* Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland; and
Science Applications International Corporation-Frederick, Inc., Frederick, Maryland
1Correspondence: Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892. E-mail: malm{at}nih.gov
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) following the irreversible elimination of CD4+ T lymphocytes by highly pathogenic SHIVDH12R. The envelope glycoproteins carried by plasma virus in CD4-depleted animals were found to contain specific alterations affecting the V2 region of gp120; similar V2 changes were observed during independent monkey infections. The altered V2 loops contained double amino acid deletions and the loss of a highly conserved N-linked glycosylation site. In contrast to the starting highly pathogenic SHIV, which is exclusively T cell-tropic, some m-phase SHIVs, bearing altered V2 regions, were able to establish spreading infections of cultured alveolar m.
Key Words: immunodeficiency • in situ hybridization • viral load • lymphocyte depletion • HIV env • V2 loop
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), a property appreciated early in the epidemic when virus-producing multinucleated giant cells were detected in the brains of infected persons [1
, 2
]. In vivo, infected m are considered to be a significant reservoir of HIV-1 because of their reported resistance to the cytopathic effects of the virus and their intrinsically long lifespan.
Unlike circulating CD4+ T cells, HIV-1-producing tissue m have been difficult to study by noninvasive techniques. Consequently, very little is known about the dynamics of virus production in cells of this lineage in vivo and whether circulating monocytes or more differentiated tissue m are the initial targets of infection. As a result, investigators have turned to an in vitro surrogate, the monocyte-derived m (MDM), to investigate parameters affecting the susceptibility of m to HIV-1.
We and others have reported that highly pathogenic simian immunodeficiency virus (SIV)/HIV-1 chimeric viruses (SHIVs) cause an extremely rapid (3–5 weeks), irreversible, and systemic depletion of CD4+ T lymphocytes following intravenous (i.v.) inoculation of rhesus monkeys [3
4
5
]. Despite the absence of significant numbers of CD4+ T cells, SHIV-infected animals survive for an additional 4–6 months and continue to generate high levels of plasma virus (>1x106 RNA copies/ml) [6
]. In situ hybridization (ISH) coupled with confocal immunofluorescence have demonstrated that m in a variety of tissues sustain high plasma virus loads in the absence of CD4+ T lymphocytes [7
]. Thus, the SHIV/macaque system has the potential of serving as a useful in vivo model of m infections, permitting a further understanding of the processes of virus entry and dissemination, disease induction, and sensitivity to antiretroviral agents when the main source of progeny virus production is tissue m.
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Env gene polymerase chain reaction (PCR)
Extraction of viral RNA from rhesus monkey plasma and reverse transcription (RT) were conducted as described [10
] using 9493(–) 5'-GCGAGTATCCATCTTCCACCTCTC-3' as cDNA primer. A 3-kbp fragment, encompassing the entire HIV-1DH12 Env coding region, was PCR-amplified using the following set of nested primers: 6428(+) 5'-GAGCCAGTAGATCCTAGACTAGAGCCC-3' and 9493(–) as outer primers and the previously designed primers 6317(+) and 9190(–) as inner primers as previously reported [3
].
V2 length analysis
A 181-bp fragment, encompassing the V2 region of gp120, was amplified by nested PCR using one of the primers in the second round of PCR labeled with the 6-carboxy-fluorescein (6-FAM) fluorophore at its 5' end [11
]. The fluorescently labeled PCR-amplified products were separated on an ABI 377 DNA sequencer (PE Applied Biosystems, Foster City, CA) and were sized with GeneScan 3.1 software (PE Applied Biosystems). The amino acid (aa) length of the V2 loop was calculated by subtracting 61 bp, representing amplified constant regions outside of the V2 loop, from the measured nucleotide fragment length obtained from the gel analysis.
Recombinant viruses
Specific V2 changes were introduced into pSHIVDH12R-CL-7 [12
] by site-directed mutagenesis using the QuickChange protocol (Stratagene, La Jolla, CA) as described [13
]. Virus stocks were prepared by transfecting plasmids into RD cells as previously reported [13
].
