viral interference in hiv-1 infected cells

Reviews in Medical Virology Rev. Med. Virol. 8: 203–211 (1998)

Viral Interference in HIV-1Infected CellsMary Jane Potash and David J. VolskyMolecular Virology Laboratory, St. Luke’s-Roosevelt Hospital, Columbia University,432 West 58th Street, New York, NY 10019, USA

SUMMARY

The study of viral interference in HIV-1 infected cells has revealed several different meanswhereby infected cells resist superinfection. The most familiar of these, down-modulationof cellular receptors for virus, can be accomplished through the independent action of atleast three HIV-1 proteins. Both the principal viral receptor CD4 and the chemokinereceptors which serve as co-receptors are subject to down-modulation as a consequence ofinfection. Elucidation of the specificity of co-receptor utilisation by HIV-1 strains is anexciting, ongoing task which has opened new avenues to the understanding of viralreplication and pathogenesis. Novel routes to resistance to superinfection have beendiscovered during HIV-1 infection and their investigation may reveal new pathways tocontrol HIV-1 and the loss of immunological function with AIDS. ? 1998 John Wiley &Sons, Ltd.

Accepted 14 April 1998

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INTRODUCTION

Viral interference was defined historically as thephenomenon through which susceptible cells acquireresistance to retroviral infection through preinfection byvirus bearing the same envelope specificity.1 HIV-1infected cells may display this form of interference,2,3

however, other routes of interference also have beenshown to protect infected cells against subsequent HIVchallenge or superinfection.4–6 This review summarisescurrent findings on interference during HIV infectionwith consideration given to events involving theprincipal virus receptor, CD4; the viral co-receptorsCXCR4 and CCR5; and receptor-independentinterference.

INTERFERENCE

Interference is generally regarded as the consequence ofblocking the cellular receptor for virus by exogenous(early interference) or endogenous (late interference) viralenvelope glycoprotein.7,8 The ability to induce inter-ference to challenge by a related retrovirus formed thebasis for retroviral classification and was found to map tothe viral envelope glycoprotein gene.9 For cells infectedby at least some retroviruses, high level production ofenvelope glycoproteins can result in the formation ofintracellular complexes between envelope and its cellular

CCC 1052–9276/98/040203–09 $17.50? 1998 John Wiley & Sons, Ltd.

receptor, preventing the surface membrane display ofreceptor necessary for binding by exogenous virus.10

These observations were made in the absence of knowl-edge of the cellular receptor determining susceptibility toinfection.

HIV AND CD4

Early in their course, studies of viral and cellular receptorinteractions during HIV-1 infection were facilitated bythe identification of CD4 as the principal cellular receptorfor HIV-1. This identification depended upon severalconverging lines of research. AIDS was recognised first asa depletion of T lymphocytes of the helper/inducerphenotype which were distinguished by their expressionof the differentiation antigen, CD4.11 Susceptibility toHIV-1 could be conferred to certain resistant human cellsthrough expression of CD412,13 and the viral envelopeglycoprotein was shown to bind CD4.14 Selected anti-CD4 antibodies were shown to inhibit HIV-1 binding andinfection of CD4 bearing T lymphocytes.15–17 Thesefindings unambiguously identified CD4 as the majorcellular receptor for HIV-1. CD4 is the major receptor forHIV-2 as well and HIV-2 has a host cell range similar toHIV-1.18 However, HIV-2 has been shown to produc-tively infect CD4-negative lymphoid cells under specificconditions as discussed below. Certain CD4-negativehuman cells of neural origin are susceptible to HIV-1,but the course of infection differs significantly from thatin lymphocytes or macrophages19–21 and will not bediscussed here.

Abbreviations used: HI, heat inactivated; L-tropic, lymphotorpic;M-tropic, macrophage-tropic; UV, irradiated with ultraviolet light.

