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Journal of Virology, April 2007, p. 4367-4370, Vol. 81, No. 8
0022-538X/07/$08.00+0     doi:10.1128/JVI.02357-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Efficiency of Human Immunodeficiency Virus Type 1 Postentry Infection Processes: Evidence against Disproportionate Numbers of Defective Virions{triangledown}

James A. Thomas, David E. Ott, and Robert J. Gorelick*

AIDS Vaccine Program, Basic Research Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland 21702

Received 27 October 2006/ Accepted 23 January 2007


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ABSTRACT
 
The vast majority of human immunodeficiency virus type 1 particles are claimed to be noninfectious, but there is disagreement as to whether they are defective or simply lack the opportunity to initiate an infection. We have examined the efficiencies of reverse transcription and integration and find that approximately 1 of every 8 virions that initiate reverse transcription form proviruses, a quantity significantly different from the commonly reported ratio of 1 in 1,000. In addition, results from two different infectivity assays demonstrate that the titers are not equivalent to the number of infectious particles. The apparent predominance of noninfectious particles is due to infrequent occurrences of successful virus-cell interactions.


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TEXT
 
The majority of retroviral particles in cell-free supernatants do not produce an infection (47). When the number of physical particles is compared with the infectious titer, one sees that the apparent ratio of infectious to noninfectious particles is typically between 1 in 1,000 and 1 in 60,000 (6, 18, 31, 35, 39, 41, 42). This difference may mean that most particles (i) are defective and cannot complete the necessary infection steps or (ii) simply never come into contact with a permissive cell. Based on experimental observations of retrovirus properties and theoretical models using murine leukemia virus vectors, the second alternative is probably more likely (2, 4, 5, 17, 30, 37). Further support for an underestimate of infectious particles comes from observations that titers can be increased by procedures such as the addition of polycations (46), vesicular stomatitis virus G (VSV-G) pseudotype formation (10, 38), or spinoculation (38). A precise understanding of this issue is important for proper biochemical analyses of infection events, especially reverse transcription and integration, where the presence of disproportionate numbers of defective particles can make it difficult to interpret results (47).

We have developed reagents for quantitation of viral DNA (vDNA) production and provirus formation using real-time PCR which have allowed examination of early infection events in great detail (9, 44, 45). For the first set of experiments, HOS cells were infected for a single round with VSV-G-pseudotyped Env– human immunodeficiency virus type 1 (HIV-1). At various times after infection, total cellular DNA was isolated (26), and specific vDNA species produced during reverse transcription and integration were quantitated as described previously (9, 44). Reverse transcription intermediates typically peaked 8 to 12 h after infection, while quantities of provirus peaked at about 24 h after infection, consistent with published results (12, 28, 44). When the maximal quantities of each vDNA intermediate were compared, it was evident that each reverse transcription step proceeded at a similar efficiency: the value for minus strand transfer (U3-U5) was 80% of that for minus strand strong stop (R-U5), the value for late minus strand synthesis (gag) was 50% of that for U3-U5, and the value for plus strand transfer (R-5' untranslated region [UTR]) was 60% of that for gag (Table 1). The overall efficiency of reverse transcription, based upon a comparison of the maximal quantities of the initial R-U5 targets to the maximal quantities of R-5' UTR, was ~30% (Table 1). Thus, more than one in four of the particles that initiate reverse transcription appear to complete it.


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  		TABLE 1. Efficiency of infection in HOS cells

Integration, directly measured by an Alu-long terminal repeat (LTR) assay (11, 44), also occurred at a similar efficiency, with ~60% of late reverse transcription intermediates (R-5' UTR) being converted to proviruses (Table 1). The overall efficiency of infection, from initial R-U5 to provirus formation, was 13%, i.e., one in eight virus particles that initiate an infection results in a provirus (Table 1). Therefore, reverse transcription and integration are not major contributors to the apparent defectiveness of HIV-1. These results are in general agreement with other published studies using Nef– HIV-1, HIV-1 vectors, and murine leukemia virus: ~10 to 30% of maximum postinfection vDNA become proviruses (3, 7, 8, 11, 12, 28, 43).