Virus replication in monkey alveolar m (AM)
Rhesus macaque AM were collected from uninfected rhesus macaques by bronchoalveolar lavage and were cultivated as described previously [14
]. Approximately 4 x 105 AM were resuspended in Dulbecco’s minimum essential medium (400 µl) and plated in LabTek II glass chamber slides (Nalge Nunc International, Rochester, NY). Following medium changes on days 1 and 2, the adherent cells were immediately infected with virus [normalized by RT activity (1x107 32P cpm)] as indicated in the text. In each case, the infected cultures were monitored for up to 25 days with complete medium changes every other day. Progeny virus production was assessed by the RT activity in the culture supernatants as described previously [3
].
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Figure 1. The genomic organizations of HIV-1, SIV, and SIV/HIV chimeric viruses. The parental, dual-tropic, primary isolate HIV-1DH12 (filled bars) and the pathogenic molecular clone of SIVmac239 (open bars) are shown at the top. The arrowheads on the SHIVDH12R genome indicate env gene changes accompanying the acquisition of the highly pathogenic phenotype, which appeared during a single passage in rhesus monkey Rh565Z. LTR, Long-terminal repeat.
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Figure 2. The emergence of highly pathogenic SHIVDH12R and SHIVDH12R-PS1 during a single passage of the nonpathogenic SHIVDH12 in rhesus monkey 565Z. The times of anti-human CD8 antibody administrations and virus inoculation are indicated. SHIVDH12R and SHIVDH12R-PS1 were recovered from peripheral blood mononuclear cells (PBMCs) and lymphoid tissue suspension collected at weeks 68 and 52, respectively. The SHIVDH12R-CL–7 molecular clone was generated from the cells infected with SHIVDH12R-PS1.
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Figure 3. Plasma viral load and peripheral blood CD4+ T-lymphocyte profiles of SHIVDH12R-infected animals. The four macaques were i.v. inoculated with the indicated amounts of SHIVDH12R. , Times of euthanasia. TCID50, Tissue culture infectious dose(50).
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The extremely rapid SHIV-induced depletion of CD4+ T lymphocytes and virus production in tissues was examined systemically by serially sacrificing SHIVDH12R-infected rhesus monkeys between days 3 and 21 postinfection. DNA PCR analyses demonstrated proviral DNA in lymph nodes on day 3 postinfection; by day 14, 2–5% of lymph node cells were producing SHIV DNA [9
]. CD4+ T cells, located in paracortical areas of lymph nodes and detected by IHC staining of formalin-fixed tissue, rapidly declined between days 10 and 14 postinoculation (Fig. 4a
and 4b
). Confocal microscopic analysis of a day-10 specimen, using a riboprobe recognizing SHIV sequences and an anti-CD3 mAb, revealed that the virus-producing cells were, in fact, T lymphocytes (Fig. 4c
4d
4e)
. No reactivity with the m-specific HAM56 mAb was detectable by ISH in samples collected during the first 3 weeks of infection (not shown). Virus production during the acute infection, monitored by DNA PCR and ISH increased systemically, peaking somewhat earlier in lymphoid tissues (days 10–14) than in PBMCs (days 14–21).
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Figure 4. Infection and depletion of CD4+ lymphocytes in the mesenteric lymph nodes of SHIVDH12R-infected animals. CD4 IHC staining of the lymph node samples collected from animals killed on days 10 (a) and 14 (b) postinfection. Confocal fluorescence microscopy of mesenteric lymph node samples from a monkey killed on day 10 postinfection using a virus-specific riboprobe (c) and an anti-CD3 mAb (e). A merged image is shown in d. Original magnifications: a, x5; b, x4; c–e, x63.
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were present throughout a mesenteric lymph node specimen. These cells were significantly larger than the background nonreactive cells, presumably of lymphocyte lineage. The precise identification of the lymph node cell type actively producing SHIV RNA was determined by confocal immunofluorescence microscopy, using a riboprobe specific for viral RNA sequences and mAb reactive with m or T lymphocytes. The results of this analysis indicated that virtually all of the ISH-positive cells stained with the m-specific mAb (Fig. 6a
6b
6c
), whereas minimal colocalization was observed when the anti-CD3 mAb was used (Fig. 6d
6e
6f)
. Quantitation of this and similar confocal analyses indicated that >95% of virus-positive cells were tissue m. In some lymph node specimens, up to 25% of the HAM56-reactive cells were producing SHIV RNA (not shown).