204 M. J. POTASH AND D. J. VOLSKY

HIV INTERFERENCE VIA CD4DOWN-REGULATION

Human T cells productively infected by HIV-1 in culturedown-regulate the surface membrane expression ofCD4.15–17,22–24 The extent of CD4 expression wasshown to be inversely proportional to the extent ofHIV-1 expression.17,22,24,25 Although other mechanismsmay also apply, one mechanism for this down-regulationis the retention of newly synthesised CD4 in the endo-plasmic reticulum in intracellular complexes withgp160.26–28 This intracellular compartmentalisation ofthe cellular receptor for virus, prohibiting surface mem-brane display, may explain the standard means of inter-ference during retroviral infection.10 One route ofresistance to HIV superinfection was shown to resultfrom this down-regulation of CD4. A set of T cell lineswhich chronically expressed HIV-1 or HIV-2, respect-ively, were tested for their susceptibility to superinfectionwith the distinct virus and for their display of surfacemembrane CD4.2 Consistent with their display of CD4,cells chronically infected with HIV-2 remained suscep-tible to HIV-1 superinfection. Conversely, HIV-1 infectedcells which had down-modulated CD4 were resistant toHIV-2 superinfection, but could produce HIV-2 upontransfection with proviral DNA. This phenomenon fulfilsthe criteria of viral interference in that susceptible cellsacquired resistance to viral infection at the level of virusentry.1 More recent studies using HIV-1 strains ofdifferent tropism reached a similar conclusion.3 Consist-ent with the ability to down-regulate surface CD4, HIV-1infected cells were protected from superinfection.Infected cells which failed to down-regulate CD4 weresusceptible to superinfection. It is noteworthy thatinfected cells which failed to down-regulate CD4 pro-duced less HIV-1 than cells which down-modulated CD4as a consequence of infection by more productive clonesof HIV-1.3 The expression of gp160 by susceptible Tcells in the absence of intact provirus was also found topartially down-regulate CD4 and rendered cells onlypartially permissive to HIV-1 challenge.29 This situationis reminiscent of the relative resistance to exogenousretroviral infection of certain strains of chickens whichendogenously express a related retroviral envelope.30

The dose dependence of interference was also illustratedin the latter study, in that the extent of the block toexogenous virus replication correlated well with theextent of envelope protein expression by chicken

30
embryos.
CD4 REGULATION VIA ENVELOPE

The dynamic relationships between CD4 expression,viral structural protein expression, and viral interferencehave been profitably studied in cells chronically infectedby intact HIV-1. In OM-10, an inducible chronicallyinfected cell line, CD4 expression and viral expressioncould be reciprocally regulated, because induction of viral

? 1998 John Wiley & Sons, Ltd.

expression reduced surface CD4 levels commensuratewith increased gp160 synthesis.31 CD4 absent from cellsurfaces could be accounted for by its appearance inintracellular complexes with gp160. This dose depen-dence may explain the surface phenotype of HIV-1infected cells in vivo; since CD4 expression is maintainedin HIV-1 infected cells in the blood, HIV-1 DNA is foundin the CD4-positive, but not the CD4-negative, sub-population of cells.32 Two features probably contributeto this finding. The majority of cells carrying HIV-1DNA probably do not express viral RNA and protein,33

and those cells which are expressing viral protein at anygiven time appear to have a very short lifetime in vivoand represent a minor fraction of cells scored as viralDNA positive.34,35 ACH-2, a chronically infected cellline, has been considered as one model for PBL whichcarry DNA but constitutively express little viral protein.These cells remain partially susceptible to superinfectionby exogenous virus, suggesting a link between viralprotein expression and the induction of interference.36,37

However, this dose dependence is not simply related tothe level of CD4 expression as discussed below.

CD4 REGULATION VIA OTHER HIV-1PROTEINS

In addition to HIV-1 envelope glycoproteins, the viralregulatory protein Nef and the accessory protein Vpuregulate expression of surface membrane CD4. Vpumodulates the efficiency of viral particle release, butis generally dispensable for HIV-1 replication.38,39

Independently of envelope, Vpu can induce intracellulardegradation of CD4 prior to transport to the mem-brane,40 in the presence of envelope this degradationmay actually facilitate transport and processing ofgp160.41 Nef, a viral protein required for pathogenesisin vivo,42 also stimulates CD4 down-modulation.43

ACH-2, despite its minimal expression of viral structuralproteins, appears to have down-regulated most of itssurface CD4,44 which may be a consequence of itsexpression of viral regulatory genes, including Nef.45 Incontrast to Vpu or envelope, Nef acts to internalise CD4resident on the membrane and promotes its degradationin lysosomes.43 HIV-1 or SIV Nef expression alone canreduce surface expression of CD4 in human cells andconcordantly protect cells from infection by HIV-1,46,47

indicating that Nef expression, under certain circum-stances, is sufficient to induce viral interference. Theseobservations have been unified in experiments whichassess the abilities of envelope, Nef, and Vpu alone or incombination to modulate surface expression of CD4.48

Each protein was able to down-modulate surface CD4expression during infection by HIV-1 constructs whichselectively expressed one or more of these genes plus amarker gene in an otherwise intact provirus. Consistentwith its expression early in HIV-1 replication, Nefinduced CD4 down-modulation prior to that induced byenvelope and Vpu. It is interesting that Nef had a greatereffect upon CD4 regulation in primary lymphocytes than

