Variable tRNA content in HIV-1IIIB

Vol. 185, No. 3, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

June 30, 1992 Pages 1005-1015

VARIABLE tRNA CONTENT IN HIV-l,,,

Min Jiang', Johnson Mak', Mark A. Wainberg', Michael A. Pamiak',

Eric Cohen', and Lawrence Kleiman'

'Lady Davis Institute for Medical Research, Sir Mortimer B. Davis -

Jewish General Hospital, 3755 Cote Ste-Catherine Road, Montreal,

Quebec, Canada H3T lE2

'Department of Microbiology, University of Montreal, Montreal, Quebec, Canada

Received April 30, 1992

SUMMARY : Low molecular weight RNA in HIV-1 is found in three size classes resembling 7s RNA, 5s RNA, and tRNA. The 2-dimensional polyacrylamide gel electrophoresis (2D PAGE) patterns of tRNA found in HIV-l have been determined in virus produced in five different cell types: H9, UHCl (a U937-derived clone), UHCB (an RT(-) derivative of U937), HeLa, and COS. The presence of the putative primer tRNA for reverse transcriptase, tRNALya,3, has also been determined either by hybridization with a tRNALYS*3-specific DNA probe or by a comparison of the electrophoretic mobility of viral tRNA species with purified humantRNALY5,3. Our results indicate the following: 1) The number of tRNA species found in infectious HIV-1 rIIB produced in different cell types varies, according to cell type, from >20 to 4, indicating that only 4 or less tRNA species are required for the viral infectious life cycle. 2) There are l-3 tRNA species tightly associated to the viral genomic RNA, depending upon the cell type producing the virus. 3) The putative primer tRNA, tRNALy',3, is detected with the tRNALYSs3- specific hybridization probe in the tRNA of HIV-l produced in H9 cells, and the tightly associated tRNA species in this virus has the same electrophoretic mobility in 1-D PAGE as purified tRNALYss3. On the other hand, we cannot detect tRNALySn3 in the tRNA of HIV-l produced in HeLa cells, and the tightly associated tRNA found in this virus does not migrate with the same electrophoretic mobility as tRNALYss3. 0 1992 Academic Press. Inc.

An initial step in the replication of retrovirus is the conversion of the

retroviral RNA genome into DNA by the enzyme reverse transcriptase. The synthesis of this DNA, which will be integrated into the infected host cell

genome, is initiated from a specific cellular tRNA which acts as the primer for

the reaction. The tRNA used differs according to the virus type. All members

of the avian sarcoma and leukosis virus group examined to date use tRNATrp as

primer for reverse transcription (1,2,3-5), whereas the murine leukemia virus

employ tRNAPro (6,7,8), and mouse mammary tumor virus utilize tRNALYss3 (9,lO).

Only the 3'-terminal 18-19 nucleotides of primer tRNA bind to a complimentary

region near the 5' end of the 35s RNA, termed the primer binding site (PBS).

The sequence of this site reported in HIV-l,,,, ( 11) suggests that the primer tRNA

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1005 Copyright 0 1992 by Academic Press, Inc.

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in this virus is also tRNALySa3, one of the two major tRNALyS isoacceptors found

in mammalian cells (12).

The primer tRNA found in retrovirus particles is selected from the host cell

tRNA population during virus assembly (6,13-16). Additional tRNAs are also

packaged along with the primer tRNA. Viral tRNAs are found in either a "free"

state or associated with the viral genome (1,2,10,14). The primer tRNA for

reverse transcriptase has been found to be more tightly associated with the

genome than the other tRNAs, perhaps because of its ability to hybridize to the

PBS (1,6,7,9,14). In investigating the cellular and viral factors responsible

for the select incorporation and genomic placement of primer tRNA in HIV-l,,,,,

we have found a cell type-dependent variability in both the number of free tRNA

species found in the virus and in the number and type of tRNAs tightly associated

with the viral genome. The function of non-primer tRNAs is not known, and our

results inducate that many of the non-primer tR.NAs incorporated into the virus

are not necessary for viral infection and replication. Our results also suggest

that tightly associated tRNA in HIV-1 is not always tRNALySs3.