One concern with this type of experiment is that HIV-1 entry mediated by VSV-G occurs through the acidified endosome pathway rather than direct fusion with the plasma membrane mediated by HIV Env (14). Because of this difference, VSV-G pseudotyping can rescue various mutant viruses that are blocked at steps prior to reverse transcription initiation (1, 14, 19, 20, 27, 34). To address this concern, we compared the relative levels of vDNA 24 h after infection, using either HOS cells with VSV-G-pseudotyped Env– HIV-1 or H9 cells with Env+ HIV-1 (Fig. 1). The potential for reinfection of H9 cells with Env+ HIV-1 precludes time course data; thus, our comparisons of quantities are at 24 h rather than maximal quantities. Comparing the ratios of R-5' UTR to R-U5 or provirus to R-5' UTR at 24 h between the VSV-G- and Env-mediated infections did not reveal any real difference between these two samples (Fig. 1). Thus, once reverse transcription begins, the efficiencies are not affected by the mode of entry.


Figure 1
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  		FIG. 1. Effects of envelope type on infection efficiencies. HOS cells (5 x 105) or H9 cells (1 x 106) were infected with VSV-G-pseudotyped Env– HIV-1 or Env+ HIV-1, respectively, for 4 h. Total cellular DNA was isolated 24 h after infection, and vDNA species were quantitated. Each quantity is expressed as a percentage of the efficiency of either the reverse transcription (R-5' UTR to R-U5), the integration (provirus to R-5' UTR), or the overall (provirus to input virus [gag RNA]) process. The bars represent the averages for seven (VSV-G) or six (Env) experiments, with the error bars representing the standard errors of the means.

Our data did reveal an inefficient step in the infection process. Comparison of the vDNA quantities produced within cells relative to the amount of RNA genomes in the inoculum (44) showed that only 5% of virus particles present actually initiated reverse transcription, with 0.41% of the genomes proceeding all the way to provirus (Table 1). It is important to note that the efficiency of viral binding and entry is likely higher with VSV-G-pseudotyped virus than with an Env-CD4-mediated interaction: VSV-G pseudotyping increases the effective number of receptors for entry, allowing a greater number of particles to initiate infections. The receptor for VSV-G has not been identified (16), but it is ubiquitous and is most likely expressed at a higher level than CD4. Figure 1 shows that the overall efficiency of infection (RNA to provirus) is about 13-fold lower with HIV-1 Env than with VSV-G-mediated infections, as expected.

The discrepancy between total virus particles and those particles that initiate infections could be due to the majority of virions being defective and unable to enter cells or to virus-target cell encounters being quite infrequent. To differentiate between these two alternatives, we measured titers with a MAGI-like single-cycle ß-galactosidase (ß-Gal) assay (29) using HCLZ cells (23, 36). If the majority of the virions were entry defective, we would expect a decrease in titer after serial transfer of infectious supernatant, because the infectious particles would be depleted. Conversely, if cell-virion contact is limiting, the majority of infectious particles should remain in the supernatant and be available for subsequent infections. To address this aspect (Fig. 2), two experiments in which the stock was used only once were performed. In an additional experiment, the stock was transferred sequentially between three sets of naïve HCLZ cells. Comparison between experiments (Fig. 2) shows that although the titers in the third experiment are somewhat lower than the others, they did not decrease even after multiple serial transfers. Based on virus particles in the supernatant, the first two experiments required approximately 103 particles per infectious event. This increased twofold in experiment 3 but remained nearly constant over the three successive sequential transfers (Fig. 2B). Therefore, the limitation in infections would appear to be that only a minor fraction of the virions actually encounter cells. This result also implies that the virus stocks in the assays contain many more virus particles than will ever come into contact with target cells, thus implying that titer determinations underestimate the true number of infectious particles.


Figure 2
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  		FIG. 2. Single-cycle infection of LTR-ß-Gal containing HCLZ cells. HCLZ cells (22) were infected with VSV-G-pseudotyped Env– HIV-1 as described previously (23). HCLZ cells (1 x 105) were seeded into each well of 24-well tissue culture plates the day prior to infection. On the day of infection, media were removed and replaced with undiluted or serially diluted, clarified, virus-containing supernatants obtained by CaPO4/DNA coprecipitation of Env– pNL4-3 and pHCMV-g as described previously (26), with 2 µg/ml Polybrene. (A) HCLZ titers from each experiment, in numbers of BCFU per ml. (B) Titers corrected for input virus for each experiment, reported as ratios of viral genomes to BCFU (genome quantifications are described in the Table 1 footnotes). The white bars show the results from the two different experiments where viral supernatants were applied and left on the cells as usual. The black bars show results from a third experiment, where virus-containing supernatants were incubated for 4 h at 37°C with the HCLZ cells (P1), removed from the first set of cultures and transferred to another set of wells containing naïve HCLZ cells and incubated for 4 h (P2), and then transferred a third time to new HCLZ cultures and incubated for 4 h (P3). "P" indicates the passage number. Error bars indicate standard deviations. Media were used to replace the virus-containing supernatants in each case. The reverse transcriptase activities (amounts of [3H]TMP incorporated per ml per minute) were determined as described previously (24) and were 7.4 x 105 for experiment 1, 8.7 x 105 for experiment 2, and 8.2 x 104 for experiment 3. The amounts of virus in genomes per ml were 5.1 x 109 for experiment 1, 8.4 x 109 for experiment 2, and 8.0 x 108 for experiment 3.