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Figure 5. Viral RNA production during the m phase of SHIVDH12R infection. In situ RNA hybridizations were performed on tissues collected during the m phase of infection. a, Mesenteric lymph node (original magnification, x10); b, lung (x10); c, cerebellum (x20); and e, liver (x20). (d) Histosection adjacent to c, stained with the m-specific mAb, HAM56.
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Figure 6. Tissue m are the major virus-producing cells during the m phase of SHIVDH12R infection. The mesenteric lymph from a late-stage animal node was subjected to confocal microscopy-based in situ RNA hybridization/immunohistochemistry. (a and d) Viral riboprobe hybridization; b, m (HAM56) immunohistochemistry; e, T lymphocyte (CD3) immunohistochemistry; c, merged image of a and b. Note that colocalization of the signals detected in a and b merge to yellow in c; f, merged image of d and e. Original magnification, x40.
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. Multinucleated cells, with a morphology characteristic of AM and producing viral RNA, were present in histosections of the lung (Fig. 5b)
. Viral RNA was detected in cells having morphological features of m/microglia in various parts of the central nervous system (CNS). These virus-positive cells were scattered throughout the brain parenchyma and were not necessarily located in perivascular regions. A cerebellum sample, collected at week 22 postinfection, contained cells that stained with the SHIV riboprobe and the HAM56 mAb (Fig. 5 c and 5d)
. Flat cells, lining sinusoids of the liver and exhibiting the morphology of Kupffer cells, were also virus-positive (Fig. 5e)
.
As noted earlier, SHIV-positive tissue m were not detectable by ISH during the peak of virus production between weeks 1 and 3. However, when it became apparent that plasma viremia gradually increased after weeks 4–5 postinfection (Fig. 3)
, lymph node specimens were re-examined by confocal fluorescence microscopy to ascertain the types of virus-producing cells maintaining the high virus loads. As shown in Figure 7
, SHIV-positive T lymphocytes and m were present in the sample collected at week 6 postinfection. Taken together, these results indicate that the initial burst of virus production in inoculated rhesus monkeys occurs in CD4+ T lymphocytes (T cell phase of infection), and the high levels of viremia measured following the systemic depletion of CD4+ T cells are sustained by tissue m (the m phase of infection). It is likely that some m also become infected during the T cell phase but only become detectable as the number of CD4+ T lymphocytes markedly declines.
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Figure 7. The transition from the T cell to the m phase of the SHIVDH12R infection. An inguinal lymph node, biopsied from a SHIVDH12R-infected animal at 6 weeks postinfection, was subjected to confocal fluorescence microscopy using a riboprobe recognizing SHIV sequences (a and d, visualized in green) and immunostaining specific for CD3+ T lymphocytes (c, visualized in red) or m (f, visualized in red). Merged images of a and c (b), or d and f (e). Original magnification, x63.
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phase [12
]. For this purpose, a total of seven rhesus monkeys were infected with virus: five with SHIVDH12R and two with SHIVDH12R-PS1 (Fig. 2)
. All seven animals produced high levels of plasma viral RNA (>107 copies/ml) within 2 weeks of infection and experienced irreversible depletion of their CD4+ T cells (data not shown). Complete env gene segments (3 kbp in size) were RT-PCR-amplified from plasma samples obtained at week 2 (the T cell phase) and following the systemic loss of CD4+ T lymphocytes (the m phase) between weeks 12 and 35, depending on the animal. Independent PCR clones from the two time points were sequenced for each of the infected macaques. Although both of the uncloned virus stocks used for animal inoculations were genetically heterogeneous, sequence analysis indicated that at the time of peak plasma viremia (week 2 postinfection), they had become quite homogeneous (data not shown). The consensus env gene sequence at week 2 was similar to a recently obtained molecular clone of SHIVDH12R, designated SHIVDH12r-CL-7 [12
], which also induces rapid and irreversible CD4+ T cell loss in rhesus monkeys.
