Viruses in the mammalian male genital tract and their effects on the reproductive system.

This review describes the various viruses identified in the semen and reproductive tracts of mammals (including humans), their distribution in tissues and fluids, their possible cell targets, and the functional consequences of their infectivity on the reproductive and endocrine systems. The consequences of these viral infections on the reproductive tract and semen can be extremely serious in terms of organ integrity, development of pathological and cancerous processes, and transmission of diseases. Furthermore, of essential importance is the fact that viral infection of the testicular cells may result not only in changes in testicular function, a serious risk for the fertility and general health of the individual (such as a fall in testosteronemia leading to cachexia), but also in the possible transmission of virus-induced mutations to subsequent generations. In addition to providing an exhaustive account of the data available in these domains, this review focuses attention on the fact that the interface between endocrinology and virology has so far been poorly explored, particularly when major health, social and economical problems are posed. Our conclusions highlight the research strategies that need to be developed. Progress in all these domains is essential for the development of new treatment strategies to eradicate viruses and to correct the virus-induced dysfunction of the endocrine system.

HIV-1 replication in CD4+ T cell lines: the effects of adaptation on co-receptor use, tropism, and accessory gene function.

We studied the replication of HIV-1 macrophage-tropic CCR5-using strains (R5) in CD4+ T cell lines to better understand the switch in co-receptor use of such strains during disease progression and to assess resulting changes in cell tropism. We found that the majority of R5 strains cannot replicate in CD4+ T cell lines without adaptation by serial passage. A small minority of primary R5 isolates, however, were able to infect two T cell lines, Molt4 and SupT1. This expanded tropism was due to the use of undetectable levels of CCR5 rather than CXCR4 or alternative receptors. In contrast, HIV-1sF162 adaptation for replication in the C8166 T cell line was due to the emergence of variant strains that could use CXCR4. Of two variants, one was dual-tropic and one T-tropic, although both could use CCR5 as well as CXCR4. A single mutation in the start codon of the accessory gene vpu accounted for the T-tropic phenotype of the second variant, indicating that a non-functional vpu impairs macrophage tropism. Thus, in vitro and in the absence of an immune response, R5 strains naturally adapt to infect CXCR4+ T cell lines. Such adaptation resembles the rare R5 to X4 switch that occurs in vivo. Mutations in accessory genes (e.g., vpu) not required for replication in rapidly dividing cell lines may also occur in vitro, abrogating replication in primary cell types such as macrophages. Such mutations, however, are normally selected against in vivo.

Expanded tropism of primary human immunodeficiency virus type 1 R5 strains to CD4(+) T-cell lines determined by the capacity to exploit low concentrations of CCR5.

Human immunodeficiency virus type 1 (HIV-1) non-syncytium-inducing (NSI) strains predominantly use the chemokine receptor CCR5, while syncytium-inducing (SI) strains use CXCR4. In vitro, SI isolates infect and replicate in a range of CD4(+) CXCR4(+) T-cell lines, whereas NSI isolates usually do not. Here we describe three NSI strains that are able to infect two CD4(+) T-cell lines, Molt4 and SupT1. For one strain, a variant of JRCSF selected in vitro, replication on Molt4 was previously shown to be conferred by a single amino-acid change in the V1 loop (M.T. Boyd et al., J. Virol. 67:3649-3652, 1993). On CD4(+) cell lines expressing different coreceptors, these strains use CCR5 predominantly and do not replicate in CCR5-negative peripheral blood mononuclear cells derived from individuals homozygous for Delta32 CCR5. Furthermore, infection of Molt4 and SupT1 by each of these three strains is potently inhibited by ligands for CCR5, including 2D7, a monoclonal antibody specific for CCR5. CCR5 mRNA was present in both Molt4 and SupT1 by reverse transcription-PCR, although CCR5 protein could not be detected either on the cell surface or in intracellular vesicles. The expanded tropism of the three strains shown here is therefore not due to adaptation to a new coreceptor but due to the capacity to exploit extremely low levels of CCR5 on Molt4 and SupT1 cells. This novel tropism observed for a subset of primary HIV-1 isolates may represent an extended tropism to new CD4(+) cell types in vivo.