A concern with the experiment whose results are presented in Fig. 2 is that VSV-G pseudotyping may eliminate the possibility of gp120 shedding from HIV-1 particles, which would certainly render virions defective. However, gp120 has been shown to be attached to particles reasonably tightly; multiple freeze-thaw cycles or purification through sucrose gradients does not dislodge it, but heating (>50°C) for 1 h causes removal of gp120 from particles (15). In addition, a systematic study of HIV-1 inactivation found that spontaneous shedding of gp120 with a half-life of ~30 h occurs at 37°C (31), which is longer than the 12-h duration of this experiment. In fact, when we performed single-cycle ß-Gal experiments with Env+ virus and compared the ratios of the numbers of blue CFU (BCFU)/ml to the numbers of virus particles/ml between VSV-G-mediated and Env-mediated infections, we find that they differ by only ~6.5-fold (0.017 ± 0.0062 [Env] versus 0.11 ± 0.060 [VSV-G]), which likely reflects the difference in receptor availability on the cell surface.

If the rate-limiting step of infection truly is virus-cell contact, then methods that increase these encounters should increase infectious titers. We have examined this issue by measuring the limiting dilution titer that gave rise to spreading infections in H9 cells (22, 24). Tenfold serial dilutions of a wild-type NL4-3 virus stock were used to infect H9 cells, by either incubation in the presence of Polybrene (Sigma Aldrich, St. Louis, MO), spinoculation (38), or Magnetofection (OZ Biosciences, Marseille, France). In two independent experiments, we observed at least a 10-fold increase in infectious titer with spinoculation or Magnetofection over that observed with the standard incubation (Table 2). Our results show that 104 particles were needed to initiate a spreading infection, which was reduced to 103 by either spinoculation or Magnetofection and even dropped to 101 in one experiment (Table 2). Thus, when the incidence of virus-cell contact is increased, infectious titer also increases.


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  		TABLE 2. Enhancement of replicative titer by different methods of infection of H9 cells

It is important to note that the properties of our transfection-generated virus may be different from those of virus in the host, which should contain cellular proteins obtained from the cells from which it was derived (13, 21). The presence of these cellular proteins may increase infectivity. A somewhat related issue is the difference between molecular clones of HIV-1, but it has been reported that diverse clinical isolates of HIV-1 do not differ much in replication kinetics or general physical properties (33, 35).

It is therefore important that the following distinction is made: the ratio of particles to infectious events is not the same as the ratio of physical particles to infectious particles, as virions that do not have a chance to infect are not inherently defective. The true ratio of infectious to defective particles is likely several orders of magnitude higher, i.e., 1 in 8 to 1 in 20 rather than 1 in 1,000 to 1 in 60,000 (2, 25, 30). Extending this property to Env-mediated infections may be reasonable as Env is fairly stable on viruses (15). These results also provide an explanation for why infections in the presence of cell-to-cell contact are far more efficient than infections with cell-free virus particles—the most inefficient or infrequent step (i.e., viruses encountering cells) is bypassed (32, 40).


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ACKNOWLEDGMENTS
 
We thank Steven Hughes (NCI-Frederick), Alan Rein (NCI-Frederick), and Jeffery Lifson (SAIC-Frederick, Inc.) for their comments and critiques of this work. We also thank Tracy Gagliardi and Laurie Queen for their technical assistance.

This project has been funded in whole or in part with federal funds from the National Cancer 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: Building 535, 4th Floor, National Cancer Institute, Frederick, MD 21702. Phone: (301) 846-5980. Fax: (301) 846-7119. E-mail: gorelick{at}ncifcrf.gov Back

{triangledown} Published ahead of print on 31 January 2007. Back


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Journal of Virology, April 2007, p. 4367-4370, Vol. 81, No. 8
0022-538X/07/$08.00+0     doi:10.1128/JVI.02357-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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