Cell-to-ceU transmission of human

Journal of General Virology(1993), 74, 33 38. Printedin Great Britain

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Cell-to-ceU transmission of human immunodeficiency virus infection induces two distinct phases of viral RNA expression under separate regulatory control Tuckweng Kok, ~* P e n g Li ~ and Christopher BurrelP ,z 1National Centre for HIV Virology Research, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5000 and 2Department of Microbiology and Immunology, The University of Adelaide, North Terrace, Adelaide, South Australia 5000, Australia

A cell clone persistently infected with human T celllymphotrophic virus type IIIB (H3B cells) contained mainly the multiply spliced (2 kb) and singly spliced (4-3 kb) species of human immunodeficiency virus (HIV) RNA. When H3B cells were co-cultured with susceptible HUT78 cells, cell fusion occurred within 4 h of cell mixing and was accompanied by a marked increase of the unspliced full-length (9.2 kb) HIV RNA. This first phase of viral RNA induction (4 to 12 h post-infection) was followed by a second phase of viral RNA synthesis from 24 h p.i. in which there were significant increases in all three species of HIV RNA. Reverse transcriptase (RT) inhibitors such as azidothymidine (AZT) at concentrations that abolished de novo HIV DNA synthesis, abolished the first phase but not the second phase of viral RNA synthesis in our model system. A comparable

one-step cell-free virus infection showed a pattern of viral RNA synthesis similar to that of the cell-to-cell transmission of infection. However, viral RNA synthesis following cell-free virus infection was totally inhibited by RT inhibitors. The early phase (4 to 12 h) expression of 9'2 kb HIV RNA is likely to use newly synthesized HIV DNA as template; during this phase, HIV RNA and DNA syntheses occur simultaneously, with each process being dependent on the other for maximal yield. During the later (24 to 48 h) phase, all three HIV RNA species may be transcribed at least in part from proviral DNA from the original donor cells. This later phase may privide one of the mechanisms for natural spread of virus to new cells and for enhanced viral gene expression in vivo, despite the presence of AZT.

Introduction

interpreted as demonstrating a programmed temporal shift from spliced to unspliced RNA transcripts as the infection process develops. We have developed a synchronous cell-to-cell transmission model using H3B cells [a clone of H9 cells persistently infected with human T cell-lymphotropic virus type IIIB (HTLV-IIIB)] as 'donor cells' and HUT78 cells as recipients. H3B cells contain two copies of integrated proviral DNA/cell and episomal HIV DNA corresponding to one or two genome equivalents/ 1000 cells (Li et al., 1992). Following cell-to-cell transmission, episomal HIV DNA appears at 4 h p.i., and both linear and circular forms then accumulate with kinetics essentially similar to those seen in cell-free virus infections (Li & Burrell, 1992). In this system, three potential templates for HIV RNA transcription may be considered: proviral D N A present in the donor cells, newly synthesized episomal DNA, and newly synthesized and integrated proviral DNA. We report here that viral RNA synthesis following cell-to-cell transmission comprises two distinct phases that are quite different to those

Infection of a cell with human immunodeficiency virus (HIV) may be initiated by cell-free virus particles or by transmission of infection from an infected cell (Li & Burrell, 1992; Li et al., 1992). Both processes are assumed to occur in the infected host; however cell-to-cell transmission may be particularly significant once neutralizing antibody has developed. Kim et al. (1989, 1990) reported conditions for infection of H9 cells with cell-free virus and demonstrated that full-length linear episomal HIV DNA was first detected 4 h post-infection (p.i.) and increases in amount to 12 h p.i. Circular DNA forms were detected later and in smaller amounts, and in the cell nucleus only. In contrast, viral RNA was first detected 16 h p.i., and increased progressively to 48 h p.i. The first RNA species found were the spliced 2 kb and 4-3 kb forms encoding regulatory and envelope proteins, but significant amounts of unspliced 9.2 kb RNA (capable of providing both new genomic RNA and gag-pol mRNA) were not seen until 24 h p.i. These findings have been 0001-1199 © 1993 SGM