MATERIALS AND METHODS

Cell Lines and Virus: Mouse mammary tumor virus (MMTV) and HIV-l,rI, produced in H-9 cells, was obtained from Advanced Biotechnology Institute (Bethesda, MD). Infectious HIV-1 IlIB produced in the U937-derived cell line UHCl and mutant (RT(- I), non-infectious, HIV-l,,,, p reduced in the cell line UHC8, were obtained from cultures as previously described (17) - in short, 4 x 10' cells were seeded in 400 ml culture medium and grown for 4-5 days, and the virus was harvested from the medium by centrifugation. HIV-l produced in COS and CD4(+) HeLa cells were obtainedby viral DNA transfection. COS-7 cells were obtained from ATCC. CD4(+) HeLa cells were obtained through the AIDS Research Reference Reagent program, division of AIDS, and were contributed by Dr. Richard Axe1 (28). HIV-l produced in COS-7 and CD4(+) HeLa cells were obtained by viral DNA transfection. The HIV proviral clone HXBc2 derived from the 1llB isolated was provided by Drs. R.C. Gallo and F. Wong Staal (11,29). The proviral DNA was cloned into an SV-40 based plasmid vector as previously described (30). The COS-7 cells and CD4(+)-HeLa cells were grown at 37°C in 5% CO, as monolayer cultures in DMEM media (Dulbecco's modified Eagle's medium containing high glucose, with glutamine, without sodium pyruvate) containing 10% fetal calf serum and 1% penicillin- streptomycin. Twenty four hours before transfection, exponentially growing cells were harvested by trypsinization, and replated at a density of 1 x lo6 cells/ml in 100 mm tissue culture dishes. Ten pg of viral DNA was added to each dish as a DNA-CaCl, suspension. To prepare this suspension, the DNA was first mixed with 50 pl of 2.5 M CaCl,, and the colume brought to 500 pl with water. This was then added dropwise to 500 ~1 of 2X HBS (Hepes-buffered saline: 280 mM NaCl, 50 mM Hepes, 1.5 mM NazHPO,, pH 7.05), and the resulting precipitate was allowed to form for 20-30 minutes at room temperature. The suspension was then transferred to cells in the tissue culture dish, and the cells were incubated for another 16 hours. Old medium was then removed, and the cells were washed with 5 ml of phosphate buffered saline twice. Seven ml of fresh medium was added back to each plate, and virus were isolated from the supernatant after a further 63 hours incubation. The supernatant was first centrifuged in a Sorval SS-34 rotor at 3,000 rpm for 30 minutes, and the virus were then pelleted from the resulting supernatant by centrifuging in a Beckman Ti45 rotor at 40,000 rpm for one hour. The viral pellet was then purified by centrifugation at 26,000 rpm for 1 hour through 15% sucrose onto a 65% sucrose cushion, using a Beckman SW41 rotor.

