9 E32 mutant that expresses 31 amino acids of the p9 N terminus was replication competent, while the p9 K30 mutant expressing two fewer amino acids was replication defective. To confirm the reported phenotypes for the p9 mutant constructs in transfection experiments, we infected ED cells with equivalent amounts of K30, E32, and parental EIAVUK virus stocks, respectively. Viral production in cell-free supernatants of infected ED cells was monitored by measuring extracellular RT activity, as shown in Fig. 1A. Consistent with our previous results, we observed that the virus production in E32 mutant-infected cells was similar to that observed with the parental EIAVUK virus infection; virus production by E32 infection was only about 20% less than that obtained with parental EIAVUK infection. In contrast, there was no detectable virus production in cells infected with the K30 mutant during the 30-day observation period. Thus, these results confirm the replication-competent phenotype of the E32 p9 mutant and the replication-defective phenotype of the K30 p9 truncation mutant and validate the use of these mutants in studies to define the defective step in K30 replication compared to E32 and parental EIAVUK.
Effects of p9 truncations on Gag polyprotein incorporation and processing in virions. Since the p9 gene sequences overlap with the viral protease gene, we next sought to examine if the replication deficiency observed in the K30 mutant resulted from defective Gag polyprotein processing in virions from transfected cells. Thus, we compared the extent of Gag protein processing in virions produced from COS-7 cells transfected with the K30, E32, and EIAVUK proviral plasmids, respectively (Fig. 1B). Virus particles were then pelleted from the supernatant of each transfected cell culture by centrifugation, and the virion content was normalized by RT content. Equal quantities of each virion preparation were then fractionated by SDS-PAGE, and the Gag protein composition was visualized by Western blot analysis using a reference equine immune serum. As shown in Fig. 1B, the replication-defective K30 and the replication-competent E32 and EIAVUK virions contained a similar predominant content of mature p26 capsid protein, indicating efficient processing of the precursor Gag polyprotein in each virion preparation. The immunoblots in Fig. 1B also revealed similar minor levels of intermediate Gag polyprotein cleavage products (p47 and p40) and negligible uncleaved Gag polyprotein (p55), confirming the relatively equal Gag processing in replication-competent and -defective virus particles. Thus, these data on virion Gag protein composition indicate that the defective nature of the K30 virions evidently cannot be attributed to a defect in Gag polyprotein incorporation or processing.
Characterization of early and late RT DNA production. To identify which step in the virus life cycle was blocked in the K30 mutant, we examined the sequence of virus replication postentry using a quantitative TaqMan-based real-time PCR assay with specific primer and probe combinations, as described in Materials and Methods. Early reverse transcripts and late reverse transcripts were analyzed after infection of ED cells with p9 mutants or the parental EIAVUK virus stocks. At specific time intervals after infection, cells were harvested by trypsinization. A fraction of the cells was cultured continuously for sampling at later time points, while another two fractions were used for total cellular DNA and high-molecular-weight DNA isolation, respectively.
The presence of early RT DNA products (minus-strand strong-stop EIAV DNA) was analyzed by specific primers as detailed in Materials and Methods. Thus, ED cells were infected with an equal dose of EIAVUK, E32, and K30 virus stocks based on virion RT content. Total cellular DNA was then isolated at 0, 3.5, 7, 15, 24, and 144 h postinfection. The synthesis of minus-strand strong-stop DNA (early RT), the first major intermediate of reverse transcription, was determined as shown in Fig. 2A. The results revealed that the infection of ED cells with the replication-defective K30 produced levels of early RT DNA products at 3.5 h similar to those observed for the replication-competent E32 and EIAVUK virus infections, about 10 early RT DNA copies per cell. The levels of early RT DNA product increased for all three viruses at 7 h and 15 h postinfection and then markedly declined at 24 h to similar levels of about 7.0 copies per cell. The data in Fig. 2A also revealed a lower level of early RT DNA products detected at 144 h (6 days) in the K30-infected ED cells (0.5 copies per cell), compared to the cells infected with the E32 virus (9.1 copies per cell) or EIAVUK virus (20.8 copies per cell).