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T. Kok, P. Li and C. Burrell

described above for cell-free virus infection. An early phase (4 to 12 h p.i.) was marked by a dramatic increase in only the full-length 9.2 kb viral RNA. This early phase was not seen in cells treated with azidothymidine (AZT) suggesting that it uses newly synthesized viral DNA as template. A second phase (24 h p.i. onwards) involved a large overall increase in all viral RNA species, and was largely independent of de novo viral DNA synthesis, suggesting that in part it uses integrated proviral DNA from the original donor cells as templates.

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Methods Cell-to-cell infection. HUT78 cells, an uninfected CD4 + lymphoblastoid cell line, were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. H3B cells are a locally derived laboratory clone of H9 cells persistently infected with the HTLV-IIIB strain of HIV. Each H3B cell contains two copies of integrated proviral HIV DNA/cell and secretes into the culture supernatant approximately 0-01 TCIDs0 virus/h (Li & Burrell, 1992). A synchronous one-step infection model was used in which 1.6 x 106 washed HUT78 cells were infected with 0.4 x 106 washed H3B cells in a volume of 10 ml of RPMI 1640 culture medium supplemented with 10% foetal bovine serum. Cell-free virus infection. A cell-free virus inoculum was harvested from the clarified culture supernatants of H3B cells as described previously (Li & Burrell, 1992). HUT78 cells (2x 106) were then infected at a nominal multiplicity of 0.5 TCIDa0 virus/cell using a centrifugation enhancement method (Pietroboni et aI., 1989). After infection, the cells were washed three times before seeding into culture wells at a concentration of 2 x l0 b cells/ml.

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RNA preparation. At various times after infection, the cells were harvested, washed once in PBS (4 °C) and then lysed with 0-65 % NP40. Nuclei were removed by centrifugation at 2500g for 1 min. The cytoplasmic R N A was then extracted in the presence of 7 M-urea and 1% SDS (Gough, 1988). After precipitation with alcohol, the R N A samples were washed once in 70% ethanol and dissolved in R N A loading buffer (Sambrook et al., 1989). Gel electrophoresis and hybridization. R N A samples from the equivalent of 2 x 106 cells were loaded into each lane of 0-8 % agarose gels containing 0.7 M-formaldehyde (Sambrook et al., 1989). After electrophoresis, R N A was transferred onto a Hybond-N + nylon membrane (Amersham) in 20 x SSC. For Northern blot hybridization, the SstI fragment of plasmid pBH10 (Hahn et aL, 1984) was labelled with 32p using the Amersham Multiprime D N A labelling system, according to the manufacturer's instructions. The typical specific activity of the hybridization probe was 2 x 108 to 3 x 108 c.p.m./gg. All Northern blot results are from R N A extracted separately from duplicate culture tubes, pooled and loaded in amounts equivalent to 2 x 106 cells/lane.

Fig. 1. (a) Kinetics o f H I V R N A production between 0 and 72 h p.i. of HUT78 cells with H3B cells. The figures at the top of each lane refer to the time (h) at which R N A was extracted. Lane H, R N A extracted from 2 x 106 H3B cells. Each lane contains an amount of RNA equivalent to that from 2 x 106 cells representing the average R N A extracted from duplicate cell cultures. (b) Patterns of HIV RNA from H3B cells. Each lane contains the average amount of R N A extracted from duplicate cell cultures of 0.4 x 106 H3B cells. The figures at the top of each lane refer to the time (h) at which the R N A was extracted.