RT-PCR and sequence analyses of plasma viral RNA, collected during the m phase of independent SHIV infections in seven animals, were remarkable in that the only consistent changes identified within the entire Env coding region were located in the V2 domain of gp120. The aa sequence of some of these altered V2 loops are shown in Figure 8
(upper) compared with the consensus SHIVDH12R V2 sequence present in plasma virus during the T cell phase of infection. The predominant and unexpected finding was the presence of double aa deletions mapping to two specific regions of the V2 loop (residues 164–165 and 186–187). Among this "M" (m) series of V2 regions, one (M-4) contained both sets of double aa deletions. Several of the m-phase V2 segments had also lost a potential N-linked glycosylation site (at position 197) in addition to the 2 or 4 aa deletion.
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Figure 8. Replication of m phase-derived viruses in monkey AM. The aa sequence of the V2 regions from representative, independent RT-PCR, full-length, m-phase env genes is shown (upper). Dashes indicate aa identity; dots denote deleted residues. The positions of deleted aa are indicated (lower); aa changes resulting in a loss of the N-glycosylation site at residue 197 are boxed. Site-directed mutagenesis of SHIVDH12R-CL-7 was used to prepare the M series of m-phase SHIVs containing the indicated V2 changes. The infectivity of three of these SHIVs (indicated by shaded circles) was tested in rhesus AM (lower). Rhesus monkey AM (5x105 cells) were infected with  8000 TCID50 of each virus stock, and progeny virus production was monitored for 23 days by the RT activity released into the culture fluid. CPM, Counts per minute.
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phase were smaller in size (Fig. 9
, right panels). In three of the monkeys (181A, 5980, 96N093), the 2-aa deleted V2 variant was the only species detected. Together with the sequence data presented in Figure 8
, these results confirm the emergence of m-phase SHIVs with shortened V2 loops.
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Figure 9. V2 loop-length polymorphism analyses of plasma virus present during the T cell and m phases of SHIVDH12R infections. The CD4+ T lymphocyte counts (solid) and plasma viral RNA (dashed) profile from a representative infection are depicted (upper). The gp120 V2 regions from five SHIVDH12R and two SHIVDH12R-PS1-infected animals were subjected to the length polymorphism analysis. Note that the V2 region during the T cell phase of infection in the SHIVDH12R stock and during the T cell phase is 40 aa in length.
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-phase gp120 V2 regions shown in Figure 8 (upper) were able to infect cultured monkey PBMCs, generating peak levels of p27 ranging from 30 (M-11) to 300 (M-14) ng/ml by day 8 postinfection. The possible acquisition of m tropism by SHIV variants emerging in monkeys with markedly depleted numbers of CD4+ T cells was examined by measuring their infectivity in rhesus AM, collected by bronchoalveolar lavage from uninfected macaques. As controls for this experiment, the m-tropic SIV strain, SIVmac316 [25
], exhibited robust replication kinetics in AM, whereas the T cell-tropic SIVmac239 failed to generate detectable progeny virions (Fig. 8
, lower). The highly pathogenic SHIVDH12R was also unable to infect macaque AM (not shown). In contrast, three of the m-phase SHIVs (M-3, M-4, and M-11) readily established infections in rhesus monkey AM; in a similar experiment, SHIV M-14 failed to replicate in AM (data not shown). |
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[7
]. During the primary infection, virus production occurs almost exclusively within CD4+ T cells. As this lymphocyte subset is eliminated, a second wave of viral progeny is generated, this time in tissue m. In one animal, at least, virus-producing T lymphocytes and m could be demonstrated in an inguinal lymph node biopsied at 6 weeks postinoculation (Fig. 7)
, marking the transition to the m phase of infection. At later times, tissue m become the predominant source of viremia, releasing large amounts of progeny virions into the plasma that reach levels of 106–107 RNA copies/ml (Fig. 3)
.