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Viral RNA isolation and fractionation: Total viral RNA was extracted from viral pellets using the guanidinium isothiocyanate procedure (18), the yield being approximately 50 pg total viral RNA per 1O'l viral particles. Total viral RNA containing both high molecular weight genomic RNA and low molecular weight tRNA was fractionated using commercial Nucleobond AX-20 columns (Nest Group, Southboro, MA). This column binds total viral RNA in a low salt buffer (0.2 M KC 1, 15% EtOH, 100 mM Tris/PO,, pH 6.3). Low and high molecular weight RNA are eluted sequentially with higher salt buffers (19). tRNA is eluted with a 0.8 M KC1 buffer (0.8 M KCl, 15% EtOH, 100 mM Tris/PO,, pH 6.3), while genomic RNA and any tP.NA associated with it is eluted next using a 1.3 M KC1 buffer (1.3 M KCl, 15% EtOH, 100 mM Tris/PO,, pH 6.3). The fractionation of free and total associated tRNA was done as follows: An AX-20 column was equilibrated with 1 ml 0.2 M KC1 buffer. Solutions were passed through the column by gravity. Twenty pg of total viral RNA was dissolved in 500 ~1 0.2 M KClbuffer and loaded on the column. The column was then sequentially washed, first with 2 ml 0.8 M KC1 buffer, which elutes free viral tRNA, and then with 2 ml 1.3 M KC1 buffer, which elutes viral genomic RNA and the tRNA associatedwith it, termed total associated tRNA. Each 2 ml wash was collected as 4 - 500 /Al fractions in 1.5 ml microcentrifuge tubes. After the addition of l- pg carrier DNA (pBR322), 400 ~1 isopropanol was added to each tube, and these were immediately centrifuged at top speed in an Eppendorf microcentrifuge for 30 minutes. (The addition of carrier DNA to the sample prior to loading it on the AX-20 column is unnecessary, since it does not improve recovery of the sample from the column.) Pelleted samples were washed 1X with 70% EtOH, 1X 95% EtOH, and dried in vacua. >80% of total viral RNA is recovered using this procedure. To isolate tightly associated tRNA total viral RNA was heated for 3 minutes at 65°C in dissociation buffer (20 mM Tris, pH 7.5; 200 mM NaCl; 2 mM EDTA-Na,), which will cause the release of loosely associated tRNA from the genomic RNA. After quick-cooling on ice, the RNA sample was fractionated with an AX-20 column, isopropanol precipitated in the presence of 10 pg carrier DNA, and washed and dried, as described above, The 0.6 M KC1 buffer will elute the freed loosely-associated RNA along with the initially free RNA, while the 1.3 M KC1 buffer will elute the genomic RNA along with the RNA still tightly associated to it.

Purification of human tRNALYS*1*2 and tRNALYsr3: lo-20 mg of total tRNA can be isolated from 200 g of human placenta, using the method of Roe (20), which uses standard phenol-chloroform extractions to isolate total RNA, followed by DEAE cellulose chromatography to isolate total tRNA. The tP.NA was aminoacylated with 3H-lysine, and tRNALys fractions were isolated using standard chromatographic methods for tRNA purification (sequentially, DEAE-Sephadex A-50, reverse phase chromatography (RPC-S), and Porex C4 chromatography (12,21). A final purification step used 2 dimensional polyacrylamide gel electrophoresis (2D PAGE) as described below. The tRNALYs isoacceptor spot was eluted from gel slices by soaking in water overnight, and concentrated by ethanol precipitation.

RNA labeling: The fractionated RNA samples were labeled using the 32pCp 3' end-labeling technique (22). 32pCp was made as follows: 5 mCi of gamma-32P-ATP (specific activity 3,000 Ci/mmole, DuPont Canada) was dried down in a microcentrifuge tube using N,. One hundred ~1 of the following reaction solution was added (reaction solution: 50 mM Tris-HCl, pH 9.2, 5 mM MgCl,, 3 mM dithiothreitol, 5% bovine serum albumin, 1 PM 3'-cytidine monophosphate, and 10 units T4 kinase). The reaction was incubated at 37°C for 3 hours, and the conversion of 3'-CMP to 32pCp was monitored using PEI thin layer chromatography in 0.8 M NH2S0,, which separates 32pCp from ATP3' (22).

Labeling of the RNA with 32pCp was as previously described (19,22). Free viral tRNA was labeled without further treatment. Total associated tRNA was first heated to 65°C for 3 minutes, quick-cooled and then labeled. Labeled tightly-associated tRNA was obtained by fractionating a similar amount of labeled, total associated tRNA on an AX-20 column, as described above. After labeling the various RNA fractions, free 32pCp was removed from the labeled macromolecules either using G-50 Sephadex (Pharmacia) home-made spin columns, equilibrated with TE buffer (10 mM Tris, pH 7.5; 1 mM EDTA), or during the electrophoresis run. Before analysis by polyacrylamide electrophoresis, the samples were heated at 90°C for 2 minutes.