Rev. Med. Virol. 8: 203–211 (1998)

VIRAL INTERFERENCE IN HIV-1 INFECTED CELLS 205

it had in a transformed T cell line,48 which may reflectthe greater requirement for Nef in primary than intransformed cells.49,50

INTERFERENCE INDEPENDENT OF CD4MODULATION

One consistent theme of these findings is that the loss ofexpression of surface membrane CD4 by susceptible cellsleads to loss of their permissivity to exogenous HIV-1infection. However, CD4-independent mechanisms alsofunction in HIV-1 infected cells to limit superinfection.The spread of infection by DNA and RNA viruses isreduced by co-infection with defective-interfering par-ticles51,52 and defective clones of HIV-1 have beendescribed which block the ability of wildtype virus toreplicate efficiently. In one example, a defective HIV-1clone effectively competed with exogenous virus forinfection of susceptible cells in a primary or reinfectionformat.4 Another series of studies described a defectiveHIV-1 clone which upon introduction into susceptiblecells protected them against wildtype virus challenge.5,53

The mechanism of this form of interference has not beenfully defined but the CD4-negative cell line from whichthe virus was cloned showed defects in both viral entryand completion of reverse transcription upon superinfec-tion with a replication competent virus at a high multi-plicity.53 Interference could also be established withoutaffecting CD4 expression by transduction of the defectivevirus into susceptible cells.5

We have investigated the development of viral inter-ference during wildtype HIV-1 replication in transformed


and primary cells and found that they acquire resistanceto exogenous virus challenge 1–2 days after primaryvirus infection, at a time when CD4 expression is highand viral envelope synthesis is low.6 Reverse transcrip-tion of the primary infecting virus is necessary andsufficient for the establishment of this form of interferenceas shown in two experimental systems. We infectedmacrophages with N1T-A, a lymphotropic clone ofHIV-1 which synthesises viral DNA but not protein inmacrophages54 and attempted to superinfect the cellswith macrophage-tropic ADA (Figure 1). Cells exposedto biologically active N1T-A, but not heat-inactivatedN1T-A, were resistant to productive infection by ADA.In addition, we were able to protect T cells againstsuperinfection by primary infection in the presence of theTat inhibitor Ro 5-3335, which also permits reversetranscription but not viral protein expression.55 In thiscase, infection and superinfection were monitored byviral DNA synthesis using strain-specific primers (notshown). These studies demonstrate that infection byintact or defective HIV-1 can induce interference bymeans other than down-modulation of the principal virusreceptor, CD4.

Figure 1. Induction of interference by non-productive infection of primary macrophages. Primaryhuman macrophages were exposed to biologically active or inactivated lymphotropic HIV-1/NIT-A as indicated and were later infected with monocytotropic HIV-1/ADA as indicated. HIV-1replication was measured by core antigen p24 production 10 days after ADA infection.

FUSION CO-RECEPTORS

More than a decade ago, CD4 was shown to benecessary but not sufficient for HIV-1 binding andentry.12 Vesicular stomatitis virus-HIV-1 envelopepseudotypes failed to replicate in murine cells despitetheir expression of human CD4. This failure was postu-lated to arise from the absence of a human membrane

Rev. Med. Virol. 8: 203–211 (1998)


protein which was required in addition to CD4 for HIV-1entry into cells. A plethora of recent studies haveidentified a set of other cellular receptors which servewith CD4 as co-receptors for HIV.56–61 These serpentinereceptors belong to the chemokine receptor family andare found, in varying amounts, on resting and activatedlymphocytes and macrophages, among other cells.62–64

The first of these co-receptors to be identified, fusin orCXCR4, is recognised by gp120 from laboratory adaptedlymphotropic (L-tropic) clones of HIV-1. It was identifiedby evaluating the ability of human cDNA clones toconfer susceptibility to HIV-1 fusion to human CD4-positive murine cells.56 CXCR4 is displayed on cellswhich can be productively infected by these virus strains,including primary lymphocytes and many transformedcell lines.56,61 The second major co-receptor, CCR5,recognises gp120 from many macrophage tropic(M-tropic) strains and is found on lymphocytes andmacrophages.57,58,60 Other chemokine receptors whichserve as HIV-1 co-receptors have also been classifiedin terms of their recognition of M-tropic or L-tropicenvelopes.61,62 These primary definitions of co-receptorutilisation by HIV and SIV strains have been used as thebasis for a new classification system for primate immuno-deficiency viruses.65 Of particular clinical significance,individuals lacking functional CCR5 are generally resist-ant to HIV-1 infection and both their lymphocytes andmacrophages maintain this resistance to HIV-1 infectionin vitro.66–68 A parsimonious model based on thesefindings states that the tropism of an HIV-1 strain isdetermined by its gp120 binding of distinct viralco-receptors on specific host cells.57 If persons or specificcell types lack this co-receptor they are resistant to virusentry and infection, a form of inherited interference.