In parallel to the preceding assays for early RT DNA products, we assayed the levels of late RT DNA production based on amplification of the EIAV gag gene as detailed in Materials and Methods. The results of these assays are presented in Fig. 2B. These data indicate that about 0.05 copies of late RT DNA per cell was observed at 3.5 h in all three virus infections and that detectable levels of late RT DNA products increased by at least 50-fold at 7 h postinfection for all three viruses. At this time postinfection, the ED cells infected with the K30 or E32 viruses contained about two late RT DNA copies per cell, while the EIAVUK infection yielded about five late RT DNA copies per cell. Interestingly, the levels of late RT DNA products in all of the infections were less than 10% of the level of early RT DNA products observed at 7 h postinfection. In the case of the E32 and EIAVUK infections, the levels of late RT DNA products observed increased at 15 h and were sustained at 144 h postinfection. In contrast, the levels of late RT DNA products observed in the K30-infected cells increased to about four copies per cell at 15 h but then declined to one copy per cell at 24 h and to undetectable levels at 144 h postinfection. The relatively similar levels of early RT DNA products postentry in cells infected with the replication-defective K30 virus compared to cells infected with replication-competent E32 or EIAVUK virus evidently indicate similar efficiencies of virus entry in ED cells, presumably via receptor-mediated uptake.
Taken together, these data demonstrate that the replication-defective K30 virus was able to enter target ED cells with efficiency equal to those of the replication-competent E32 and EIAVUK viruses, as evidenced by the similar amounts of early and late RT DNA products at 3.5 h postinfection, by the increasing early and late RT DNA product levels up to 15 h postinfection, and by the similar early RT DNA product levels at 24 h postinfection. Moreover, the high levels of early and late RT DNA products observed in the E32 and EIAVUK infections compared to the K30 infection at 144 h postinfection are compatible with multiple cycles of infection produced by the replication-competent E32 and EIAVUK viruses, compared to single-cycle infection by the replication-defective K30 virus.
Characterization of nuclear viral DNA. It has been established that translocation of retroviral DNA from the cytoplasm to the nucleus is accurately reflected in the formation of 2-LTR DNA circles within the nucleus of the infected cell (6, 7). Therefore, the efficiency of translocation of viral DNA from cytoplasmic to nuclear compartments of the various infected ED cells was monitored by measuring 2-LTR circular DNA species using primers that span the junction between the 5'-LTR R region and 3'-LTR U5 region as created during formation of the 2-LTR circle. The results of these assays are summarized in Fig. 2C. These data demonstrated detectable levels of 2-LTR DNA circles in the E32- and EIAVUK-infected ED cells in the range of 0.004 to 0.03 copies per cell at 7 to 24 h postinfection, with levels increasing to an average of about 0.08 to 0.11 copies per cell at 144 h postinfection. In marked contrast, no detectable 2-LTR DNA circles were observed in the K30-infected ED cells, even at 144 h postinfection. To exclude the possibility that the observed block of K30 integration was due to insufficient amount of virus input, ED cells were infected with 16-fold-more K30 virus than in the infections described above and analyzed daily for late viral DNA and 2-LTR circle DNA production. As summarized in Fig. 3, even at the higher viral infectious dose, the K30 infection failed to produce detectable 2-LTR DNA circles, despite a 22-fold increase in late DNA transcripts produced at 24 h postinfection in these infected cells compared to the level observed above in cells infected with the lower infectious dose. Thus, these data indicate that, in contrast to the replication-competent E32 and EIAVUK viruses, the replication-defective K30 mutant is deficient in the production of 2-LTR DNA circles from the linear DNA products observed postinfection.
Integration of EIAV viral DNA in ED cells infected with p9 mutants or wild-type virus. To complete the comparative analysis of provirus production by the three test viruses, we next evaluated the production of integrated proviral DNA in ED cells infected with the K30, E32, or EIAVUK virus by quantifying the amount of EIAV DNA contained in purified high-molecular-weight chromosomal DNA. These data are summarized in Fig. 2D. In the case of the E32 and EIAVUK infections, the first integrated proviral DNA was reproducibly detected at 15 h postinfection at levels of about 0.0064 copies per cell. The level of integrated proviral DNA increased at 144 h postinfection to an average of about 0.17 copies per cell for E32 and 0.33 copies per cell for EIAVUK infections. The level of provirus integration observed in the ED cells infected with the E32 or EIAVUK virus represented about 5% of the total viral DNA contained in the infected cell. This level of integration is very similar to that reported for other lentiviruses (8, 9, 29). In contrast to these replication-competent EIAV stocks, there was no detectable integrated proviral DNA in ED cells infected with the K30 virus, consistent with the absence of precursor 2-LTR DNA circles observed in the preceding assays.