HIV coreceptors, cell tropism and inhibition by chemokine receptor ligands.

HIV is a persistent virus that survives and replicates despite an onslaught by the host's immune system. A strategy for cell entry, requiring the use of two receptors, has evolved that may help evade neutralizing antibodies. HIV and SIV usually require both CD4 and a seven transmembrane (7TM) coreceptor for infection. At least eleven different 7TM coreceptors have been identified that confer HIV and/or SIV entry. For HIV-1, the major coreceptors are CCR5 and CXCR4, while the role of other coreceptors for replication and cell tropism in vivo is currently unclear. Polymorphisms in the CCR5 gene that reduce CCR5 expression levels, protect against disease progression, suggesting that drugs targeted to CCR5 could be effective. Such therapies however will not work if HIV simply adapts to use alternative coreceptors. In the light of these themes, this review will discuss the following topics: (i) the coreceptors used by primary HIV-1 and HIV-2 viruses, (ii) the properties and coreceptors of HIV-2 strains that infect cells without CD4, (iii) the role of coreceptors in HIV cell tropism and particularly macrophage infection and (iv) the properties of chemokine receptor ligands that block HIV infection.

CXCR4 as a functional coreceptor for human immunodeficiency virus type 1 infection of primary macrophages.

The coreceptors used by primary syncytium-inducing (SI) human immunodeficiency virus type 1 isolates for infection of primary macrophages were investigated. SI strains using only CXCR4 replicated equally well in macrophages with or without CCR5 and were inhibited by several different ligands for CXCR4 including SDF-1 and bicyclam derivative AMD3100. SI strains that used a broad range of coreceptors including CCR3, CCR5, CCR8, CXCR4, and BONZO infected CCR5-deficient macrophages about 10-fold less efficiently than CCR5(+) macrophages. Moreover, AMD3100 blocked infection of CCR5-negative macrophages by these strains. Our results therefore demonstrate that CXCR4, as well as CCR5, is used for infection of primary macrophages but provide no evidence for the use of alternative coreceptors.

Expression of interferons-alpha and -gamma in testicular interstitial tissue and spermatogonia of the rat.

The testis is divided into two compartments: the seminiferous tubules and the interstitial tissue. The latter essentially consists of the blood and lymphatic vessels, testosterone-producing Leydig cells, and testicular macrophages. In the exploration of the testicular antiviral defense system, we initially searched for interferon (IFN) production by the seminiferous tubule cells. The site of virus entry into the testis is probably the interstitial compartment; thus, it is important to know whether and how the cells in this compartment are protected against viral infection. In addition, as germ cell precursors (spermatogonia) are only partially protected by the blood-testis barrier, it was important to explore the antiviral capability of these cells. In this study we searched for IFN production by Leydig cells, testicular macrophages, and spermatogonia after exposure to Sendai virus. We also investigated the effect of viral exposure on testosterone production by Leydig cells. Our results show that spermatogonia do not constitutively express IFNs and give a very poor response to the virus. In contrast, testicular macrophages constitutively produced type I IFNs, and this production was markedly stimulated by Sendai virus. Leydig cells produced twice as much type I IFNs as testicular macrophages after viral exposure, and they were the only cells producing both IFNalpha and -gamma, with these IFNs being dramatically induced/ increased in response to exposure to the virus. Furthermore, incubation of Leydig cells with the Sendai virus stimulated testosterone production. In conclusion, this study further establishes the topography of IFN expression within the testis. This allows us to hypothesize that the potential antiviral system represented by Leydig cells and, to a lesser extent, by macrophages plays a key role in protecting both androgen production and spermatogenesis.

Interferons and interferon-induced antiviral proteins in the testis.