Although attractive, this hypothesis is not fully sup-ported at the cellular level. The major discrepancyconcerns the principles governing the ability of strains ofHIV-1 to productively infect primary macrophages.Studies of chemokine receptor use as HIV-1 fusionco-factor were conducted under the assumption thatsurface display of a particular co-receptor together withCD4 should render human cells permissive to HIV-1replication.56–61 The majority of these studies employednon-human transformed target cell lines which expresshuman CD4 and one of several chemokine receptors,conferred by transfection.56–61 The implication of thisview is that the major determinant of HIV-1 host cellrange is efficient virus entry into cells. Since thelaboratory-adapted L-tropic viruses use exclusivelyCXCR4 as a co-receptor in the non-human assay systemdesigned56–59 and fail to infect macrophages as a functionof their V3 region in envelope,69,70 it was proposed thatmacrophages lack surface membrane expression ofCXCR4.57 However studies of chemokine receptor dis-play uniformly report that CXCR4 is present on humanmacrophages.63,64,71 The hypothesis was revised to statethat CXCR4 on macrophages is non-functional for virusentry,72 but this proposal was refuted in a particularlyinformative report.67 HIV-1/89.6, like many primaryisolates, can bind and enter cells through CD4 together


with either CCR5 or CXCR4,67 and can productivelyinfect macrophages.73 Macrophages carrying thehomozygous deletion in CCR5 were shown to be infect-able by 89.6 and this infection was shown to utilise the12G5 determinant on CXCR4, indicating a display ofCXCR4 on macrophages similar to that on lym-phocytes.63 This study clearly demonstrates that CXCR4can mediate efficient HIV-1 entry into macrophages. Inanother study investigating the functionality of CXCR4on macrophages, clones were isolated from a transformedmacrophage cell line which were either susceptible orresistant to fusion with L-tropic HIV-1.74 However therewas no correlation between display of CD4 or CXCR4and permissivity to L-tropic clones.

Examining the converse case, that is the elementsnecessary for efficient HIV-1 infection of macrophages,one might expect that any infectious HIV-1 which usesCD4 and CCR5 for entry would infect macrophagesbecause macrophage tropic strains use CD4 andCCR5.57,58 However, two studies using related virusesof different tropism described replication competentclones which utilise CCR5 as a co-receptor in cell linesbut fail to productively infect macrophages.72,75 Onesimple explanation of these findings is that certain strainsof HIV-1 bind, fuse, and enter primary macrophages butfail in a post-entry phase of replication, as we and othersoriginally showed.54,76,77 It is worth noting that theinteraction of HIV-1 gp120 with CCR5 can activatecells,78 raising the possibility that signals transduced byspecific co-receptors may alter the intracellular milieu toaffect the subsequent efficiency of HIV-1 replication in aparticular host cell type.

EFFECTS OF CHEMOKINES

Another level of complexity regarding the strain-specificcontrol of HIV-1 infection concerns the use of commoncellular receptors by HIV-1 and specific chemokines. Priorto the identification of specific chemokine receptors asfusion co-factors, certain â-chemokines produced by CD8bearing lymphocytes were shown to inhibit M-tropicHIV-1 infection.79 It was suggested that the resistanceto HIV-1 of certain primary CD4 bearing T cell clonesmay arise from their constitutive synthesis of thesechemokines.58,80 Later studies demonstrated that thesecells carried the homozygous deletion in CCR5, abrogat-ing expression of intact protein.66 However, these resultsfrom isolated cell culture systems raise the possibility thatchemokine imbalances arising from the absence of theirreceptor may contribute to the HIV-1 resistance ofindividuals carrying the CCR5 deletion, a congenitallimitation of virus susceptibility currently attributed tothe absence of a receptor. Moreover, there are con-siderable discrepancies regarding the ability of the âchemokines which inhibit HIV-1 infection of lym-phocytes to inhibit infection of macrophages.81–84 Thismay reflect virus strain specific differences in the particu-lar utilization of CCR5, as has been reported forCXCR4,71 rather than a cell type specific difference in the