Results Two phases of H I V RNA expression following cell-to-cell transmission of infection Steady state H3B cells contained predominantly 4.3 and 2 kb HIV RNA species in the cytoplasmic fraction, with a barely detectable shadow at 9'2 kb (Fig. la, lane H). Quantification of the content of cytoplasmic HIV RNA,

by comparison of cytoplasmic RNA with known HIV DNA standards, by dot-blot hybridization yielded an approximate estimate of 20 copies of HIV RNA/H3B cell. This is likely to be a minimum estimate because it was not corrected for differences in length. Fig. 1 a shows the changes in cytoplasmic HIV RNA during the period

Cell-to-cell transmission-induced H I V RNA between 0 and 72 h, after mixing 0.4 x 106 H3B cells and 1.6 x 106 HUT78 cells. Four hours after mixing there was a significant increase in genomic-length 9.2 kb R N A as compared to that seen at 0 h (just after mixing) or that seen with a fivefold greater number of H3B cells (2 x 106 cells; Fig. 1 a, lane H). This increased level of 9-2 kb R N A , and near constant or slightly increased levels of 4-3 kb and 2 kb spliced RNAs, was maintained until 24 h when a second large increase in all three R N A species was seen. Finally, by 72 h p.i. large amounts of 4.3 kb and 2 kb R N A were present but 9.2 kb R N A was barely detectable. The R N A signals were shown not to be due to contaminating HIV D N A because treatment with 100 lag/ml RNase abolished all Northern blot signals; this treatment has previously been shown not to affect D N A hybridization in Southern blots (Li & Burrell, 1992). We also showed that the above two phases of HIV R N A induction were not due to manipulation and culture of H3B cells. When 0.4 x 106 H3B cells alone were washed and then cultured as above, there was no induction of 9-2 kb HIV R N A and the levels of 4.3 kb and 2 kb R N A remained essentially the same throughout a 24 h period. During this time the number of H3B cells increased two- to three-fold (Fig. 1 b).

The early, but not the later, phase of H I V R N A expression is inhibited in AZT-treated cells Fig. 2 shows the kinetics of cytoplasmic R N A expression in a similar experiment to that in Fig. 1 (a) except that both H3B and HUT78 cells were treated with 20 gMA Z T from 18 h prior to infection. The drug was renewed at the time of infection and maintained throughout the 72 h infection period. Conditions of infection and R N A extraction were identical to those in Fig. 1 (a), and the Northern blots were hybridized with the same probe mix. A Z T treatment of H3B cells alone led to a minimal increase in 4.3 kb and 2 kb HIV RNA, but did not induce 9.2 kb RNA. After infection, the early phase of 9.2 kb R N A synthesis was not seen, but 24 to 48 h p.i. marked induction of all three HIV R N A species was seen, with a similar profile and time course but at lower levels than without AZT.

H I V R N A transcription following infection with cell-free virus Fig. 3 shows the induction of HIV R N A from 0 to 72 h, after infection of HUT78 cells with cell-free virus at a nominal multiplicity of 0.5 TCIDs0 virus/cell. The use of centrifugally enhanced inoculation would have resulted in an effective multiplicity of 5 TCIDs0 virus/cell (Pietroboni et al., 1989). The 9.2 kb R N A species appeared from

H

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Fig. 2. Kinetics of HIV RNA production in the presence of 20 ~.IMAZT between 0 and 72 h p.i. of HUT78 cells with H3B cells. The figures at the top of each lane refer to the time (h) at which the RNA was extracted. Lane H, RNA extracted from 2 × 106H3B cells (treated with 20 ~tM-AZT). Each lane contains an amount of RNA equivalent to that from 2 × 106 cells representing the average RNA extracted from duplicate cell cultures. 2

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Fig. 3. Kinetics of HIV RNA production between 0 and 72 h p.i. of HUT78 cellswith cell-freeHIV at a nominalmultiplicity of 0.5 TCIDs0. The figures at the top of each lane refer to the time (h) at which the RNA was extracted. Each lane contains an amount of RNA equivalent to that from 2 x 106 infected HUT78 cells. 8 h p.i. onwards, whereas significant 4.3 kb and 2 kb R N A species were seen from 24 h p.i. onwards. The detected 9-2 kb R N A was not from input virions, as 2 to 4 h samples did not produce visible bands corresponding to genomic RNA. These results are quite different from the findings of Kim et al. (1989). At 24 h p.i. a large increase in all three R N A species was seen, and by 72 h p.i. the level of 9.2 kb R N A was lower than that at 8 h

36

T. Kok, P. L i and C. Burrell

p.i. whereas the spliced RNA species remained at high levels. When 20 ~tM-AZT was added to a duplicate experiment 18h prior to infection and maintained throughout, HIV RNA was not detected at any time (data not shown).