Despite the dramatic development of sexually transmissible diseases, the antiviral capabilities of testicular cells have not yet been explored. Interferons (IFNs) are proteins playing a key role in the antiviral defense system, their activity being mediated by several IFN-induced proteins. In the present study, we have investigated both the expression of IFN and of the three main IFN-induced proteins by isolated testicular cells. The highest responders to a viral stimulation in terms of IFN production are the Leydig and the Sertoli cells, followed by peritubular cells and testicular macrophages, while germ cells are devoid or virtually devoid of IFN and IFN-induced protein expression. Sertoli cells constitutively expressed the three IFN-induced proteins tested, and their levels were greatly increased after exposure to Sendai virus. Peritubular cells were also able to markedly express these three proteins after viral exposure. In conclusion, we hypothesize that, for a virus coming from the blood, the first testicular line of defence is ensured by Leydig cells and testicular macrophages, the second line being ensured by the myoid cells, lining the seminiferous tubules, and by Sertoli cells. These two barriers are probably fundamental in protecting both androgen production and spermatogenesis.

The testicular antiviral defense system: localization, expression, and regulation of 2'5' oligoadenylate synthetase, double-stranded RNA-activated protein kinase, and Mx proteins in the rat seminiferous tubule.

Although the involvement of viruses in alterations of testicular function and in sexually transmitted diseases is well known, paradoxically, the testicular antiviral defense system has virtually not been studied. The well known antiviral activity of interferons (IFNs) occurs via the action of several IFN-induced proteins, among which the 2'5' oligoadenylate synthetase (2'5' A synthetase), the double-stranded RNA-activated protein kinase (PKR), and the Mx proteins are the best known. To explore the antiviral capacity of the testis and to study the testicular action of IFNs, we looked for the presence and regulation of these three proteins in isolated seminiferous tubule cells, cultured in the presence or in the absence of IFN alpha, IFN gamma, or Sendai virus. In all conditions tested, the meiotic pachytene spermatocytes and the post-meiotic early spermatids lacked 2'5' A synthetase, PKR, and Mx mRNAs and proteins. In contrast, Sertoli cells constitutively expressed these mRNAs and proteins, and their levels were greatly increased after IFN alpha or Sendai virus exposure. While peritubular cells were also able to markedly express 2'5' A synthetase, PKR, and Mx mRNA and proteins after IFN alpha or viral exposure, only PKR was constitutively present in these cells. Interestingly, IFN gamma had no effect on peritubular cells' 2'5' A synthetase and Mx production but it enhanced Mx proteins in Sertoli cells. In conclusion, this study reveals that the seminiferous tubules are particularly well equipped to react to a virus attack. The fact that the two key tubular elements of the blood-testis barrier, namely, Sertoli and peritubular cells, were found to assume this protection allows the extension of the concept of blood-testis barrier to the testicular antiviral defense.

Interferon-alpha and -gamma expression in the rat testis.

Interferon-alpha (IFN alpha), -beta, and -gamma are well known for their antiviral, antiproliferative, and immunoregulatory activities. Although several studies suggest an involvement of IFNs in the spermatogenic process, nothing is known about the possible production of these molecules within the testis. Moreover, the antiviral capabilities of testicular cells have not yet been explored despite their importance in the context of sexually transmissible diseases. Using reverse transcription-polymerase chain reaction, a cytopathic inhibition micromethod assay, and an enzyme-linked immunosorbent assay, the present study demonstrates for the first time that IFN alpha and -gamma are produced by testicular cells. IFN alpha protein and corresponding messenger RNA are expressed by peritubular, Sertoli, and germ cells. In vitro, IFN alpha production by Sertoli cells, peritubular cells, and early spermatids was inducible by the Sendai virus, whereas pachytene spermatocyte IFN alpha production was not triggered by this virus. Of all the testicular cell types tested, Sertoli cells by far produced the highest concentrations of IFN alpha/beta, followed by peritubular cells. Both IFN gamma messenger RNA and IFN gamma protein were found in early spermatids, but, in contrast, were not produced by peritubular cells, Sertoli cells, or pachytene spermatocytes. In conclusion, our study establishes the cellular distribution of IFNs within the seminiferous tubules and provides the basis for research into the possible involvement of IFNs in regulation of the spermatogenic process. To the best of our knowledge, our results afford the first insight on how the testicular antiviral defense system is organized.