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One- and two-dimensional nolvacrvlamide ael electroohoresis (1D PAGE and 2D PAGE): Size classes of low molecular weight RNA were determined on DNA sequencing gels, using Ml3 sequence fragments as size markers. Ml3 sequence fragments were produced using the standard di-deoxy chain termination method of sequencing (23).

Electrophoresis of viral RNA was carried out at 4°C using the Hoeffer SE620 gel electrophoresis apparatus. Gel size was 14 cm x 32 cm. The first dimension was run in a 10% polyacrylamide/7 M urea gel for approximately 16 hours at 800 volts, until the bromophenol blue dye was beginning to elute from the bottom of the gel. After authradiography, the piece of gel containing RNA was cut out and embedded in a second gel (20% polyacrylamide (4 M urea) and run for 46 hours (25 watt limiting), followed by autoradiography. All electrophoretic runs were carried out in 0.5 x TBE (1 X TBE - 5 mM Tris; 5 mM boric acid; 1 mM EDTA-Na,). The electrophoretic gel patterns shown in this paper show only low molecular weight RNA, since the high molecular weight viral genomic RNA cannot enter the polyacrylamide gels. Furthermore, these patterns represent only the most abundant tRNA species present, since the high specific activities of the labeled tRNAs used will reveal many more minor abundance species with longer film exposures.

Detection of tRNALyS-isoaccentors using RNA-DNA hvbridization: To detect the presence of tRNALySr3 in viral RNA, we have synthesized an 18-mer DNA oligonucleotide complimentary to the 3' 18 nucleotides of tmALYS I 3. 5'TGGCGCCCGAACAGGGAC3'. This probe hybridizes specifically with tRNALYsr3 (Figure 5), and was hybridized either to dot blots of northern blots of RNA samples on Hybond N (Amersham). Dot blotting was performed using standard techniques (27). Northern blots were performed by first heating RNA at 65°C for 15 minutes in 1X ssc, 50% deionized formamide, 7.4% formaldehyde, cooling in ice for 5'. and adding the formaldehyde-dye loading buffer (27). Samples were electrophoresed at 100 volts for l-l.5 hours in a formaldehyde-agarose gel (1% agarose, 6.5% formaldehyde, 20 mM MOPS, pH 7.0, 8 mM sodium acetate, 1 mM EDTA, pH 8.0) in running buffer (20 mM MOPS, pH 7.0, 8 mM sodium acetate, 1mM EDTA, pH 8.0), and blotted to Hybond N. The DNA oligomers were 5'-end labeled using T4 polynucleotide kinase (24) and gamma- 32P-ATP (3,000 Ci/mmol, DuPont Canada), and specific activities 10s to 10' cpm/pg were generally reached. Approximately 10' cpm oligomer was generally used per blot in hybridization reactions.

RESULTS

1. tRNA populations in HIV-l vary with the cell type producing the virus.

Figure 1 shows a size class analysis of viral low molecular weight RNA in HIV-

1 produced in H9 cells. The RNA is electrophoresed in a 6% DNA sequencing gel

along with sequencing reactions for Ml3 DNA, which is used as an approximate size

marker. In the total low molecular weight RNA (lane C), three size classes can

be seen. The smallest size class is approximately the size of tRNA (66-93

nucleotides). There are also present RNA species similar in size to 5S RNA (124

nucleotides) and 7S RNA (183 nucleotides). The amount of these latter two

species varies according to the cell type producing the HIV-l, but is always

present. 7S RNA is not found in the free viral low molecular weight RNA, and

other data (not shown) indicate that this species is found entirely associated

with the viral genomic RNA, albeit more loosely bound than primer tRNA. In this

work, we refer to the tRNA-sized RNA species as tRNA because it has been shown

in other retrovirus that this RNA can be aminoacylated by different aminoacyl

tRNA synthetases (l), and also because we have found tRNALYsg3 in this viral RNA

size class (see below). The precise size of these molecules cannot be obtained

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183-

124-

go-

74-

Fiaure 1. Size classes of low molecular weight (1.m.w.) RNA in H9 cell-produced HIV-l,,,,. A. Sequence gel of Ml3 DNA, used as a size marker. B. Free 1.m.w. RNA. C. Total 1.m.w. RNA.

from this partially denaturing gel since tRNA molecules of similar size can have

different mobilities depending upon their degree of denaturation under these

conditions.