Discussion The kinetics of cytoplasmic HIV RNA expression in the cell-to-cell infection model show a significant initial increase in 9"2 kb genomic length RNA 4 h after coculturing HUT78 and H3B cells. This time corresponded to the first appearance of episomal HIV DNA in the same model [data not shown, but see Li & Burrell (1992)]. In contrast, the levels of the 4.3 kb and 2 kb viral RNA transcripts did not show more than twofold increase during the period from 0 to 12 h p.i. We did not examine cytoplasmic and nuclear HIV R N A levels separately at each time. Cytoplasmic RNA levels may be affected by changes in RNA stability and nucleus to cytoplasm transport as well as changes in RNA transcription. However, nuclear HIV R N A in H3B cells represented only a tiny fraction of the total viral RNA and no 9.2 kb species could be detected (data not shown). Moreover, levels of singly and doubly spliced transcripts were also maintained at constant or slightly increased levels, at times when a marked increase in the 9.2 kb species was observed. Therefore, we believe that the above changes are likely to represent a true increase in HIV RNA transcription, and not merely a change in splicing, RNA transport or other forms of processing. This early induction of the 9'2 kb transcript was also seen following infection with cell-free virus, although it was somewhat delayed (8 h p.i. instead of 4 h p.i.). This also corresponded to the slightly slower appearance of episomal HIV DNA in a cell-free virus infection, compared to that in the cell-to-cell infection (Li & Burrell, 1992; Li et al., 1992). Both of the above patterns of viral RNA synthesis differ markedly from that seen in the cell-free virus infection model reported by Kim et al. (1989, 1990). These authors reported that the first major RNA species to be induced consisted of the heterogeneous group of 2 k b regulatory transcripts at 16h p.i., and that significant 9-2 kb genomic-length RNA did not appear until 24 h p.i. A similar differential RNA transcription pattern in a cell-free virus infection model has previously been reported with visna virus (Vigne et al., 1987). Equine infectious anaemia virus, another member of the lentivirus family, showed a different pattern of viral RNA synthesis, with full-length RNA appearing early as in the present study (Rasty et al., 1990). However, the kinetics of transcription of visna virus and equine infectious anaemia virus DNA were examined in more relaxed time

