Elsevier

Virus Research

Volume 94, Issue 2, August 2003, Pages 103-112
Virus Research

The effects of HHV-8 vMIP-II on SIVmac251 infection and replication competent and incompetent SIVmac239Δ3 vectors

https://doi.org/10.1016/S0168-1702(03)00138-2Get rights and content

Abstract

Human herpesvirus type 8 vMIP-II has one of the broadest ranges of chemokine receptor binding and therefore a multiplicity of biologic effects, both immunologic and antiviral. These properties make vMIP-II an attractive effector gene to be expressed from gene therapy vectors. The present studies were concerned with both therapeutic approaches: (1) an anti-simian immunodeficiency virus (SIV) biologic, and (2) an effector gene in SIV-based vectors. Regarding its antiviral properties, vMIP-II expressed from bacteria and SIV-based vectors bound the surface of CEMx174 cells and specifically suppressed SIVmac251 infection. A CCR3 monoclonal antibody partially inhibited vMIP-II binding, suggesting that both SIVmac251 and vMIP-II utilize a similar CCR3-like receptor for CEMx174 cell binding. Replication competent SIV-based vectors containing forward and reverse vMIP-II produced neither identifiable vMIP-II nor virions for the first 21 days. Virus replication occurred after this period. Significant sequence alterations in the forward vMIP-II containing replication competent vector transcripts were responsible for the failure of vMIP-II expression. The genetic basis for the initial failure to replicate virus and its later restoration was not determined but appeared in the II-PIMv containing vectors to coincide with deletions and compensatory rearrangements in nef 3′ of the polypurine tract. Cells transfected with SIVmac239Δ3ΔLTR-vMIP-II vectors expressed biologically active vMIP-II that bound CEMx174 cells and suppressed SIVmac251 infection. These data suggest that replication defective SIV vectors expressing immunobiolgic genes such as vMIP-II may prove useful in gene therapies, particularly in augmenting immune responses in chronically infected individuals.

Introduction

Human herpesvirus, type 8 (HHV-8) contains several chemokine-like genes, one of which is macrophage inflammatory protein, type II (vMIP-II). VMIP-II is around 40% analogous to human macrophage inflammatory protein, type Iα in amino acid structure (Moore et al., 1996, Dittmer, 1998). Interestingly, vMIP-II has the broadest known range of receptor binding of any known chemokine (LiWang et al., 1999a, LiWang et al., 1999b, Luttichau et al., 2000), thus endowing it with a wide range of biologic activities. The role of vMIP-II on host immune response and HHV-8 persistence has been of primary interest (Lalani and McFadden, 1999). The immunomodulatory properties of vMIP-II have been used to prolong cardiac allograft survival in mice (DeBruyne et al., 2000). Studies of vMIP-II have also concerned an additional biologic property, its ability to inhibit HIV-1 infection (Boshoff et al., 1997, Kledal et al., 1997, Kanbara et al., 2000, Luo et al., 2000, Luttichau et al., 2000, Zhou et al., 2000). VMIP-II is viewed as an important discovery in the development of a novel group of antivirals that work by preventing HIV entry (Kanbara et al., 2000, Zhou et al., 2000) or of immunobiologics that inhibit lymphocyte mediated immunity (DeBruyne et al., 2000).

The purpose of this study was to extend earlier work on vMIP-II and HIV-1 to the SIVmac239/251 infection model. The inhibitory effect of vMIP-II on SIVmac has not been studied and the availability of both molecular clones of simian immunodeficiency virus (SIV) and a relevant animal infection model lead to possible applications of SIVmac vectors that express vMIP-II. In addition to gene delivery, such constructs may have attenuated virulence and/or altered immunogenicity that may make them desirable vaccine candidates. VMIP-II is an ideal candidate for insertion into viral vectors such as SIVmac239Δ3; it is relatively small and has immune stimulatory properties.

There is precedence for the insertion of cytokine genes into SIVmac as a means to alter virulence or host immunity. Genes of immunomodulatory proteins such as IFN-γ IL-2, or IL-12 have been previously inserted into SIVmac derived vectors and tested in rhesus monkeys (Giavedoni and Yilma, 1996, Giavedoni et al., 1997, Gundlach et al., 1997, Kledal et al., 1997, Kuwata et al., 2000, Sawai et al., 2000). The rationale for this approach is that the co-expression of viral and immunomodulatory proteins during the first few weeks of infection may enhance protective Th1 immunity. While vaccinated rhesus monkeys have shown some protection, it is uncertain whether the degree of protection is more than what could be induced with SIVmac239Δ3 alone (Giavedoni et al., 1997, Linhart et al., 1997). The cellular genes are rapidly deleted in vivo, leaving behind the replication competent virus (Giavedoni et al., 1997, Sawai et al., 2000).

The studies reported herein were first concerned with determining the antiviral properties of vMIP-II on SIVmac251 and what particular receptor might be implicated in such suppression. After demonstrating that vMIP-II was intact and the protein biologically active against SIVmac251, the behavior of the gene was studied in the context of replication competent and incompetent SIVmac239Δ3-based vectors in an effort to thoroughly assess gene deletion from the context of a lentivirus as well as the expression of an SIV suppressive gene in such a setting. We report here that vMIP-II can be expressed and biologically active from SIV-based vectors that are replication incompetent and that vMIP-II can suppress SIVmac251 infection.