Figure 2 shows 2D PAGE patterns of tRNA isolated from HIV-l produced in 5 cell

types, as well as the viral tRNA found in mouse mammary tumor virus (MMTV).

There are 20 or more major tRNA species detected in virions produced continuously

in H9, UHCl, and UHC8 cell lines. There are also 15 or more tEWAs found in

virions transiently produced in HeLa cells transfected with the HIV-l,,,, genome.

However, the infectious virions transiently produced in CO.9 cells transfected

with the same HIV-l genome contain only 4 major tRNAs, indicating that the

majority of other tRNAs found in HIV-l produced in other cell types are not

necessary for viral replication or infectivity. This conclusion is not unexpected since it has previously been shown that infectious MMTV contain only

2 major tRNA species (9), and we have reproduced this observation (Figure 2,E).

Both MMTV and HIV-l are believed to use the same tRNA as primer, tRNALYSs3 (9,ll).

The pictures in Figure 2 show the tRNA species most abundant in the virus. In

all cases, longer film exposures reveal tENAs present in lower concentrations.

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Finure 2. 2D-PAGE of free tRNA of HIV-1 produced in different cell types. A) H9, 8) UHCl, C) UHCB, D) HeLa, E) COS, F) MMTV.

2. The pattern of tRNA tightly-associated with the HIV-1 genome varies with the

cell type in which the virus is produced.

Studies with other retrovirus have shown that the primer tRNA is the tRNA most

tightly-associated with the RNA genome, presumably due to base pairing with the

primer binding site. Figure 3 shows the 2D PAGE patterns fo tightly-associated

tRNA in HIV-l produced in various cell types. The number of major tightly-

associated species in HIV-1 are as follows: H9,3; UHC1,2; COS,l; HeLa,l. Figure

4A compares the electrophoretic mobility of pure tRNALYSv3 to that of the tightly

associated tRNA species found in HIV-l produced in H9 and HeLa cells. It is

clear that the tightly associated tRNA from virions produced in H9 cells has a

similar mobility to purified tRNALYSs3. The tightly associated tRNA species in

HIV-1 produced in HeLa cells does not migrate with human tRNALysJ3. (We have also

found that the tightly associated tRNA in virus produced in COS cells migrates

with purified tRNALY5*3 in 1D PAGE (data not shown).

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Figure 3. 2D-PAGE of tightly associated tRNA of HIV-lproduced in different cell types. A) H9, B) UHCl, C) COS, D) HeLa.

3. The putative primer tRNA, tRNALys*', is present in the free tRNA of HIV-L

produced in H9 cells, but not in tRNA of HIV-1 produced in HeLa cells,

We have developed an 18-mer DNA probe which specifically hybridizes with

tR.NALYsr3. The probe is complimentary to the last 18 nucleotides at the 3' end

of rabbit tRNALYs*3 (12), i.e., it has the same sequence as the primer binding

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Finure 4. Presence of tRNALyss3 in HIV-l. (A) lD-PAGE of tightly associated HIV-1 tEWA: 1) and 4) Purified human tFXALYs.3. (2) Produced in H9 cells. 3) Produced in HeLa cells. (B) Ethidium bromide-stained free tRNA electrophoresed in 1% agarose gels. (1) Produced in H9 cells. 2) Produced in HeLa cells. (C) Northern blots of the same RNA samples in (B), hybridized with the tRNALySt3 DNA probe. 1) Produced in H9 cells. 2) Produced in HeLa cells.