Specificity of viral DNA synthesis in virus-infected cells. Several experiments were performed to confirm the specificity of the viral DNA synthesis observed in the ED cells infected with the p9 mutants or the parental EIAVUK virus. To exclude the possibility that the intracellular DNA detected in virus-infected cells could be the result of contaminating DNA contained in the culture supernatants collected from the transfected cells, we assayed for viral DNA in several EIAV-nonpermissive cells (NIH 3T3, COS-7, and 293T) incubated with the EIAVUK virus preparations, respectively. In parallel, separate cultures of ED cells were incubated with the infectious virus supernatant or with supernatant containing purified proviral plasmid DNA (2 μg) for 2 h and then washed with PBS three times, as described in Materials and Methods. After 24 h, the treated cell cultures were then assayed for intracellular viral DNA using the specific primers and PCR conditions described above to detect EIAV early RT and late RT products. As summarized in Fig. 4, the results of these control experiments demonstrated a lack of detectable viral DNA in any of the nonpermissive cell lines incubated with the parental EIAVUK virus preparations. In addition, there was no detectable DNA in permissive ED cells incubated with proviral DNA. The results conclusively demonstrate the requirement for EIAV entry into target cells, as observed in the permissive ED cells, to yield detectable viral DNA transcripts, confirming the specificity of the DNA species observed by PCR assays in the preceding experiments.
Detection of the viral transcription and viral protein synthesis. To extend the analyses of the replication defect of the K30 mutant, we next evaluated the levels of virus-specific transcription and protein production in the ED cells infected with the K30, E32, and EIAVUK virus constructs. While one might not predict detectable EIAV transcription and protein synthesis in the absence of detectable K30 provirus, we reasoned that the transcription and translation assays might provide a more sensitive assay for K30 infection than the DNA amplifications used to detect various proviral products. Alternatively, these latter assays could serve to confirm the data, indicating a lack of stable provirus production after infection of ED cells by the K30 virus.
To assess the transcription levels in the respective infected cells, the Gag mRNA was quantified by RT-PCR, as described in Materials and Methods. As shown in Fig. 5A, virus-specific RNA could be detected at 7 h postinfection and at all later time points in the lysates of cells infected with the E32 or EIAVUK virus strain. In contrast, there was no detectable EIAV mRNA in the lysates of cells infected by the K30 mutant virus at all of the time points tested. These data demonstrate a lack of detectable transcription of full-length viral RNA in K30 infections, consistent with the concept of a defect in the production of stable provirus in these infected cells.
Finally, virus-specific protein expression produced by each of the virus infections was monitored by examining the intracellular levels of EIAV proteins by Western blotting of cellular lysates from infected cells at 6 days postinfection, using a reference equine immune serum. The resulting immunoblots (Fig. 5B) revealed similar amounts of EIAV proteins in lysates of ED cells infected with the E32 or EIAVUK virus, but no detectable viral proteins in cells infected with the K30 mutant virus. These protein expression data are consistent with the absence of detectable provirus transcription in K30 virus-infected cells, in contrast to the robust transcription and protein synthesis observed in cells infected with either the E32 or EIAVUK virus.