A surprising feature of the SHIV/rhesus monkey model was the emergence, during the transition from the T cell to the m phase of infection, of viral variants bearing similarly deleted gp120 V2 regions. The same double aa V2 deletions appeared during independent infections of seven macaques (Figs. 8
and 9)
[10
]. It is quite likely that in the absence of CD4+ T lymphocytes, the capacity to infect tissue m is strongly selected for in vivo. Although it is not presently clear why specific alterations of V2 would facilitate entry into m, it is known that the V1 and V2 loops of HIV-1 gp120, situated on the surface of the trimeric envelope complex, partially occlude the CD4 and chemokine coreceptor-binding sites of gp120 [26
]. The conformational changes affecting the viral envelope glycoprotein during the binding of virions to CD4 on the surface of target cells repositions the common V1/V2 stem and exposes the high-affinity gp120-binding site for the coreceptor [26
27
28
29
30
]. It is therefore possible that a deletion in V2, in combination with the loss of the highly conserved glycosylation site at residue 197 (Fig. 8)
, creates a SHIVDH12R gp120 conformation with greater affinity for CD4 and/or the chemokine coreceptor.
It is now known that the expression of CD4 and chemokine receptor is modulated, depending on the activation or differentiation status of the cell. The low levels of CD4 on the surface of freshly isolated human monocytes increase significantly during their differentiation in culture into MDM [31
, 32
]. The expression of CD4 on the surface of human and rhesus monkey AM has been reported to be extremely low-to-undetectable and does not change during in vitro cultivation [33
, 34
]. The capacity to infect AM may therefore be limited to virions carrying unique gp120s, which are able to mediate fusion with cells expressing very little surface CD4 and chemokine receptor. The use of AM, rather than MDM, may represent a more rigorous measure of m tropism, possibly identifying SHIV env gene variants with high affinities for CD4. Nonetheless, SIVmac316 and three of four m-phase SHIVs replicated in AM, whereas SHIVDH12R, which induced rapid and irreversible elimination of CD4+ T cells, did not. SHIVDH12R appears to be exclusively T cell-tropic.
Studies are currently in progress to assess which gp120 determinants confer the m-tropic properties to late-stage SHIVs. This is an important issue, as such viral envelope elements could direct the incoming virus into specialized body compartments such as the CNS. The nucleotide sequence and infectivity data obtained thus far suggest that the double aa deletion affecting residues 164/165 but not 186/187 is critical for m tropism, as SHIVs M-3, M-4, and M-11 were able to infect AM, whereas SHIV M-14 was not (see Fig. 8
). The three m-tropic SHIVs, in addition, have lost the highly conserved glycan at position 197 compared with the exclusively T-tropic SHIVDH12R. Additional experiments, examining, for example, SHIV M-5, which is deleted at residues 164/165 but has retained the downstream glycosylation site (see Fig. 8
), could reveal the relative contributions of each of these V2 motifs for m tropism. The functional implications of gp120s with aa deletions or the loss of an N-linked glycosylation site affecting V2 are presently unknown. The receptor- and coreceptor-binding properties of gp120s containing specifically altered V2 regions are in the process of being evaluated.
Preliminary results have recently been obtained indicating that the starting CD4+ T cell-depleting SHIV as well as the m-tropic SHIVs, which emerge later, use CXCR4 not CCR5 for infection of rhesus macaque PBMCs (T. Igarashi et al., in preparation). In those experiments, small molecule competitors that target specific chemokine receptors were used to block spreading virus infections. For HIV-1, infection of MDM is usually associated with CCR5 use. Nonetheless, some dual-tropic HIV-1 strains have been shown to enter MDM by using CXCR4, and a recent isolate from a CCR5
32 homozygote has been reported to use CXCR4 during MDM infections [35
36
37
38
]. Thus, similar to SIV, SHIVDH12R does not change coreceptor use as a consequence of acquiring tropism for m [39
, 40
]. The T-tropic SIVmac239 and m-tropic SIV use CCR5, and the T-tropic SHIVDH12R and the m-tropic SHIVs M-3, M-4, and M-11 use CXCR4 during productive infections of rhesus PBMCs. Analogous-blocking experiments are planned to assess coreceptor use of the m-tropic SHIVs during infections of AM.
Received May 1, 2003; accepted July 10, 2003.
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Gly) at codon 69 J. Virol. 74,10958-1096432 exhibits dual tropism by infecting macrophages and MT2 cells via CXCR4 J. Virol. 76,3114-3124This article has been cited by other articles:
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