site in HIV-l. Figure 5 shows the specificity of this probe. It hybridizes only

to RNA containing tRNALySS3. The probe hybridizes to purified tRNALYSs3 and to the If tmALyS _ rich" fraction obtained during the purification of human placental

tRNALYS using DEAE cellulose chromatography. But the oligomer does not hybridize

to purified tRNALysS1*2, nor to the "tRNALYs-poor" fraction from the DEAE cellulose

column, nor to E. coli tRNAPhe. Using this probe, we have found (Figure 4) that

the tRNA of HIV-l lIIB produced in H9 cells contains tRNALYSe3, while the tRNA of

HIV-l,,,, produced in HeLa cells does not. This supports our previous observation

that tRNALYs,3 is not present in the tightly associated tRNA in these virus.

tRNA: A B

LYS (1,2)

EColi Phe

Figure 5. Specific hybridization of the tRNA Lys,3 DNA probe to RNA dot blots. The isolation of the RNA samples, dot blotting, and hybridization were performed as described in the text. A) 10 ng RNA. B) 100 ng RNA.

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DISCUSSION

The pattern of tRNAs found in HIV-l,,,, varies according to the cell type

producing the virus (Figure 2). The different tRNA patterns in virus produced

in HeLa and COS cells does not reveal any transfection-dependent pattern.

Previous reports have shown that COS cells transfected with the HIV-l genome

efficiently produce infectious virus (26). Because only 4 major tRNA species

are found in these virus, it is likely that many of the tRNAs incorporated into

virus produced in H9, UHCl, and HeLa cells are not necessary for viral

infectivity. This conclusion is further supported by the finding that MMTV

contains only 2 major tRNA species (Figure 2; (9.19)). Also, the mechanism by

which tRNAs other than tRNALYs are incorporated into HIV-l may be different than

that used to incorporate t17NALYs isoacceptors. Thus, the RT(-) mutant virions

produced in UHC8 are still able to incorporate tRNAs, but do not appear to be

able to incorporate the primer tRNA found in wild type virions (25).

Cell-dependent tRNA patterns are also found within the population of tRNA

tightly associated with the viral genome (Figure 3). Studies with other

retrovirus have found only one major tRNA species in this compartment, which has

been identified with the primer tRNA for reverse transcriptase. The variable

number of tightly associated tRNA species in HIV-1 (l-3) appears dependent on

the type of cell producing the virus, and could reflect a heterogeneity of post-

translational modification of tRNALYSv3. The tightly-associated tRNA species in

HIV-l produced in H9 cells has electrophoretic mobility similar to tRNALyss3

(Figure 4A). On the other hand the single tight-associated tRNA species in HIV-

1 produced in HeLa cells behaves quite differently. It does not have an

electrophoretic mobility resembling tRNALyss3, and tRNALySt3 cannot be detected in

the total tRNA population of HeLa cell-produced virions using the tRNALysr3

hybridization probe. Since these virus are produced by the reinfection of HeLa

CD4(+) cells, it appears that either a uniquely modified tRNALyse3 is used as the

primer tRNA, or a primer tRNA other than tRNALYs,3 is used. The identification

of these different tightly associated tRNA species in HIV-l, by RNA sequencing,

is now underway in one of our laboratories (IX).

This data provides information about the number of tightly associated tRNA

species found in a population of virus, but not in an individual virion. For

example, individual virus produced in H9 may contain only one of the three

tightly associated tRNA species found in the total virus population, and only

one of the tRNA species may prove functional as primer. This would mean that

the viral population contains both infectious and uninfectious virus. Therefore,

to understand more fully the significance of multiple tightly associated tRNAs

in HIV-l, it will be necessary to identify which tightly associated species can

be labeled in virions by the reverse transcriptase reaction.

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ACKNOWLEDGMENTS

This work was supported in part by grants from the National Health Research

Development Program, Health and Welfare Canada. M.A.P. is a Chercheur-boursier

of the Fonds de la recherche en Sante du Quebec. Thanks to Sandy Fraiberg for

assistance in preparation of the manuscript.

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Variable tRNA content in HIV-1IIIB

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