frames. It is not clear at this stage why, in the present study using free virions, the 9'2 kb viral RNA was detected much earlier than in the previous report (Kim et al., 1989). However, Kim et al. (1989) suggested that only 10 to 20% of cells in their system were undergoing primary synchronous infection. Their observation that the concentration of genomic-length RNA only was markedly increased between 24 and 36 h p.i. would be consistent with our findings if this represented secondary infection by a cell-to-cell transmission mechanism. We have shown previously that 20 gM-AZT completely abolishes de novo episomal HIV DNA synthesis for at least 48 h in the cell-to-cell transmission model (Li et al., 1992). The present study has demonstrated that AZT has little effect on steady-state 4.3 kb and 2 kb HIV RNA levels in H3B cells alone, but completely abolishes the first phase of RNA synthesis 4 to 12 h following cell mixing. Theoretically the template for the first phase of HIV RNA induction might be integrated proviral DNA in donor cells, newly made episomal DNA or newly made and integrated proviral DNA (Panganiban & Temin, 1983; Stevenson et al., 1990). Our findings suggest that the transcriptional template for the first phase of HIV RNA expression is likely to be newly synthesized free and/or integrated viral DNA. Furthermore, and most significantly, as the copy number of 9"2 kb RNA in H3B cells is insufficient to provide templates for the full amount of newly synthesized HIV DNA seen between 4 and 12 h p.i. (50 to 100 copies/cell; Li & Burrell, 1992), our findings point to a system of simultaneously occurring HIV DNA and RNA synthesis, each dependent on the other for maximum yield of progeny molecules. The second phase (24 to 72 h p.i.) of RNA induction is more extensive and involves all three R N A species. It is reduced to some extent if de novo HIV DNA synthesis is inhibited with AZT. One conceivable interpretation is that cell fusion might provide stimulatory signals for transcription from the integrated viral sequence of the H3B cells, which is not dependent on de novo HIV DNA synthesis. The level of enhanced viral RNA synthesis observed in the AZT-treated cells represents only a portion of the second phase viral R N A synthesis observed in the AZT-ffee culture; by this stage, integration of de novo synthesized HIV DNA is well established in AZT-free cultures (T. W. Kok, unpublished results), and presumably this is also available as transcriptional template by this time. In other reports it has been shown that stimulation of the chronically infected ACH-2 and U1 cell lines with phorbol esters leads to an early (2 to 6 h) minor increase in 2 kb RNA followed by a later significant increase in levels of unspliced RNA (Pomerantz et al., 1990; Michael et al., 1991). It was suggested by both groups that the increase

Ceil-to-cell transmission-induced H I V R N A

in unspliced RNA might be a consequence of increased rev activity, but these reports differed as to whether there may have been a significant increase in de novo RNA synthesis. It is difficult at this stage to make a direct comparison between these experimental systems and the present study; however, we have found no evidence for an initial significant increase in cytoplasmic 4.3 kb or 2 kb RNA before the appearance of a 9.2 kb RNA in either the cell-to-cell or cell-free virus systems. In summary, different mechanisms may be involved in these two phases of viral RNA induction following cellto-cell transmission of HIV infection. The first phase of RNA expression is likely to use newly synthesized viral DNA as template, whereas a significant part of the second phase may be due to increased transcription from proviral DNA provided by the donor cells. Since donor cell proviral sequences are present from the onset, but no change in transcription from this template is seen during the time (4 to 12 h p.i.) when newly synthesized DNA is being transcribed, it is likely that each of these transcriptional processes is under separate control and uses different patterns of RNA processing. We have previously demonstrated unequivocally that de novo reverse transcription is an essential requirement for HIV replication following cell-to-cell transmission when analysed within the time frame of a single-step synchronized virus growth cycle (Li et al., 1992). However, prolonged incubation with AZT in different cell-free HIV infection and cell-to-cell HIV infection models is followed by 'escape' from AZT inhibition and production of progeny virus (Smith et al., 1987; Gupta et al., 1989; Li et al., 1992). In the case of cell-to-cell transmission, the second AZT-resistant phase of HIV RNA synthesis, defined in the present study, could provide the genomic viral RNA required for progressive virion production in the presence of AZT. However, escape from AZT following cell-free virus infection may be due to incomplete arrest of reverse transcription in the presence of fluctuating AZT triphosphate levels (Li et aL, 1992). In addition, prolonged incubation in a cell-free virus infection is likely to lead to secondary cell-to-cell transmission. Finally, this enhanced genomic-length RNA induction obtained in our cell-cell infection model may also be reflected in the observation that late in disease, when syncytiuminducing virus isolates dominate (Schuitemaker et al., 1992), the ratio of genomic viral RNA and viral DNA in infected cells increases by over 1000-fold when compared to the early stage of disease (Michael et al., 1992). Thus, this process may in part explain the long-term failure of AZT or other reverse transcription inhibitors in the treatment of AIDS. We thank Alice Stephenson and Lara Kuiper for excellent technical assistance, and Wendy Fleming for typing the manuscript. This work

37

was supported by the Australian Commonwealth AIDS Research Grant Programme.

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(Received 30 June 1992; Accepted 10 September 1992)