Rev. Med. Virol. 8: 203–211 (1998)

superinfection that we observe.


receptor conformation between lymphocytes andmacrophages. As in the case of the â chemokines SDF-1,the natural ligand for CXCR4, can inhibit HIV-1 infec-tion.85,86 A chemokine has been identified which blocksinfection of both lymphocytes and macrophages andblocks viruses which utilise either CXCR4 or CCR5.87

The cellular receptor(s) for this factor, macrophage-derived chemokine (MDC), has not been identified. It isnoteworthy that MDC fails to inhibit infection of atransformed T cell line in which both CXCR4 and CCR5are displayed, suggesting that the receptors are displayeddifferently on transformed cells, or that both L-tropic andM-tropic HIV-1 can utilise a third form of cellularreceptor, or that MDC operates at a level different from

54,76,77
virus entry.
FUSION CO-RECEPTORDOWN-REGULATION

The description of co-receptors which bind gp120 raisesthe possibility of an alternative route to viral interferencemediated by down-regulation of the co-receptor ratherthan the principal receptor, CD4. This possibility hasbeen examined in an experimental system in which anHIV-2 strain was selected for its ability to infect CD4-negative cells in which CXCR4 served as the primaryviral receptor.88 High level virus production wasaccompanied by CXCR4 down-modulation from the cellsurface.88 The involvement of a secondary receptor wasalso postulated during investigation of the cross-interference properties of feline leukaemia virusenvelopes.89 Envelope proteins which were shown tobind and precipitate the same cellular protein were foundto be unable to induce cross-resistance to virus challenge.The authors interpreted these results to suggest thatinterference in this system depended upon the availabilityof an unidentified second receptor.89 Because the inter-ference established during wildtype HIV-1 infection weobserved is independent of CD4 down-modulation6 weconsidered the possibility that it might involve down-modulation of a co-receptor. We investigated CXCR4expression following L-tropic HIV-1 infection of CEM


cells (Figures 2 and 3) under the conditions we haveestablished to induce post-entry interference.6 Theexpression of RNA encoding CXCR4 by CEM cells wasnot altered within 6 days of infection as determined byPCR amplification of cDNA (Figure 2). To verify themaintenance of CXCR4 protein we also examined cellsurface CXCR4 in flow cytometry using monoclonalanti-CXCR4 antibody, 12G5.88 Despite the resistance ofthese cells to superinfection 1 day after infection,6 theirsurface CXCR4 levels were comparable to those ofuninfected cells (Figure 3) as we previously reported forexpression of CD4.6 These findings rule out co-receptordown-modulation as the mechanism of resistance to

6

CONCLUSIONS

In summary, normally susceptible cells can be renderedresistant to HIV-1 infection by a variety of means.Classical viral interference involving receptor down-modulation obtains in the case of CD4 or CXCR4down-modulation in HIV infected cells. á and â chemo-kines can compete with HIV-1 for cellular receptors,effectively blocking virus entry and replication. Infectionby certain defective HIV-1 clones can protect cellsagainst superinfection by replication competent virus, butthe mechanism(s) responsible for protection remain to bedefined. We have shown that HIV-1 DNA synthesis issufficient to protect lymphocytes and macrophages fromproductive virus infection and that such resistant cellsmaintain surface CD4 expression. These distinct routes toHIV-1 inhibition and interference offer promise for thedevelopment of new therapies for HIV infection. Thechallenge remains to induce changes in cellular proteinexpression which alter their susceptibility to infectionwithout altering their immune function.

Figure 2. Expression of mRNA encoding CXCR4 by CEM cells after HIV-1 infection.CEM-cells were infected with HIV-1/KS283 exactly as described6 and harvested at thetimes indicated after infection for amplication of CXCR4 by reverse transcription PCR.cDNA levels were standardised by amplification of the cellular â-actin transcript, cDNAis visualised by ethidium bromide staining.

ACKNOWLEDGEMENTS

This work was supported by PHS grants AI 35466 andHL 43628.

Rev. Med. Virol. 8: 203–211 (1998)


Figure 3. Expression of cell surface CXCR4 by CEM cells after HIV-1 infection. CEM cells were infected withHIV-1/KS283 exactly as described6 and were subjected to staining with anti-CXCR488 under standard conditions.Panel A shows staining of uninfected cells, panel B shows staining of cells 24 h after infection when interference hasbeen established.6 The open curve shows staining by control mouse IgG, the filled curve shows staining bymonoclonal anti-CXCR4.

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