Section snippets

Cloning vMIP-II into bacterial expression systems and SIVmac239Δ3

DNA from cells infected with HHV-8 (Renne et al., 1996) were PCR amplified using primers 5′ vMIP-II Xho-1 (5′-GCG CTC GAG ATG GAC ACC AAG GGC ATC CTG CTC GTC GCT-3′) and 3′ Not-1 (5′-GCG GCG GCC GCT CAG CGA GCA GTG ACT GGT AAT TGC TGC-3′) added to a 50 μl reaction mixture (5 μl 10× PCR buffer, 8 μl dNTP mix, 40 pmol each primer, 1.25U Taq DNA polymerase, and 37.5 μl sterile DNAse-free water) and amplified (1 cycle 95C/8 min, 35 cycles 95C/30 s, 55C/30 s, 72C/45 s, followed by terminal extension

vMIP-II mediated suppression of SIVmac251 infection of CEMx174 cells

Recombinant vMIP-II-GST and vMIP-II-6xHis significantly suppressed SIV p27 expression when added at a concentration of 200 nM/well to CEMx174 cell cultures prior to infection with SIVmac251 (Fig. 2a). The suppressive effects of vMIP-II on SIVmac251 infection was dependent on the concentration and the length of time post-infection during which the cultures were exposed to the protein. The greatest suppression occurred by the addition of vMIP-II-GST or vMIP-II-6xHis daily for up to 4 days

Discussion

The application of vMIP-II to gene therapy has great potential, mainly because of the broad range of biologic properties of the protein as well as a blocking effect on CCR5 and CXCR4 coreceptors in HIV-1 (Boshoff et al., 1997, Kledal et al., 1997, Kanbara et al., 2000, Luo et al., 2000, Luttichau et al., 2000, Zhou et al., 2000). However, the effect vMIP-II may have on SIV has not been studied and there are no reports of SIV-derived vectors expressing this viral chemokine. Furthermore, the

Acknowledgements

The authors would like to thank Thomas North, Earl T. Sawai, Peter Barry, Patty LiWang, May Chen, Cheryl London, Rob Grahn and Gerhart Reubel for their continuous support and technical advice.

References (51)

  • L.A. DeBruyne et al.

    Gene transfer of virally encoded chemokine antagonists vMIP-II and MC148 prolongs cardiac allograft survival and inhibits donor-specific immunity

    Gene Therapy

    (2000)
  • R.C. Desrosiers et al.

    Identification of highly attenuated mutants of simian immunodeficiency virus

    Journal of Virology

    (1998)
  • Dittmer, D. a. D.H.K., 1998. Do viral chemokines modulate Kaposi's Sarcoma? Bioessays 20...
  • E.J. Fernandez et al.

    Comparison of the structure of vMIP-II with Eotaxin-1, RANTES, and MCP-3 suggests a unique mechanism for CCR3 activation

    Biochemistry

    (2000)
  • J.M. Frade et al.

    Characterization of the CCR2 chemokine receptor: functional CCR2 receptor expression in B cells

    Journal of Immunology

    (1997)
  • L. Giavedoni et al.

    Expression of gamma interferon by simian immunodeficiency virus increases attenuation and reduces postchallenge virus load in vaccinated rhesus macaques

    Journal of Virology

    (1997)
  • L.D. Giavedoni et al.

    Construction and characterization of replication-competent simian immunodeficiency virus vectors that express gamma interferon

    Journal of Virology

    (1996)
  • J.S. Gibbs et al.

    Construction and in vitro properties of SIVmac mutants with deletions in ‘nonessential’ genes

    AIDS Research and Human Retroviruses

    (1994)
  • B.R. Gundlach et al.

    Construction, replication, and immunogenic properties of a simian immunodeficiency virus expressing interleukin-2

    Journal of Virology

    (1997)
  • H. Heath et al.

    Chemokine receptor usage by human eosinophils

    The Journal of Clinical Investigation

    (1997)
  • S. Hibbitts et al.

    Coreceptor ligand inhibition of fetal brain cell infection by HIV Type 1

    AIDS Research and Human Retroviruses

    (1999)
  • P.O. Ilyinskii et al.

    The role of upstream U3 sequences in the pathogenesis of simian immunodeficiency virus-induced AIDS in Rhesus Monkeys

    Journal of Virology

    (1994)
  • K. Kanbara et al.

    A study of anti-HIV compounds which interfere with the virus entry via coreceptor CXCR4

    Kansenshogaku Zasshi

    (2000)
  • F. Kirchhoff et al.

    Upstream U3 sequences in simian immunodeficiency virus are selectively deleted in vivo in the absence of an intact nef gene

    Journal of Virology

    (1994)
  • F. Kirchhoff et al.

    Simian immunodeficiency virus variants with differential T-cell and macrophage tropism use CCR5 and an unidentified cofactor expressed in CEMx174 cells for efficient entry

    Journal of Virology

    (1997)
  • View full text