DISCUSSION
We previously reported that a p9 truncation mutant of EIAV, designated E32, expressing the N-terminal 31 amino acids of the p9 protein, was replication competent, whereas the deletion of two additional amino acids from the p9 protein produced a provirus (designated K30) that could produce virus particles in transfected COS-1 cells but was replication defective in permissive ED cells (11). Since proviral DNA transfection produced evidently normal virus particles, we deduced that the p9 protein might perform a critical role in EIAV infectivity leading to integrated provirus that was defective in the K30 mutant provirus. The current study tested this hypothesis by examining the sequential steps of EIAV infection, including early and late reverse transcription; viral DNA nuclear localization; and provirus integration, transcription, and translation. The results of these studies clearly demonstrated that the K30 virus is able to produce similar levels of early and late viral DNA from the infecting virion genomic RNA postentry, compared to the replication-competent E32 and EIAVUK viruses assayed in parallel. In contrast to the replication-competent viruses, however, the K30 infection evidently failed to effectively produce 2-LTR DNA proviral circles, indicative of nuclear localization of late DNA products, resulting in a lack of detectable integrated provirus, EIAV mRNA, and viral proteins in the K30 virus-infected cells. Thus, the current data for the first time reveal a critical role for the EIAV p9 protein in early steps of infection necessary for the production of stable integrated provirus and gene expression required for a productive infection. The function of EIAV p9 in early steps of infection is very similar to that of Moloney murine leukemia virus p12, which is known to be important for both late and early events in the virus life cycle (65, 66).
The precise role of EIAV p9 in virus infection of target cells remains to be defined. However, based on the premise that 2-LTR circular DNAs (3, 21, 47, 54, 63) are a reliable indicator of transport of retroviral DNA PICs from the cytoplasm to the nucleus, we propose that EIAV p9 performs a critical role in the efficient progression of cytoplasmic linear DNA to nuclear circular DNA. The role for p9 may be in stabilizing or mediating transport of cytoplasmic viral DNA. In this regard, studies with other retroviruses, including HIV-1, have implicated a number of virion proteins as important components of the viral DNA PIC, including the viral integrase (4, 6, 57), matrix (18, 20, 26, 28, 33, 61), capsid (10, 23, 64), and Vpr (5, 31, 42, 56). While the EIAV p9 protein and other lentivirus homologs like HIV-1 p6 have been shown to supply important L-domain functions in viral assembly and budding, this appears to be the first evidence for a role of this lentivirus Gag protein in the early steps of virus infection, as described previously for the Moloney murine leukemia virus p12 (65, 66). Therefore, it will be important to determine if HIV-1 and simian immunodeficiency virus p6 and the homologous Gag proteins of other animal lentiviruses supply infection functions in addition to assembly functions during viral replication.
How might the EIAV p9 protein mediate the progression of the PIC from the cytoplasm to the nucleus following completion of reverse transcription postentry? One possible model is that the EIAV p9 protein is necessary to stabilize the linear viral DNA products to prevent degradation during translocation from the cytoplasm to the nucleus. A second model is that the p9 protein may provide a nuclear localization signal (NLS) to target the PIC to the nucleus. The possibility of p9 providing an NLS is supported by the location of a number of lysine residues in the sequences surrounding the K30 and E32 mutation sites that differentiate replication competence and differ by only two amino acids, 30KKEYNVKEK38. To determine whether the basic lysine residues in this sequence would be predicted to form an NLS sequence, we analyzed the p9 open reading frame using a computer program (PSORT II) to identify the potential for NLS motifs. However, no NLS-like sequence in the p9 protein was predicted by this computer modeling. Also arguing against the role of a p9 NLS function is the previous study from this laboratory (11) demonstrating that the mutation of the lysine residues at positions 31 and 32 to methionine residues resulted in a replication-competent provirus construct, indicating that these two lysines are dispensable for virus replication. As a third model, we postulate that p9 protein association with other viral proteins and specific cellular factors may direct nuclear import of PICs. It has been reported that HIV-1 p6 protein serves as an anchor to incorporate the Vpr proteins into the virion and that the Vpr protein is necessary for viral DNA nuclear localization (34, 37, 48). Since EIAV does not encode an apparent Vpr-like protein, the proposed role of EIAV p9 in the nuclear import of viral DNA might be similar to the function of the accessory protein Vpr of HIV-1 translocation of HIV PICs (24, 31). Experiments are currently in progress to examine the EIAV PIC and to determine the role of EIAV p9 in viral DNA stabilization and nuclear transport.
ACKNOWLEDGMENTS
This work was supported by the grant R01 CA49296 from the National Cancer Institute of the National Institutes of Health.
We thank Shannon Durkin for critical editing of the manuscript.
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